Purified multivalent protein-hyaluronic acid polymer conjugates

Abstract

The present invention relates to purified hyaluronic acid conjugates containing bioactive peptides. The present invention further relates to pharmaceutical compositions of the conjugates and methods for their preparation.

Description

[Technical field]

[0001] Not applicable CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 331,548, filed April 15, 2022, which is incorporated by reference in its entirety for all purposes. Sequence Listing

[0002] The contents of the attached Sequence Listing are incorporated herein by reference in their entirety. 2023-4-11 Sequence_Listing_ST26 The attached file named 052566-507001WO.xml was created on April 11, 2023 and is 187,801 bytes in size. [Background technology]

[0003] 2. Background of the Invention The use of biopolymers to modify the properties of bioactive agents is a recurring theme across a wide range of medical and biological applications. Various chemical linkers can be used to attach bioactive peptides or proteins to biopolymers to modify the pharmacological properties of the resulting conjugates for use as drugs that can provide optimal treatment for specific diseases. The use of peptide-polymer conjugates, which contain multiple copies of one or more species of peptides attached to a single biopolymer chain, provides distinct improvements in the pharmacological properties of the peptides, including: (1) higher binding affinity to biological targets, (2) slower diffusivity through target tissues, and (3) inhibition of proteases that can inactivate the bioactivity of the peptide or protein.

[0004] These improved pharmacological properties of peptide-polymer conjugates are particularly useful for the delivery of potent drugs delivered directly to diseased tissues. Because the drug is administered locally to the target tissue, the dose delivered directly to the tissue can be lower than that required to achieve the same therapeutic effect after systemic administration. It is also possible to administer drugs to tissues that have poor transport properties from the blood. Specific examples of tissues where direct drug administration is common include the posterior chamber of the eye using intravitreal injections and the joints using intra-articular injections.

[0005] However, local tissue administration requires a specialist to safely provide the necessary injections, which increases the burden and cost of administration compared to systemic administration. When peptide drugs are administered as part of a peptide-polymer conjugate, the frequency of drug administration can be significantly reduced, thereby reducing the burden on patients to receive effective treatment. Furthermore, reducing the number of local injections reduces the risk of local tissue damage and injection side effects. Finally, the need to reduce administration frequency can shorten the time that drug concentrations in target tissues fall below therapeutic concentrations, thereby improving the overall effectiveness of the drug. Based on these advantages, there is a strong motivation to develop protein-polymer pharmaceuticals for various diseases.

[0006] The synthesis of multivalent Sonic hedgehog proteins conjugated to hyaluronan via an N-ε-maleimidocaproic acid hydrazide linker has been reported. See Wall, ST et al. Bioconjugate Chemistry 2008, 19, 806-812.

[0007] To properly formulate a peptide-polymer conjugate as a drug product, one must achieve a sufficiently high drug concentration to allow proper administration to a patient. One must also prepare purified peptide-polymer conjugates that exhibit high bioactivity and storage stability, for example by being able to remain in solution for up to two years from the date of manufacture to the date of clinical use. Interactions between peptide-polymer conjugates can adversely affect the ability to achieve any of these drug-enabling properties.

[0008] The method used to link the polymer and peptide can have a substantial effect on the pharmacological properties, intra-conjugate interactions, and inter-conjugate interactions of the conjugate.Therefore, there is a need to develop purified peptide-polymer conjugates using specific linker methods that achieve favorable pharmacological properties for a given disease and allow successful formulation into pharmaceutical products.The present invention meets this and other needs. Summary of the Invention

[0009] BRIEF SUMMARY OF THE PRESENTINVENTION In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa and has the following formula III: [ka] {In the formula, each X is independently a 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 following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; Each R 1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 is independently H or C1-C6 alkyl; each Z 3a is independently 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; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 100 and 10,000.

[0010] In some embodiments, the conjugate has a molecular weight of about 0.8 MDa and has the following formula IIIa: [ka] {In the formula, each X 1 is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO:67; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21); Each Y has the structure: [ka] is an organic linker having the formula: each X 1 -X 2 -YZ 1 The moiety has the following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; each Z 3a is independently OH or Y'; Each Y' has the structure: [ka] having; Each R 1 and R 2 is ethyl or -(CH2)3-NMe2; subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; the subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 1000 and 3000.

[0011] In some embodiments, the pharmaceutical composition comprises a conjugate described herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient.

[0012] In some embodiments, the methods of the present invention are methods of treating an ocular disease in a subject in need thereof, comprising administering to the subject a conjugate described herein.

[0013] In some embodiments, the methods of the invention include a method of treating a joint disease or disorder in a subject in need thereof, comprising administering to the subject a conjugate described herein.

[0014] In some embodiments, the method for preparing a conjugate of the present invention comprises the following steps: (a) a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa, about 0.1 to about 2 equivalents of a coupling agent per hyaluronic acid monomer, and a compound of the formula HN-R Y {In the formula, R Y below: [ka] and and subscript m is an integer from 1 to 300; thereby forming a first reaction mixture containing an organic linker agent of formula IV: [ka] forming an intermediate polymer having a monomer of (b) forming a second reaction mixture comprising the intermediate polymer and a peptide having a molecular weight of about 5 kDa to about 200 kDa, wherein the peptide comprises one or more -SH groups; thereby preparing a conjugate; Includes. [Brief description of the drawings]

[0015] [Figure 1A] FIG. 1A shows the expressed open reading frame (ORF). [Figure 1B] FIG. 1B shows the soluble expression (mAU*mL) of Hu2H10 or 2H10 containing various peptide linkers. [Figure 1C]FIG. 1C shows protein expression by SDS-PAGE of Hu2H10_5MUT (SEQ ID NO: 55), Hu2H10_5MUT_CYS (SEQ ID NO: 141), or Hu2H10_5MUT_aH_CYS (SEQ ID NO: 142). [Figure 1D] FIG. 1D shows the soluble expression (fold process yield) of HuNb42_A88P (SEQ ID NO: 67) or HuNb42_A88P aH_CYS (SEQ ID NO: 145).

[0016] [Diagram 2] FIG. 2 shows the amino acid sequences of 2H10 and point mutation variants.

[0017] [Diagram 3] Figure 3 shows protein expression of Hu2H10 mutants. The Y-axis shows immobilized metal affinity chromatography (IMAC) peak area (mAU*mL). The X-axis shows protein expression of Hu2H10_5MUT ("WT(5MUT)") (SEQ ID NO: 55), Hu2H10_5MUT_R86K_A87P ("R86K_A87P") (SEQ ID NO: 56), Hu2H10_5MUT_L115Q ("L115Q") (SEQ ID NO: 57), Hu2H10_5MUT_R86K_A87P_L115Q ("R86K_A87P_L115Q") (SEQ ID NO: 58).

[0018] [Figure 4] FIG. 4 shows the amino acid sequences of Nb42 and point mutation variants.

[0019] [Diagram 5] FIG. 5 shows Coomassie Brilliant Blue (CBB) staining of E. coli cell extracts expressing HuNb42 and point mutation variants.

[0020] [Figure 6] FIG. 6 shows the relative E. coli cell culture yields of HuNb42 (SEQ ID NO: 61) and HuNb42 A88P (SEQ ID NO: 67) point mutation variants in the cytoplasm and periplasm.

[0021] [Figure 7] FIG. 7 shows the relative humanity of Nb42 (SEQ ID NO: 61), HuNb42 (SEQ ID NO: 62), and HuNb42_A88P (SEQ ID NO: 67) compared to caplacizumab, bevacizumab, and ranibizumab.

[0022] [Figure 8] FIG. 8 shows Coomassie Brilliant Blue (CBB) staining of E. coli cell extracts expressing aTNFaMu (SEQ ID NO: 71) or aTNFaMu_3MUT (SEQ ID NO: 72) at room temperature, 50°C, 60°C, 70°C, and 80°C.

[0023] [Figure 9] FIG. 9 shows the relative protein expression of E1-1 (SEQ ID NO: 81) and point mutants E1-1 F11L (SEQ ID NO: 82), E1-1 S49A (SEQ ID NO: 83), E1-1 F11L / S49A (SEQ ID NO: 84), and E1-1 CDR (SEQ ID NO: 85).

[0024] [Figure 10] Figure 10A shows Coomassie Brilliant Blue (CBB) staining of purified HuNb42 protein (SEQ ID NO: 62) induced at 18°C ​​and 37°C. Figure 10B shows purified Hu2H10_5MUT protein (SEQ ID NO: 55) induced at 16°C, 27°C, 30°C, and 37°C.

[0025] [Figure 11A] FIG. 11A shows two preparations of HuNb42 A88P (SEQ ID NO: 67), with ("220119") or without ("220204") EDTA treatment. [Figure 11B]FIG. 11B shows the recovery of TNFα3MUT (SEQ ID NO: 104) before ("Input") and after treatment with EDTA and filtration through a 50 kDa ("50 kDa FT") or 100 kDa ("100 kDa FT") polyethersulfone membrane (left side), and endotoxin levels before ("Input") and after filtration through a 50 kDa ("50 kDa FT") or 100 kDa ("100 kDa FT") polyethersulfone membrane (right side). [Figure 11C] FIG. 11C shows an SDS-PAGE gel of purified aAng2_D4_aH_CYS (sequence number 120).

[0026] [Figure 12] Figure 12 shows the Rh distribution of DARPin multivalent proteins (MVPs) prepared by method 1 (conjugate 5, conjugate 2) and method 5 (conjugate 3, conjugate 6, conjugate 7). MVPs prepared using method 5 showed a smaller MVP radius.

[0027] [Figure 13A-13B] 13A-13B show the activity of MVP. [Figure 13A] Figure 13A shows biolayer interferometry (BLI) VEGF binding and dissociation curves of anti-VEGF MVPs made using various intermediates and anti-VEGF E1-1 peptide. Top: binding and dissociation curves for method 2 conjugate 10 (0.123 KD = nM). Bottom: binding and dissociation curves for method 5 conjugate 8 (0.184 KD = nM) showing baseline, and for various concentrations of conjugate 8 (x-axis: time in seconds, y-axis: BLI signal). Both conjugates show similar binding kinetics curves and theoretically calculated dissociation constants. [Figure 13B]Figure 13B shows the VEGF binding affinity ("KD", nM) of two peptides compared to the corresponding MVP. First bar: non-binding 2H10_5MUT_aH_CYS (SEQ ID NO: 142); second bar: non-binding HuNb42_A88P_aH_CYS (SEQ ID NO: 145); third bar: MVP-containing 2H10_5MUT_aH_CYS (SEQ ID NO: 142); fourth bar: MVP-containing HuNb42_A88P_aH_CYS (SEQ ID NO: 145).

[0028] [Figure 14] Figure 14 shows SEC traces of MVP stability samples at various ages (X-axis: retention time, Y-axis: absorbance at 280 nm). Examples of SEC retention time changes for anti-VEGF DARPin MVPs synthesized using intermediates made using Method 5 (conjugate 1, top) or Method 1 (conjugate 2, bottom) and stored at 37°C for up to 71 days. MVPs synthesized using Method 5 (top) showed a greater loss of radius upon degradation over time.

[0029] [Figure 15] FIG. 15 shows the change in radius of gyration of DARPin MVPs produced using intermediate method 1 (conjugate 2, upper line) or method 5 (conjugate 3, lower line) after aging under accelerated conditions for 28 and 32 days at 37° C.

[0030] [Figure 16] Figure 16 shows the VEGF binding constants (KD) of BI VHH anti-VEGF MVPs made using intermediate method 1, method 2 or method 5 before and after aging at 37°C. By day 4 of the stability study, MVPs synthesized using the intermediate of method 5 no longer bound VEGF. The limit of detection (LOD) of BLI is 0.001 nM. The samples listed in the figure with this value were below the LOD of BLI for KD.

[0031] [Figure 17]17 shows the change in binding constant (K on ) of DARPin MVPs made using intermediate method 1 (conjugate 2, squares, upper line) or method 5 (conjugate 1, triangles, lower line) after aging under accelerated conditions for 28 and 32 days at 37° C. A lower K on initially and upon aging and slower binding was observed for conjugate 1, suggesting that the low impurity polymer of conjugate 2 stabilizes and enhances the K on .

[0032] [Figure 18] Figure 18 shows the in vivo half-life extension of VHH and MVP synthesized using method 1 intermediate following intravitreal injection in rabbits. Rabbit intravitreal pharmacokinetic (IVT PK) study of conjugate 12 anti-TNFα VHH MVP (method 1 intermediate), >2X half-life extension vs. unbound. n=3 eyes per time point. All eyes received 50 μg of VHH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Invention I. Overview The present invention provides purified peptide-hyaluronic acid polymer conjugates, in which each peptide is covalently attached to the polymer using a linker, and a method for preparing the same. The purified peptide-hyaluronic acid conjugates exhibit higher stability compared to previously described conjugates. II. Definition

[0034] Unless otherwise specified, 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. Furthermore, 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:

[0035] When referring to values, "about" includes the stated value + / - 10% of the stated value. For example, about 50% includes the range of 45% to 55%, whereas about 20 molar equivalents includes 18 to 22 molar equivalents. Thus, when referring to ranges, "about" refers to the respective stated value + / - 10% of the stated value at each end of the range. For example, the range of about 1 to about 3 (wt / wt) includes the range of 0.9 to 3.3.

[0036] "Alkyl" refers to a straight or branched chain saturated monovalent or divalent hydrocarbon. For example, an alkyl group can be any group having 1 to 10 carbon atoms (i.e., C 1-10 alkyl) or 1 to 8 carbon atoms (i.e., C 1-8 alkyl) or 1 to 6 carbon atoms (i.e., C 1-6 alkyl) or 1 to 4 carbon atoms (i.e., C 1-4Examples of alkyl groups include, but are not limited to, 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-B u, s-butyl, -CH(CH3)CH2CH3), 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 (-CH2CH2CH(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(C H3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, and octyl (-(CH2)7CH3).

[0037] "Cycloalkyl" means an alkyl group having 3 to 20 cyclic carbon atoms (i.e., C 3-20Cycloalkyl) refers to a single saturated or partially unsaturated all-carbocyclic ring, e.g., having 3 to 12 ring atoms, e.g., 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. The term "cycloalkyl" also includes polycondensed, saturated and partially unsaturated all-carbocyclic ring systems (e.g., ring systems containing 2, 3 or 4 carbocyclic rings). Thus, cycloalkyl includes polycyclic carbocyclic rings, such as bicyclic carbocyclic rings (e.g., bicyclic carbocyclic rings having about 6 to 12 annular 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 annular carbon atoms). The rings of a polycondensed ring system may be connected to each other through fused, spiro, and bridged bonds, if permitted by valence requirements. 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.

[0038] As used herein, "organic linker" refers to a chemical moiety that directly or indirectly covalently links a peptide to a polymer. Organic linkers useful in the present invention are about 100 Da to 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. One of skill in the art will appreciate that other types of organic linkers are useful in the present invention.

[0039] "Thiol" refers to the -SH functional group.

[0040] "Heteroalkyl" refers to an alkyl group of any suitable length and having 1-6 heteroatoms, such as N, O, and S. Additional heteroatoms, including but not limited to B, Al, Si, and P, may also be useful. Heteroatoms may also be oxidized, such as but not limited to -S(O)- and -S(O)2-. For example, heteroalkyls may include ethers, thioethers, and alkylamines. The heteroatom portion of a heteroalkyl may replace a hydrogen on an alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom portion may be the linking atom or inserted between two carbon atoms.

[0041] As used herein, "coupling agent" refers to a reagent that effects a reaction between a carboxylic acid (-(C=O)-OH) and an amine (-NH2) group to form an amide (-(C=O)-NH-).

[0042] "Peptide", "polypeptide", and "protein" are used interchangeably herein to 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 naturally occurring amino acids. The term "peptide" encompasses fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; immunologically tagged proteins, and the like. Peptides further encompass post-translationally modified polypeptides.

[0043] As used herein, "VHH" refers to a single domain heavy chain antibody.

[0044] "DARPin" refers to designed ankyrin repeat proteins, which are engineered antibody-mimetic proteins that can exhibit high specificity and high affinity target protein binding.

[0045] An "alpha-helix" or "α-helix" is a common motif in protein secondary structure, a right-handed helical conformation in which every backbone NH group hydrogen is bonded to a backbone C=O group of an amino acid located four residues prior along the protein sequence. An alpha-helix is ​​also called a Pauling-Corey-Branson α-helix or 3.6 (meaning an average number of residues per turn of the helix, with 13 atoms involved in the ring formed by hydrogen bonds). 13 An alpha-helix is ​​also known as an alpha-helix. A peptide that contains an alpha-helix is ​​said to be alpha-helical. Such a peptide may be partially or completely alpha-helical. As understood in the art, an alpha-helix has at least four amino acid residues. In some embodiments, an alpha-helix has between 4 and 40 amino acids.

[0046] Pharmaceutically acceptable salts of the peptides or conjugates described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms, and other materials that are useful for preparing pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0047] As used herein, "pharmaceutical composition" refers to a product that contains specific ingredients in specific amounts, as well as any product that results directly or indirectly from the combination of specific ingredients in specific amounts. Pharmaceutical compositions are generally safe for bioavailability.

[0048] As used herein, "pharmaceutical acceptable excipient" refers to a substance that aids in the administration of an active agent for absorption 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 pharmaceutical carriers and / or excipients are useful in the present invention.

[0049] The compounds described herein may be prepared and / or formulated as pharmaceutically acceptable salts, or as free bases, as appropriate. Pharmaceutically acceptable salts are non-toxic salts of the free base form of the compounds that possess the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids or bases. For example, compounds containing basic nitrogen may be prepared as pharmaceutically acceptable salts by contacting the compounds with inorganic or organic acids. 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, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne, hexyne-1,4-dioate ... Examples of suitable pharma- ceutically acceptable salts include, but are not limited to, 1,6-dioate, 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.

[0050] Examples of "pharmaceutically acceptable salts" of the conjugates described herein include alkali metal (e.g., sodium, potassium), alkaline earth metal (e.g., magnesium), ammonium, and NR4 +Also included are salts derived from appropriate bases, such as, for example, where R is C1-C4 alkyl. Also included are base addition salts, such as sodium or potassium salts.

[0051] As used herein, "therapeutically effective amount" refers to the dosage that produces the therapeutic effect that it is administered. The exact dosage depends on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (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 amount may be lower than the usual therapeutically effective amount in non-sensitized cells.

[0052] As used herein, "Inhibition," "inhibits," and "inhibitor" refer to a compound or method that prevents a particular action or function.

[0053] As used herein, "treatment" or "treat" or "treating" refers to an approach for obtaining 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 worsening of symptoms associated with a disease or condition. In one embodiment, "treatment" or "treating" includes one or more of: 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) delaying or preventing the onset of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, slowing the progression or worsening of a disease or condition); and c) relieving a disease or condition, e.g., causing regression of clinical symptoms, improving a disease state, slowing progression of a disease, improving quality of life, and / or prolonging survival.

[0054] "Prophylaxis" refers to preventing or slowing the progression of clinical disease in a patient suffering from a disease.

[0055] A "subject" of the present invention is a mammal, which may be a human or a non-human mammal, such as a companion animal, such as a dog, cat, rat or the like, a livestock animal, such as a horse, donkey, mule, goat, sheep, pig, or cow or the like. In some embodiments, the subject is a human.

[0056] As used herein, "joint" refers to a fibrous or cartilaginous joint, which is a fibrous or cartilaginous area where two or more bones connect to one another.

[0057] As used herein, "diffusion half-life" refers to the time it takes for the initial concentration of a conjugate in a given volume or space to decrease by half, where the decrease in concentration is a function of the concentration gradient.

[0058] As used herein, "intra-articular half-life" refers to the time it takes for the initial concentration of a conjugate in a particular joint to decrease by half, and transport out of the joint is via convection, which is a combination of transport by diffusion and advection, whereas advection is the transport of a substance by bulk motion. III. Peptides

[0059] In some embodiments, the peptides of the present invention offer advantages over comparable peptides in the art, such as a high degree of humanity, high solubility, high stability, low tendency to aggregate in solution, and / or high expression levels in convenient systems such as E. coli.

[0060] In some embodiments, the peptide has the following formula (I): [ka] A peptide having the formula: CDR1, CDR2, and CDR3 are each independently a complementarity determining region; FR1 is the following: X 10 VQLX 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1) {In the formula, X 10 is Q, E, or D, X 11 is V, Q, A, or E; X 12 is S or T, X 13 is L, S, or V; X 14 is V or A, X 15 is P, A, or T, X16 is G, D, or R, X 17 is A, V, T, or E; and X 18 is A or V} having an amino acid sequence comprising: FR2 is the following: X 20 X 21 WX 22 RQX 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2) {In the formula, X 20 is M, I, V, or L; X 21 is G, S, or A, X 22 is F, Y, or V; X 23 is A, V, P, or T, X 24 is E, G, A, or Q, X 25 is R or L, X 26 is F, G, W, or L; X 27 is A, G, or S; and X 28 is A, S, or G} having an amino acid sequence comprising: FR3 is: X 30 DSVKGRFTISX 31 DX 32 X 33 KX 34 X 35 VX 36 LQMX 37 X 38 LRX 39a EDTAX 39b YYCAA (SEQ ID NO: 3) {In the formula, X30 is A, G, S, or T, X 31 is R or Q, X 32 is N, S, or D, X 33 is S, A, or D, X 34 is N or K, X 35 is T or M, X 36 is Y, D, or S; X 37 is N or D, X 38 is S or N, X 39a is P or A, and X 39b is V, M, L or I} having an amino acid sequence comprising: FR4 is: YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4) {In the formula, X 40 is Q or K, X 41 is L or Q} The peptide has an amino acid sequence comprising:

[0061] In some embodiments, X 13 is L.

[0062] In some embodiments, X 27 is A.

[0063] In some embodiments, X 30 is A.

[0064] In some embodiments, X 39a is P.

[0065] In some embodiments, X 40 is Q.

[0066] In some embodiments, FR1 has an amino acid sequence comprising QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5).

[0067] In some embodiments, FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6).

[0068] In some embodiments, FR3 has an amino acid sequence comprising YADSVKGRFTISRDNSKNTVYLQMNSLRPE DTAVYYCAA (SEQ ID NO:7).

[0069] In some embodiments, FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0070] In some embodiments, FR1 has an amino acid sequence comprising QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5); FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6); FR3 has an amino acid sequence comprising: YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7); and FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0071] In some embodiments, CDR1, CDR2, and CDR3 are complementarity determining regions from an antibody or a cytokine, respectively. In some embodiments, the antibody 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, the complementarity determining regions are specific for vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-α), programmed cell death protein 1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), cluster of differentiation 40 (CD40), cluster of differentiation 134 (CD134), cluster of differentiation 137 (CD137), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), V-domain immunoglobulin inhibitor of T cell activation (VISTA), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), lymphocyte activation gene 3 (LAG3), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), or interleukin 15 (IL-15), respectively. In some embodiments, the complementarity determining regions are each specific for vascular endothelial growth factor (VEGF).

[0072] In some embodiments, the peptide consists of Formula I.

[0073] In some embodiments, the peptide has one or more of the following: (a) a CDR1 that is 7 amino acids in length; (b) a CDR2 that is 7 or 8 amino acids in length; and / or (c) a CDR3 that is 9-16 amino acids in length.

[0074] In some embodiments of the peptide, (a) CDR1 has an amino acid sequence comprising FAYSTYS (SEQ ID NO: 9), CDR2 has an amino acid sequence comprising NSGTFRLW (SEQ ID NO: 10), and CDR3 has an amino acid sequence comprising RAWSPYSSTVDAGDFR (SEQ ID NO: 11); or (b) CDR1 has an amino acid sequence comprising RRFSIEA (SEQ ID NO: 12), CDR2 has an amino acid sequence comprising DSGGSTD (SEQ ID NO: 13), and CDR3 has an amino acid sequence comprising IGGSWYGRGLD (SEQ ID NO: 14); or (c) CDR1 has an amino acid sequence comprising GTFSSII (SEQ ID NO: 15), CDR2 has an amino acid sequence comprising SWSGGTTV (SEQ ID NO: 16), and CDR3 has an amino acid sequence comprising RPYQKYNWASASYNV (SEQ ID NO: 17); or (d) CDR1 has an amino acid sequence comprising GGSDAGT (SEQ ID NO: 18), CDR2 has an amino acid sequence comprising SWAGTAWR (SEQ ID NO: 19), and CDR3 has an amino acid sequence comprising LGSYEMDHH (SEQ ID NO: 20).

[0075] In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-98, 101-109, 111-131, and 141-170. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 142. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 145.

[0076] In some embodiments, the peptide has an amino acid sequence comprising any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-95, 101-106, and 111-118. In some embodiments, the peptide has an amino acid sequence comprising any one of SEQ ID NOs: 73, 81, 91, and 92. In some embodiments, the peptide has an amino acid sequence comprising any one of SEQ ID NOs: 101-106. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 67. IV. Conjugates

[0077] In some embodiments, the conjugate has the following formula IIa: [ka] {In the formula, each X 1 is independently a peptide described herein; each X 2 are independently a peptide linker having a length of 3 to 100 amino acids; each Y is independently an organic linker; Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; and The subscript n is an integer from 1 to 1500. It is a conjugate of.

[0078] In some embodiments, the conjugate has the following formula IIb: [ka] {In the formula, each X 1 are independently peptides having a molecular weight of about 5 kDa to about 200 kDa; each X 2A is, independently, a peptide linker comprising an α-helix; each Y is independently an organic linker; Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; and The subscript n is an integer from 1 to 1500. It is a conjugate of.

[0079] In some embodiments, each X 1 are independently peptides of the invention.

[0080] In some embodiments, each peptide linker is independently between 7 and 100 amino acids in length. In some embodiments, each peptide linker is independently between 10 and 30 amino acids in length.

[0081] In some embodiments, each peptide linker independently comprises the following amino acid sequence: 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 (SEQ ID NO:27), Includes.

[0082] In some embodiments, each peptide linker comprises one of the following: AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21), The amino acid sequence comprises:

[0083] Each peptide can be linked to a biocompatible polymer by various organic linkers commonly known in the art to form antibody-drug conjugates, such as those provided by Conju-probe or BroadPharm of San Diego, CA, or Creative Biolabs of Shirley, NY. Methods for forming bioconjugate bonds are described in Bioconjugate Techniques, 3 rd Edition, Greg T. Hermanson. The organic linkers can be reactive with amines, carbonyls, carboxyls and activated esters, can react by click chemistry (with or without copper) or can be reactive with thiols.

[0084] 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, generally having a molecular weight of about 100 Da to about 500 Da, including two functional groups consisting of maleimide and either an amine or a hydrazide. In some embodiments, the peptide is covalently attached 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 includes a 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.

[0085] Exemplary organic linkers include, but are not limited to, the following: [ka] Examples include:

[0086] In some embodiments, the organic linker is N-epsilon-maleimidocaproic acid hydrazide (EMCH): [ka] It is.

[0087] In some embodiments, the organic linker has the following 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.

[0088] In some embodiments, the organic linker has the following structure: [ka] has.

[0089] In some embodiments, the preparation of the conjugates 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 conjugates of the present invention, unreacted organic linkers are present on the biocompatible polymer. The structure of the unreacted organic linkers depends on the organic linker and will be understood by those skilled in the art.

[0090] Representative free organic linkers include, but are not limited to, the following: [ka] Examples include:

[0091] In some embodiments, the unreacted organic linker has the following structure: [ka] has.

[0092] In some embodiments, the unreacted organic linker has the following 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.

[0093] In some embodiments, the unreacted organic linker has the following structure: [ka] has.

[0094] In some embodiments, the biocompatible polymer is a polysaccharide.

[0095] In some embodiments, the biocompatible polymer is a glycosaminoglycan.

[0096] In some embodiments, the biocompatible polymer is hyaluronic acid.

[0097] In some embodiments, the biocompatible polymer has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, the biocompatible polymer has a molecular weight of about 0.7 MDa to about 1.5 MDa. In some embodiments, the biocompatible polymer has a molecular weight of about 0.8 MDa.

[0098] In some embodiments, the subscript n is an integer from 1 to 1500. In some embodiments, the subscript n is an integer from 5 to 1000. In some embodiments, the subscript n is an integer from 10 to 400. In some embodiments, the subscript n is an integer from 10 to 100.

[0099] In some embodiments, the conjugate has the following formula IIa: [ka] {In the formula, each X 1 is independently a peptide described herein; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21); Each Y has the structure: [ka] is an organic linker having the formula: Z is a biocompatible polymer that is hyaluronic acid having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript m is an integer from 1 to 300; and The subscript n is an integer from 1 to 1500. It is a conjugate of.

[0100] In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa and has the following formula III: [ka] {In the formula, each X is independently a 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 following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; Each R 1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 is independently H or C1-C6 alkyl; each Z 3a is independently 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; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 100 and 10,000. The conjugate is a random polymer of

[0101] In some embodiments, each X is a peptide having an amino acid sequence comprising any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-98, 101-109, 111-131, and 141-170. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 142. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 145.

[0102] In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-95, 101-106, and 111-118.

[0103] In some embodiments, the conjugate has the structure of Formula IIIa: [ka] {In the formula, each X 1 are independently peptides having a molecular weight of about 5 kDa to about 200 kDa; each X 2 is a peptide linker containing an α-helix} has.

[0104] In some embodiments, each X 1 is represented by the following formula (I): [ka] and a peptide having the formula: CDR1, CDR2, and CDR3 are each independently a complementarity determining region; FR1 is the following: X 10 VQLX 11 EX 12 GGGX13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1) {In the formula, X 10 is Q, E, or D, X 11 is V, Q, A, or E; X 12 is S or T, X 13 is L, S, or V; X 14 is V or A, X 15 is P, A, or T, X 16 is G, D, or R, X 17 is A, V, T, or E; and X 18 is A or V} having an amino acid sequence comprising: FR2 is the following: X 20 X 21 WX 22 RQX 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2) {In the formula, X 20 is M, I, V, or L; X 21 is G, S, or A, X 22 is F, Y, or V; X 23 is A, V, P, or T, X 24 is E, G, A, or Q, X 25 is R or L, X 26is F, G, W, or L; X 27 is A, G, or S; and X 28 is A, S, or G} having an amino acid sequence comprising: FR3 is: X 30 DSVKGRFTISX 31 DX 32 X 33 KX 34 X 35 VX 36 LQMX 37 X 38 LRX 39a EDTAX 39b YYCAA (SEQ ID NO: 3) {In the formula, X 30 is A, G, S, or T, X 31 is R or Q, X 32 is N, S, or D, X 33 is S, A, or D, X 34 is N or K, X 35 is T or M, X 36 is Y, D, or S; X 37 is N or D, X 38 is S or N, X 39a is P or A, and X 39b is V, M, L or I} and FR4 is: YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4) {In the formula, X 40 is Q or K, X41 is L or Q} The peptide has an amino acid sequence comprising:

[0105] In some embodiments, X 13 is L.

[0106] In some embodiments, X 27 is A.

[0107] In some embodiments, X 30 is A.

[0108] In some embodiments, X 39a is P.

[0109] In some embodiments, X 40 is Q.

[0110] In some embodiments, FR1 has an amino acid sequence comprising QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5).

[0111] In some embodiments, FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6).

[0112] In some embodiments, FR3 has an amino acid sequence comprising YADSVKGRFTISRDNSKNTVYLQMNSLRPE DTAVYYCAA (SEQ ID NO:7).

[0113] In some embodiments, FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0114] In some embodiments, FR1 has an amino acid sequence comprising QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5); FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6); FR3 has an amino acid sequence comprising: YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7); and FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0115] In some embodiments, CDR1, CDR2, and CDR3 are complementarity determining regions from an antibody or a cytokine, respectively.

[0116] In some embodiments, the antibody 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, the antibody is a monoclonal IgG. In some embodiments, the antibody is an IgG fragment. In some embodiments, the antibody is a single domain heavy chain VHH. In some embodiments, the antibody is a DARPin.

[0117] In some embodiments, the complementarity determining regions are specific for vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-α), programmed cell death protein 1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), cluster of differentiation 40 (CD40), cluster of differentiation 134 (CD134), cluster of differentiation 137 (CD137), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), V-domain immunoglobulin inhibitor of T cell activation (VISTA), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), lymphocyte activation gene 3 (LAG3), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), or interleukin 15 (IL-15), respectively. In some embodiments, each of the complementarity determining regions is specific for vascular endothelial growth factor (VEGF). In some embodiments, each of the complementarity determining regions is specific for tumor necrosis factor alpha (TNF-α). In some embodiments, each of the complementarity determining regions is specific for interleukin-1β (IL-1β).

[0118] In some embodiments, the peptide consists of Formula I.

[0119] In some embodiments, the peptide has one or more of the following: (a) a CDR1 that is 7 amino acids in length; (b) a CDR2 that is 7 or 8 amino acids in length; and / or (c) a CDR3 that is 9-16 amino acids in length.

[0120] In some embodiments of the peptide, (a) CDR1 has an amino acid sequence comprising FAYSTYS (SEQ ID NO: 9), CDR2 has an amino acid sequence comprising NSGTFRLW (SEQ ID NO: 10), and CDR3 has an amino acid sequence comprising RAWSPYSSTVDAGDFR (SEQ ID NO: 11); or (b) CDR1 has an amino acid sequence comprising RRFSIEA (SEQ ID NO: 12), CDR2 has an amino acid sequence comprising DSGGSTD (SEQ ID NO: 13), and CDR3 has an amino acid sequence comprising IGGSWYGRGLD (SEQ ID NO: 14); or (c) CDR1 has an amino acid sequence comprising GTFSSII (SEQ ID NO: 15), CDR2 has an amino acid sequence comprising SWSGGTTV (SEQ ID NO: 16), and CDR3 has an amino acid sequence comprising RPYQKYNWASASYNV (SEQ ID NO: 17); or (d) CDR1 has an amino acid sequence comprising GGSDAGT (SEQ ID NO: 18), CDR2 has an amino acid sequence comprising SWAGTAWR (SEQ ID NO: 19), and CDR3 has an amino acid sequence comprising LGSYEMDHH (SEQ ID NO: 20).

[0121] In some embodiments, each X 1 is a peptide having an amino acid sequence including any one of SEQ ID NOs: 51 to 58, 61 to 73, 81 to 85, 91 to 95, 101 to 106, and 111 to 118. 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 67. 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 73. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO:91.

[0122] In some embodiments, each X 2 below: 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 (SEQ ID NO:27), is a peptide linker having an amino acid sequence comprising:

[0123] In some embodiments, each X 2 below: AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21), is a peptide linker having an amino acid sequence comprising:

[0124] In some embodiments, the organic linker has the following structure: [ka] has.

[0125] In some embodiments, the organic linker is N-epsilon-maleimidocaproic acid hydrazide (EMCH): [ka] It is.

[0126] In some embodiments, the organic linker has the following 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.

[0127] In some embodiments, the organic linker has the following structure: [ka] The organic linker having the above structure is known as MP2H.

[0128] In some embodiments, the random polymer of formula III 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.

[0129] In some embodiments, each R 1 and R 2 are 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; and each R 2 is -(CH2)3-NMe2. In some embodiments, each R 1 is -(CH2)3-NMe2; and each R 2 is ethyl.

[0130] In some embodiments, each R 3 and R 4 is independently C1-C3 alkyl. In some embodiments, each R 3 and R 4 is methyl.

[0131] 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.

[0132] In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa and has the following formula III: [ka] {In the formula, Each X is independently one of the following: QVQLQES GGGLVQPGGS LRLSCAASGR TFSDHSGYTY TIGWFRQAPG KEREFVARIY WSSGNTYYAD SVKGRFAISR DIAKNTVDLT MNNLEPEDTA VYYCAARDGI PTSRSVESYN YWGQGTQVTV SSPSTPPTPS PSTPPGGCDD DDK (SEQ ID NO: 101), QVQLQES GGGLVQPGGS LRLSCAASGR TFSDHSGYTY TIGWFRQAPG KEREFVARIY WSSGNTYYAD SVKGRFAISR DIAKNTVDLT MNNLEPEDTA VYYCAARDGI PTSRSVESYN YWGQGTQVTV SSAEAAAKEA AAKEAAAKAG C (SEQ ID NO: 102), QVQLQDS GGGLVQAGGS LRLSCAASGG TFSSIIMAWF RQAPGKEREF VGAVSWSGGT TVYADSVLGR FEISRDSARK SVYLQMNSLK PEDTAVYYCA ARPYQKYNWA SASYNVWGQG TQVTVSSAEA AAKEAAAKEA AAKAGC (SEQ ID NO: 103), QVQLQES GGGLVQAGGS LRLSCAASGG TFSSIIMAWF RQAPGKEREF VGAVSWSGGT TVYADSVKGR FTISRDSARK SVYLQMNSLK PEDTAVYYCA ARPYQKYNWA SASYNVWGQG TQVTVSSAEA AAKEAAAKEA AAKAGC (SEQ ID NO: 104), CGGGVDNKFN KEVGWAFGEI GALPNLNALQ FRAFIISLWD DPSQSANLLA EAKKLNDAQA PK (SEQ ID NO: 105), or EIVMTQS PSTLSASVGD RVIITCQASQ SIDNWLSWYQ QKPGKAPKLL IYRASTLASG VPSRFSGSGS GAEFTLTISS LQPDDFATYY CQNTGGGVSI AFGQGTKLTV LGGGGGSGGG GSGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCTASGFS LSSAAMAWVR QAPGKGLEWV GIIYDSASTY YASWAKGRFT ISRDTSKNTV YLQMNSLRAE DTAVYYCARE RAIFSGDFVL WGQGTLVTVS SSPSTPPTPS PSTPPGGC (SEQ ID NO: 106); an anti-TNF-α or anti-IL-1β peptide comprising: Each Y has the structure: [ka] is an organic linker having the formula: each XYZ 1 The moiety has the following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; Each R 1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 is independently H or C1-C6 alkyl; each Z 3a is independently OH or Y'; Each Y' has the structure: [ka] is an unreacted organic linker having subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 100 and 10,000.

[0133] In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa and has the following formula III: [ka] {In the formula, each X is independently a 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 following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; Each R 1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 is independently H or C1-C6 alkyl; each Z 3a is independently 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; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 100 and 10,000.

[0134] In some embodiments, each X is a peptide having an amino acid sequence comprising any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-98, 101-109, 111-131, and 141-170. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 142. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 145.

[0135] In some embodiments, the conjugate has a molecular weight of about 0.8 MDa and has the following formula IIIa: [ka] {In the formula, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO:55; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21); Each Y has the structure: [ka] is an organic linker having the formula: each X 1 -X 2 -YZ 1 The moiety has the following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; each Z 3a is independently OH or Y'; Each Y' has the structure: [ka] having; Each R 1 and R 2 is ethyl or -(CH2)3-NMe2; subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; the subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 1000 and 3000.

[0136] In some embodiments, the conjugate has a molecular weight of about 0.8 MDa and has the following formula IIIa: [ka] {In the formula, each X 1 is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO:67; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21); Each Y has the structure: [ka] is an organic linker having the formula: each X 1 -X 2 -YZ 1 The moiety has the following structure: [ka] having; each Z 2 has the following structure: [ka] having; each Z 3 may independently have the structure: [ka] having; each Z 3a is independently OH or Y'; Each Y' has the structure: [ka] having; Each R 1 and R 2 is ethyl or -(CH2)3-NMe2; subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; the subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of the subscripts n, p, and q; and The subscript q is an integer between 1000 and 3000.

[0137] In some embodiments, the conjugates of the invention exhibit a half-life in vivo of about 12 hours to about 24 hours, about 1 day to about 3 days, about 3 days to about 7 days, about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, or about 1 month to about 6 months.

[0138] In some embodiments, the conjugates of the invention exhibit a therapeutically effective residence time in vivo of about 12 hours to about 24 hours, about 1 day to about 3 days, about 3 days to about 7 days, about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, about 1 month to about 3 months, or about 3 months to about 6 months.

[0139] The bioactivity of the conjugate is enhanced compared to the activity of the corresponding peptide in soluble form, e.g., compared to the activity of the peptide not conjugated to a polymer. In some embodiments, the bioactivity of the conjugate is at least about 25%, at least 50%, at least 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold, or greater than 1000-fold, than the bioactivity of the peptide in soluble (unconjugated) form. V. Composition

[0140] In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition comprising a conjugate described herein and a pharma- ceutically acceptable excipient. A. Formulations

[0141] For preparing pharmaceutical compositions from the conjugates of the present invention, pharma- ceutically acceptable carriers can be either solid or liquid. Solid formulations include powders, cachets, and dispersible granules. A solid carrier can be one or more substances that can also act as diluents, binders, preservatives, disintegrants, or encapsulating materials. Details of formulation and administration techniques are well explained in the scientific and patent literature. See, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington").

[0142] In powders, the carrier is a finely divided solid, which is mixed 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.

[0143] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions.For parenteral injection, solutions can be formulated in solution in aqueous polyethylene glycol solution.

[0144] 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 made 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 acacia, and dispersing or wetting agents, such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and 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 preparation may be adjusted for osmotic pressure.

[0145] Also included are solid form preparations intended to be converted immediately prior to use into liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, and the like.

[0146] Oily suspensions can be formulated by suspending the conjugate of the present invention in a vegetable oil, such as arachis oil, olive oil, sesame oil, or coconut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Oily suspensions can contain a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol, or sucrose. These preparations can be preserved by the addition of an antioxidant, such as ascorbic acid. For examples of injectable oily vehicles, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The formulation 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 acacia and gum tragacanth, natural phosphatides such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, condensation products of these partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents, as in the case of the formulation of syrups and elixirs. Such formulations may also contain a demulcent, preservative, or coloring agent.

[0147] The composition 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 for sustained subcutaneous release (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995); as biodegradable and injectable gel formulations (see Gao Pharm. Res. 12:857-863, 1995); or as microspheres for oral administration (see Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).Both transdermal and intradermal routes allow constant delivery for weeks or months.

[0148] In another embodiment, the compositions of the present invention can be formulated for parenteral administration, for example, intratumoral administration, intravitreal administration into the eye, or into a body cavity, such as the intra-articular space of a joint. Formulations for administration will generally comprise a solution of the compositions 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, isotonic sodium chloride. 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, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables as well. These solutions are sterile and generally free of undesirable matter. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharma-ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the composition of the present invention in these formulations can vary widely and is selected mainly based on the volume of body fluids, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the patient. For IV, intratumoral or intravitreal administration, the formulation may be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol.

[0149] In another embodiment, the formulation of the composition of the present invention can be delivered by using liposomes that fuse with cell membranes or are endocytosed, i.e., by using ligands attached to the liposomes or directly attached to the oligonucleotides that bind to the surface membrane proteins of the cell's receptors that cause endocytosis.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 directed to a particular organ (see, for example, 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).

[0150] Lipid-based drug delivery systems include lipid solutions, lipid emulsions, lipid dispersions, self-emulsifying drug delivery systems (SEDDS), and self-microemulsifying drug delivery systems (SMEDDS). In particular, SEDDS and SMEDDS are isotropic mixtures of lipids, surfactants, and co-surfactants that disperse spontaneously in aqueous media to form fine emulsions (SEDDS) or microemulsions (SMEDDS). Lipids useful in the formulations of the present invention include any natural or synthetic lipid, including, but not limited to, sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®. B. Administration

[0151] The conjugates and compositions of the invention can be delivered by any suitable means, including oral, parenteral and topical methods. In some embodiments, the delivery method is intra-articular. In some embodiments, the delivery method is intra-articular. In some embodiments, the delivery method is intravitreal. In some embodiments, the delivery method is intratumoral.

[0152] The pharmaceutical preparation is preferably in unit dosage form.In such form, the preparation is subdivided into unit doses containing appropriate amounts of the conjugate and composition of the present invention.The unit dosage form may be a packaged preparation, the package containing discrete amounts of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules.

[0153] The conjugates and compositions of the present invention can be co-administered with other agents. Co-administration includes administration of the conjugates or compositions of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the other agent. Co-administration also includes administration at the same time, about the same time (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or consecutively 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 provide a preferred daily dosage level.

[0154] In some embodiments, simultaneous administration can be achieved by co-formulation, i.e., preparing a single pharmaceutical composition containing the conjugates and compositions of the invention and any other agents, or alternatively, the various components can be formulated separately.

[0155] The conjugates and compositions of the present invention, as well as 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, disease state, etc. 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, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg. The compositions may also include other compatible therapeutic agents. The conjugates described herein can be used in combination with each other, with other active agents known to be useful for modulating the glucocorticoid receptor, or in combination with adjuvants that are not effective alone but may contribute to the effectiveness of the active agent. VI. Treatment method

[0156] In some embodiments, the present invention relates to methods and / or uses comprising the conjugates or compositions described herein for the treatment of a disease or disorder in a subject in need thereof.

[0157] In some embodiments, the method comprises multiple administrations of the conjugate. In some embodiments, the method comprises administering the conjugate daily, daily, every third day, or weekly. In some embodiments, the method comprises administering the conjugate weekly, every two weeks, every three weeks, or every month. 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 per year. In some embodiments, the method comprises administering the conjugate annually. A. Eye disorders

[0158] In some embodiments, the methods of the present invention are methods of treating an ocular disease in a subject in need thereof, comprising administering to the subject a conjugate described herein.

[0159] In some embodiments, the methods include administering the conjugate intravitreally.

[0160] In some embodiments, the methods comprise administering the conjugate every month, every two months, or every three months.

[0161] In some embodiments, the intravitreal half-life of the conjugate is at least 2, 3, 4, 5, 10, 20, 50, or at least 100 times longer than the half-life of the unconjugated peptide, in some embodiments, the intravitreal half-life of the conjugate is at least 5 times longer than the half-life of the unconjugated peptide.

[0162] The eye disorders that can be treated using the method of the present disclosure include, but are not limited to, uveitis, macular degeneration also known as age-related macular degeneration (AMD), choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, and ocular inflammation.In some embodiments, the macular degeneration is wet macular degeneration.In some embodiments, the macular degeneration is dry macular degeneration.

[0163] Ocular diseases that may be treated using the methods of the present disclosure include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting a posterior ocular site or location; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusion, retinal detachment, and uveoretinopathy; sympathetic ophthalmia; Vogt-Koyanagi-Harada syndrome; uveal diffusion; ocular posterior ocular conditions caused or affected by laser treatment for the treatment of vision problems; posterior ocular conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorder; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome; cone-rod dystrophy; Stargardt disease (fundus flammeus); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; total choroidal atrophy; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic atrophy; retinopathy of prematurity; and color vision disorders such as achromatopsia, protanopia, deutzia, and tritanopia.

[0164] In some cases, the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, diabetic retinopathy, or achromotopsia or color blindness.

[0165] In some cases, the composition comprising the conjugate is administered via intravitreal, transscleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intralacrimal routes. In some cases, the composition comprising the conjugate is administered intravitreally. In some cases, the composition is delivered intravitreally or very close to the posterior segment. In some cases, the composition is administered intravitreally by injection. In some cases, the composition comprising the conjugate is administered by intraocular injection. B. Joint Disease

[0166] In some embodiments, the methods of the invention include a method of treating a joint disease or disorder in a subject in need thereof, comprising administering to the subject a conjugate described herein.

[0167] In some embodiments, the methods include administering the conjugate intra-articularly.

[0168] In some embodiments, the intra-articular half-life of the conjugate is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or at least 100-fold longer than the half-life of the unconjugated peptide. In some embodiments, the intra-articular half-life of the conjugate is at least 5-fold longer than the half-life of the unconjugated peptide.

[0169] The present invention also provides a method for treating diseases and disorders of articular tissues using the conjugates of the present invention.Examples of diseases and disorders of articular tissues include, but are not limited to, rheumatoid arthritis, wear-related osteoarthritis, age-related osteoarthritis, post-traumatic osteoarthritis, psoriatic arthritis, and aseptic implant loosening, joint effusion, ankylosing spondylitis, bursitis, gout, reactive arthritis, synovitis, and avascular necrosis.In some embodiments, the disease or disorder is rheumatoid arthritis, wear-related osteoarthritis, age-related osteoarthritis, post-traumatic osteoarthritis, psoriatic arthritis, and aseptic implant loosening, joint effusion, ankylosing spondylitis, bursitis, gout, reactive arthritis, synovitis, or avascular necrosis.

[0170] Many polypeptides have been used as drugs to attenuate immune cell function, which has considerable utility in the treatment of many joint disorders. Articular tissues are particularly susceptible to injury and disease, as the typical cellular response to these attacks, i.e., upregulation of inflammatory mediators, is also a signal that promotes catabolism of articular cartilage and resorption of underlying bone tissue. Degeneration of articular surfaces promotes the worsening of damage to articular tissues, further promoting the regulation of inflammatory mediators. Over time, these mechanisms generate a feed-forward loop, resulting in cumulative damage to articular tissues.

[0171] Any joint in the human or animal body can be treated using the methods and conjugates of the present invention. Representative joints include, but are not limited to, fibrous joints, cartilaginous joints, synovial joints, facet joints, immobile joints, hemijoints, and mobile joints. The joints can be simple joints with two articular surfaces, compound joints with three or more articular surfaces, or complex joints with two or more articular surfaces and joint knee or meniscus. Anatomical joints that can be treated using the conjugates and methods of the present invention include, but are not limited to, the hand joints including the fingers, elbow joints, wrist joints, shoulder joints, sternum and clavicle joints, spinal joints, jaw and skull joints, pelvis and hip joints, knee joints, ankle joints, and ankle joints including the toes. Joints can also be classified as planar joints, ball and socket joints, hinge joints, pivot joints, condylar joints, and saddle joints. The conjugates and methods of the present invention can be used to treat tissues of joints including, but not limited to, connective tissue, cartilage, articular surfaces, synovial cavities, menisci, etc.

[0172] Examples of drugs designed to attenuate immune cell function include antibodies that may interfere with tumor necrosis factor-α and IL-1β, IL-6, or interferon-γ. Other examples include selective antibody inhibitors of T cell and B cell function. These antibodies may be monoclonal IgG antibodies, IgG antibody fragments, single chain scFv antibodies, single domain heavy chain VHH antibodies, or engineered antibody-like scaffolds such as adnectins, affibodies, anticalins, DARPins, and engineered Kunitz-type inhibitors. Other examples also include decoy receptors for immunomodulatory cytokines such as tumor necrosis factor-α and IL-1β, IL-6, or interferon-γ.

[0173] One of the common side effects of using anti-inflammatory drugs such as those mentioned above is a high risk of infection. Because they weaken the body's immune response, the immune system is compromised to fight bacteria, viruses, and parasites. Therefore, the benefits of systemic use of these drugs must be carefully weighed against the risks associated with systemic immunosuppression. In the case of diseases in which the entire body is affected by hyperimmune disorders such as rheumatoid arthritis, systemic use of immune attenuating drugs may be justified. However, in the case of conditions that only affect one or a limited number of joints, the systemic risk of infection often does not justify systemic use of these drugs.

[0174] Alternatively, intra-articular (IA) administration of immunomodulatory drugs has been proposed to prevent or inhibit the long-term effects of inflammation associated with osteoarthritis. However, these drugs are rapidly cleared from the joint cavity and do not provide an adequate therapeutic window following IA administration. After IA injection, the half-life of anti-inflammatory proteins in the synovium is short (<1.5 hours). This is evident from clinical studies in which anti-inflammatory drugs, including infliximab and etanercept, were administered by IA injection to humans for various joint disorders. Some of these studies reported significant reductions in joint inflammation but acknowledged that frequent (e.g., weekly) administration was necessary for successful outcomes. Thus, IA anti-inflammatory therapy using existing drugs is limited by the high cost and inconvenience of frequent IA administration. Clearly, methods are needed to extend the bioactivity of anti-inflammatory drugs within the synovial fluid to enable this therapeutic approach to treat joint disorders.

[0175] The main symptoms associated with joint disorders are pain, effusion, limited range of motion, and pathologic remodeling of the joint anatomy. The efficacy of a treatment for treating a joint disorder may include pain relief measured by a generalized assessment, such as a visual assessment score. Efficacy can also be determined based on improved scores using systems specific to the particular joint disorder, such as the WOMAC score for osteoarthritis, the ACR20 for rheumatoid arthritis, the psoriatic arthritis quality of life for psoriatic arthritis, or the SASSS for ankylosing spondylitis. Efficacy can also be measured using functional outputs, such as increased pain-free walking distance or increased range of joint motion. Efficacy can also be measured based on radiographic evidence showing restoration of normal joint anatomy.

[0176] The conjugate can be administered at any suitable frequency or amount, as described above. In some embodiments, the conjugate is injected into the joint about once a month or less. In some embodiments, the conjugate is injected into the joint about once a month to once every six months. In some embodiments, the conjugate is injected into the joint about once every two months or once every three months. 1. Osteoarthritis

[0177] In 2015, an estimated 7.75 million Americans experienced symptoms of osteoarthritis (OA) that may be related to known joint damage. Post-traumatic OA (PTOA) accounts for at least 15% of all OA cases, although it has been postulated that many other OA diagnoses may also be related to prior joint trauma. In the absence of disease-modifying therapies, joint replacement is often the only treatment option to relieve associated discomfort and restore mobility. However, PTOA is often diagnosed in younger patients for whom joint replacement is not a viable option. Overall, the cost of treating these PTOA patients is over $4 billion in healthcare costs each year.

[0178] Short-term suppression of injury-related inflammation limits the long-term symptoms of PTOA. Many types of joint injury are associated with PTOA, including dislocations, ligament ruptures, meniscal injuries, and intra-articular fractures. Although the initial injury may be acute, the damage is sufficient to initiate a cascade of inflammatory mediators. The resulting chronic, whole-joint inflammation can promote catabolism of articular cartilage, leading to further tissue damage that accumulates over time and manifests as PTOA. TNFα and IL-1β have well-known roles in mediating joint inflammation. These cytokines interact to promote cartilage destruction, both by downregulating the expression of cartilage matrix components and by upregulating the expression of matrix metalloproteinases (MMPs). TNFα also stimulates the recruitment of osteoclasts and induces apoptosis of bone-forming osteoblasts in an inflammatory environment. This contributes to the erosion of articular cartilage tissue. TNFα and IL-1β are compelling targets for mitigating the inflammatory response to joint injury. Inhibition of these important acute inflammatory cytokines in the joint environment has been proposed for early intervention to slow the progression of PTOA. 2. Inflammation caused by immune response to intra-articular microparticles

[0179] Wear occurring between articular surfaces of joints can generate micron-scale particles that cause joint inflammation and osteolysis. Wear particles can be generated by abrasion between endogenous surfaces such as ossified cartilage lesions, osteophytes (bone spurs) or exposed subchondral bone lesions. This type of wear particle generation occurs frequently in the later stages of OA, causing severe joint pain and immobility. This additional inflammatory response accelerates the rate of degeneration of joint tissue in OA.

[0180] Wear particles may also form between the surfaces of artificial joints. In 2015, over 7 million Americans were living with an implanted artificial joint. Nearly a quarter of a million of these individuals ultimately required revision surgery due to osteolysis of the bone surrounding the device, which ultimately leads to loosening and failure of the device.

[0181] Wear-related inflammation results from a foreign body reaction to inert particulates released from the articular surfaces. Macrophages within the synovial lining readily recognize wear particulates as foreign and release proinflammatory factors that recruit other active immune cells to the synovium, stimulating osteoclast expansion while simultaneously inhibiting bone formation. Thus, persistent inflammation triggers a feed-forward cycle in which cartilage degeneration and osteolysis increase friction between the articular surfaces, which in turn increases movement and physical stress, resulting in increased particulates.

[0182] In some embodiments, the peptide modulates the activity of an immune cell function. In some embodiments, the peptide inhibits tumor necrosis factor-α, interleukin-1β, interleukin-6, or interferon-γ. In some embodiments, the peptide inhibits tumor necrosis factor-α.

[0183] Tumor necrosis factor (TNFα) is a compelling target for controlling foreign body responses. TNFα has a well-known role in mediating joint inflammation. TNFα also stimulates osteoclast recruitment and induces apoptosis of bone-forming osteoblasts in an inflammatory environment, leading to osteolysis of subchondral bone. Inhibition of TNFα using a systemically administered receptor antagonist (etanercept) has been shown to reduce bone resorption induced by wear particles in mice, but the risks associated with systemic anti-TNFα are generally not considered acceptable in localized conditions. As an alternative, IA anti-TNFα therapy has been proposed to prevent or inhibit osteolytic responses to intra-articular wear particles.

[0184] In some embodiments, the use of the present invention is the use of a conjugate as described herein for the preparation of a medicament for a method of treating a disease or disorder in a subject.

[0185] In some embodiments, the subject is a human.

[0186] In some embodiments, the use of the present invention is for treating a disease or disorder, comprising a conjugate or pharmaceutical composition as described herein.

[0187] In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition for use to treat a disease or disorder, comprising a conjugate as described herein.

[0188] In some embodiments, the conjugate of the invention is for use in treating a disease or disorder described herein. VII. Preparation method

[0189] In some embodiments, a method is provided for preparing a peptide of the invention, comprising: (a) transferring a genetic sequence encoding the peptide into a bacterium in a first reaction mixture; and (b) removing endotoxins from the first reaction mixture by forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA); thereby preparing the peptide.

[0190] In some embodiments, a method is provided for preparing a peptide of the invention, comprising: (a) transferring a genetic sequence encoding the peptide into bacteria in a first reaction mixture; (b) forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA); and (c) filtering the second reaction mixture; thereby preparing the peptide. In some embodiments, the second reaction mixture further comprises sodium chloride. In some embodiments, the second reaction mixture further comprises sodium citrate. In some embodiments, the second reaction mixture further comprises sodium citrate pH 5.5.

[0191] In some embodiments, the bacterium is E. coli.

[0192] In some embodiments, the second reaction mixture comprises about 0.1 mM to about 5 mM EDTA, hi some embodiments, the second reaction mixture comprises about 0.2 mM to about 1 mM EDTA.

[0193] Filtration of the second reaction mixture is accomplished by any method known in the art. In some embodiments, filtration of the second reaction mixture comprises a membrane filtration. In some embodiments, the membrane filtration comprises polyethersulfone (PES) or regenerated cellulose. For example, the membrane filtration comprises a 50 kDa or 100 kDa PES membrane.

[0194] In some embodiments, the method for preparing a conjugate of the present invention comprises the following steps: (a) a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa, about 0.1 to about 2 equivalents of a coupling agent per hyaluronic acid monomer, and a compound of the formula HN-R Y {In the formula, R Y below: [ka] and and subscript m is an integer from 1 to 300; thereby forming a first reaction mixture containing an organic linker agent of formula IV: [ka] forming an intermediate polymer having a monomer of (b) forming a second reaction mixture comprising the intermediate polymer and a peptide having a molecular weight of about 5 kDa to about 200 kDa, wherein the peptide comprises one or more -SH groups; thereby preparing a conjugate; Includes.

[0195] In some embodiments, the hyaluronic acid polymer has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, the hyaluronic acid polymer has a molecular weight of about 0.7 MDa to about 1.5 MDa. In some embodiments, the hyaluronic acid polymer has a molecular weight of about 0.8 MDa.

[0196] In some embodiments, the first reaction mixture includes from about 0.2 to about 1.5 equivalents of coupling agent per hyaluronic acid monomer. In some embodiments, the first reaction mixture includes from about 0.2 to about 1 equivalent of coupling agent per hyaluronic acid monomer.

[0197] In some embodiments, the coupling agent comprises a carbodiimide. In some embodiments, the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1,3-diisopropylcarbodiimide, or dicyclohexylcarbodiimide, or a salt thereof. In some embodiments, the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or a salt thereof.

[0198] In some embodiments, R Y below: [ka] It is.

[0199] In some embodiments, the first reaction mixture comprises about 0.2 to about 6 equivalents of organic linker agent per hyaluronic acid monomer.

[0200] In some embodiments, the first reaction mixture includes a catalyst. In some embodiments, the catalyst is ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), hydroxybenzotriazole, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), or 1-hydroxy-7-azabenzotriazole, or a salt thereof. In some embodiments, the catalyst is hydroxybenzotriazole.

[0201] In some embodiments, the second reaction mixture comprises about 0.5 to about 1.5 equivalents of peptide per organic linker.

[0202] In some embodiments, the method for preparing a conjugate of the present invention comprises the following steps: (a) a hyaluronic acid polymer having a molecular weight of about 0.8 MDa, about 0.2 to about 1 equivalent of a coupling agent per hyaluronic acid monomer, and a compound of the formula HN-R Y {where, R Y below: [ka] 4, whereby a first reaction mixture is formed containing an organic linker agent of the following formula IV: [ka] forming an intermediate polymer having a plurality of monomers of wherein the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or a salt thereof; and the first reaction mixture comprises about 0.2 to about 6 equivalents of an organic linker agent per hyaluronic acid monomer; and (b) forming a second reaction mixture comprising the intermediate polymer and a peptide having a molecular weight of about 5 kDa to about 200 kDa, where the peptide comprises one or more -SH groups; thereby preparing a conjugate; Includes.

[0203] The following structure: [ka] and known as 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-(3-hydrazinyl-3-oxopropoxy)ethoxy)ethyl)propanamide, also known by the abbreviation MP2H. References to "MP2H" as an organic linker agent or organic linker as used herein are understood in light of its usage by those of skill in the art. EXAMPLES

[0204] VIII. Working Examples Certain abbreviations and acronyms are used in describing the Experimental Details, 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. Table 1. List of Abbreviations and Acronyms [Table 1]

[0205] The general methods were used in the following examples. Addition of peptide linkers for protein / conjugate stability

[0206] In-frame fusion between the therapeutic protein and the C-terminal peptide linker was achieved by two methods. E. coli codon optimized nucleotides encoding the peptide linker were added to the therapeutic ORF (with a single C-terminal cysteine ​​residue for MVP conjugation) and ordered as a linear Geneblock (IDT or similar) with overhangs compatible for direct cloning into a protein expression plasmid. Alternatively, an oligonucleotide primer complementary to the therapeutic ORF containing the codon optimized sequence encoding the peptide linker was extended, amplified by PCR reaction, and generated a linear amplicon that was directly cloned into a protein expression plasmid. Sanger sequencing was performed on all isolated plasmids to confirm the correct substitution and integrity of the ORF containing the therapeutic fused to the peptide linker. All ORFs were expressed by an IPTG-inducible T7 promoter in a commercially available T7-adapted E. coli strain. Cytoplasmic expression in E. coli

[0207] To measure the amount of soluble protein expressed in E. coli, the protein was expressed under the control of an IPTG-inducible T7 promoter (NEB Shuffle T7 Express) and grown to an OD of 0.6 in Terrific Broth. 600nm The cultures were grown to 0.5 mL and induced with 0.5 mM IPTG for a period of 4 hours at 37° C. Culture sizes varied depending on needs and objectives, but ranged from 10 mL to 1 L. Expression of E. coli periplasm

[0208] For expression in the E. coli periplasm, the periplasmic targeting sequence MalE for E. coli was added to the N-terminus of the ORF. All subsequent expression and downstream purification techniques were performed without modification. IMAC and affinity tag removal

[0209] E. coli pellets from 1 L cultures were lysed by sonication in 25 mM HEPES, 20 mM imidazole, 400 mM sodium chloride, 0.5 mM EDTA, 5% glycerol, 0.01% Tween 20 (pH 7.5), clarified at 20 k*g, and applied to a GE Ni NTA HisTrap™ column. Non-specific proteins were washed off with the above buffer containing an additional 40 mM imidazole. Proteins of interest were then eluted using an FPLC with a gradient to 260 mM imidazole. Purity was confirmed by SDS-PAGE, and elution peak areas identified by AKTA Unicorn software were used as a comparison measure for culture output. In some cases, proteins were expressed with an N-terminal TEV cleavable IMAC affinity tag (removed after IMAC purification). Protein A Purification

[0210] In cases where a polyhistidine affinity tag was not used, Protein A resin (JSR Life Sciences, Amsphere A3) was used for capture of sdAb from clarified E. coli lysate in 20 mM Tris, 25 mM sodium chloride, 0.5 mM EDTA, pH 8.5. The immobilized sdAb was then washed with fresh lysate buffer and then eluted with 50 mM sodium citrate pH 5, 25 mM NaCl, 1 mM EDTA. Advanced purification by chromatography

[0211] For further purification of the protein, the pooled IMAC eluate was diluted 5-fold with nanopure water and applied to a GE HiTrap Q HP column pre-equilibrated with 20 mM Tris, 25 mM sodium chloride, 0.5 mM EDTA, pH 8.5. These conditions were adequate to remove contaminating E. coli proteins from the affinity chromatography eluate pool with the target protein remaining in the column effluent. The Q column effluent was further diluted 2-fold with nanopure water, adjusted to pH 5 with acetic acid, and applied to a GE HiTrap SP HP column pre-equilibrated with 10 mM sodium citrate, 0.25 mM EDTA, pH 5.0. The purified protein was eluted using a gradient to 25 mM sodium citrate, 0.5 M sodium chloride, 1 mM EDTA, pH 5.5. Purity was confirmed by SDS-PAGE, and elution peak areas identified by AKTA Unicorn software were used as a comparison measure of protein yield. Endotoxin removal and protein polishing

[0212] Pure SP eluate fractions were pooled and the material was passed twice through a 100 kDa regenerated cellulose spin concentrator to remove endotoxins. The 100 kDa spin concentrator effluent protein solution was then concentrated with a 3 kDa regenerated cellulose spin concentrator to a protein concentration of >175 mg / mL. Sterile glycerol was then added to 10% CF (v / v) before flash freezing and storage at -80°C.

[0213] Alternatively, a final concentration / purification step was performed by cation exchange chromatography after Q anion exchange chromatography. Proteins were bound at pH 5 and eluted through a gradient from solution A (10 mM sodium citrate pH 5, 0.25 mM EDTA) to solution B (25 mM sodium citrate pH 5.5, 1 M NaCl, 1 mM EDTA), typically eluting with 10-25% B. Peak fractions were then pooled and these pooled protein solutions were then passed through a 100 kDa membrane filter (either PES or regenerated cellulose) at 3000*g. The pure protein was then further concentrated by a 10 kDa membrane filter. Gel analysis of free cysteine

[0214] To confirm that a single free cysteine ​​was available for conjugation to a biopolymer, ∼20 equivalents of a 1.2 kDa PEG-maleimide moiety were incubated at 42°C for 45 min and run on a 4-20% SDS-PAGE to confirm that the mobility shifted by a single 1.2 kDa gel mobility shift. Additional protein / conjugate stabilizing mutations

[0215] Protein sequences were searched by BLAST queries against the PDB database. The MSA procedure was performed using the Jalview program, which included sequence alignment using Clustal Omega, manual curation of sequences from the alignment to include only sdAbs with the desired topology, and removal of sequence redundancy such that only ∼100 sequences remained in the MSA. Positions in the MSA with a conservation score of 9 or higher were considered as consensus and incorporated into the sdAb sequence, including a C-terminal α-helical linker peptide between the ORF and the conjugation cysteine. Site-directed mutagenesis

[0216] Complementary oligonucleotide pairs containing the desired codon-optimized amino acid substitution mutations were designed according to the guidelines published in the Agilent QuikChange site-directed mutagenesis kit protocol and purchased from IDT. SDM PCR reactions were performed on ∼10 ng of plasmid DNA according to the manufacturer's protocol. Freshly isolated plasmids were subjected to Sanger sequencing to confirm the appropriate amino acid substitution(s). Heat precipitation of soluble proteins

[0217] 25x volume of lysis buffer 25mM HEPES, 20mM imidazole, 400mM sodium chloride, 0.5mM EDTA, 5% glycerol, 0.01% Tween 20, pH 7.5 was added to E. coli pellets harvested from 25mL TB cultures and sonicated 5 times on ice at 40% power using a probe sonicator. Upon lysis, whole cell extracts were clarified at 10K*g, 100μL of supernatant was aliquoted and incubated at 50°C, 60°C, 70°C, and 80°C for 15 minutes, followed by incubation on ice for 10 minutes. Heat precipitated proteins were removed at 10K*g for 5 minutes, and the soluble extract fractions were immediately combined with Laemmli sample buffer, denatured, and run on a 4-20% SDS-PAGE to assess yield and stability. Example 1. Evaluation of peptide linkers

[0218] Peptides containing a bioactive peptide of interest attached to a peptide linker prior to attachment to the polymer were expressed in E. coli based on the expression open reading frame (ORF) in Figure 1 A. Various peptide linkers connecting the bioactive peptide to the polymer were evaluated (Table 2). Table 2. Peptide linkers [Table 2]

[0219] Figure IB shows soluble expression of an exemplary protein, 2H10, in E. coli. Improved yield of the soluble fraction was observed with 2H10 variants containing an α-helical peptide linker. The presence of the α-helical peptide linker improved expression when a reactive cysteine ​​was present at the C-terminus of the protein.

[0220] Figure 1C shows that the increase in soluble expression is evident in SDS-PAGE analysis of the comparative peptides. Cultures (5 mL) containing autoinduction medium and carbenicillin were grown to saturation overnight at 37°C. Saturated cultures were harvested by centrifugation, washed with 1 mL PBS, and harvested by centrifugation again. Supernatants were aspirated and cultures were frozen at -80°C. Cells were lysed by probe sonication on ice, clarified using centrifugation, normalized for protein content, and run on a 4-20% SDS-PAGE. Protein expression of Hu2H10_5MUT (SEQ ID NO: 55) and Hu2H10_5MUT_CYS (SEQ ID NO: 141), evidenced by a band around 15 kDa, was lower than that of Hu2H10_5MUT_aH_CYS (SEQ ID NO: 142) containing the α-helical peptide linker of SEQ ID NO: 21 at the C-terminus (band at approximately 17 kDa).

[0221] The increased soluble expression was not limited to the above proteins. The anti-VEGF protein HuNb42 also showed increased soluble expression with the addition of a C-terminal α-helical peptide. Figure 1D shows the soluble expression of HuNb42_A88P (SEQ ID NO: 67) ("null") compared to HuNb42_A88P aH_Cys (SEQ ID NO: 145) ("+aH_CYS"). The total process yield per liter of culture medium with the α-helical linker was about 4-fold higher than the yield of the corresponding protein without the C-terminal α-helical peptide. Example 2. Effect of humanization on soluble expression

[0222] A higher degree of humanness is desirable in therapeutic peptides and proteins to reduce the risk of immunogenicity. However, certain residues in single domain antibodies affect humanness and at the same time reduce stability. Therefore, a systematic evaluation of specific point mutations in framework regions associated with humanness and stability was performed.

[0223] Sequence humanization was performed using computational resources from the Abysis antibody analyzer and the T20 score analyzer from LakePharma. For sdAb targeting human proteins, specific amino acids within the consensus sequence were altered to achieve a T20 framework-constrained score of 85 or higher.

[0224] Figure 2 shows the amino acid sequences of 2H10 and point mutation variants tested for expression in E. coli. As illustrated in Figure 3, variants Hu2H10 R86K A87P (SEQ ID NO: 56) and Hu2H10 R86K A87P L115Q (SEQ ID NO: 58) had higher protein expression than Hu2H10 5MUT (SEQ ID NO: 55) or Hu2H10 L115Q (SEQ ID NO: 57).

[0225] Figure 4 shows the amino acid sequences of Nb42 and point mutation variants tested for expression in E. coli. Figure 5 shows that the HuNb42 A88P variant (SEQ ID NO: 67) exhibits enhanced soluble expression. Figure 6 shows that the higher relative expression was independent of the cellular compartment, where both the cytoplasmic and periplasmic compartments showed higher relative levels of soluble expression.

[0226] Figure 7 shows the humanity of Nb42 (SEQ ID NO: 61), HuNb42 (SEQ ID NO: 62), and the HuNb42_A88P variant (SEQ ID NO: 67) compared to caplacizumab, bevacizumab, and ranibizumab. HuNb42 (SEQ ID NO: 62) and the HuNb42 A88P variant (SEQ ID NO: 67) were comparable to literature humanized antibodies in terms of humanity as measured by Z-score or T20 score, respectively.

[0227] 8 shows Coomassie Brilliant Blue (CBB) staining of E. coli cell extracts expressing aTNFaMu (SEQ ID NO: 71) or aTNFaMu_3MUT (SEQ ID NO: 72) at room temperature, 50° C., 60° C., 70° C., and 80° C. The staining illustrates that three point mutations in the aTNFaMu protein enhanced yield and thermal stability up to approximately 70° C.

[0228] 9 shows the effect of specific point mutations of E1-1 on protein expression yield. E1-1 S49A (SEQ ID NO: 83), E1-1 F11L S49A (SEQ ID NO: 84), and E1-1 CDR (SEQ ID NO: 85) showed approximately 10-fold greater relative protein expression compared to E1-1 (SEQ ID NO: 81) or E1-1 F11L (SEQ ID NO: 82).

[0229] Figure 10 shows a gel shift analysis consistent with fewer reactive cysteines in the final protein preparation when protein synthesis was performed at higher temperatures (upper graph). Fewer reactive cysteines would indicate that naturally occurring disulfide bridges were formed in the antibody, thus resulting in a more stable product. A similar gel shift (lower graph) showed a gradient of disulfide bridge formation from low to high as the temperature of protein synthesis was increased. Example 3. Endotoxin removal

[0230] Endotoxin is undesirable in protein preparations because it passes through the drug conjugation step and is a source of contamination in animal studies (endotoxin triggers an immune response).

[0231] Endotoxin removal relied on the presence of EDTA in the buffer, which aggregates endotoxins to a specific size, making them filterable, while at the same time minimizing protein loss during filtration. Figures 11A-11B show the endotoxin removal process, in which >99.5% of the endotoxins in the sample were removed.

[0232] FIG. 11B shows the removal of endotoxin using 50 and 100 kDa filters and their recovery of anti-TNFα 3MUT VHH(mouse)- aH (SEQ ID NO: 104) content. Method: Protein solutions of ∼15 mg / mL in 25 mM sodium citrate pH 5.5, 100 mM NaCl, 1 mM EDTA were passed through 50 and 100 kDa polyethersulfone membrane (PES) filters at 15 K*g for 10 minutes at room temperature. Protein concentration was measured using A280 spectrophotometry (nanodrop) and endotoxin was measured using Charles River Endosafe LAL cartridges. method:

[0233] 5 mL cultures were grown to saturation at 37° C. in TB-autoinduction medium + antibiotics. Cells were harvested by centrifugation at 4000 RPM at 4° C. for 10 seconds. Pellets were washed with 1 mL PBS and transferred to eppendorf tubes. Cells were pelleted at 14000 RPM at 4° C. for 2.5 minutes and the supernatant was aspirated and frozen. Frozen cell pellets were thawed on ice and sonicated on ice in lysis buffer (50 mM HEPES pH 7.5, 20 mM imidazole, 400 mM NaCl, 5% glycerol, 0.01% tween-20, and 0.5 mM EDTA) using a small tip sonicator at 40% power for a total of 60 seconds with 5 seconds pulse on and 5 seconds pulse off. Cell lysates were normalized to total protein content using Nanodrop A280 and run on a 4-20% SDS-PAGE under denaturing and reducing conditions. Gels were stained with InstaBlue protein stain and extensively destained with water. % densitometric signal was calculated using ImageJ software and normalized across all gel lanes. Overexpression is achieved when >10% of the total lane protein signal is due to a band at the approximate predicted molecular weight and was not seen in the uninduced sample control. An exemplary SDS-PAGE gel is shown in FIG. 11C.

[0234] A summary of expression level measurements for certain peptides of the invention is shown in Table 3 below. Table 3. Expression concentration measurements [Table 3] § Each sequence listed was covalently linked to a C-terminal alpha helical peptide of SEQ ID NO:21, except where indicated with an asterisk ("*"). Example 4. Consensus sequences for protein expression of single domain antibodies

[0235] Based on the data presented in the examples above, the following framework sequences allow single domain antibodies to be expressed more stably and / or more human. Tables 4 and 5 show exemplary framework regions with tolerated amino acid substitutions. Table 4. Framework regions [Table 4] Table 5. Permitted substitutions in exemplary peptides [Table 5-1] [Table 5-2] Example 5. Preparation of purified thiol-reactive hyaluronic acid conjugate intermediate

[0236] Sodium hyaluronate (HA, 830 kDa) was suspended at 4 mg / mL in water or 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer pH 5.7 by mixing with gentle rotation or nutation overnight at room temperature. To 3 mg (3.6 nmol, amount varies based on polymer composition and MW) of HA in solution was added hydroxybenzotriazole (HOBt) hydrate as a stock solution of ~5-100 mg / mL in DMSO, a thiol-reactive linker agent (e.g., hydrazide-X-thiol reactive group such as MP2H) in 10-100% DMSO (stock solution of 10-100 mg / mL), and a coupling agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) in 0.1 M MES buffer pH 5.7. The molar equivalents of each reactant per mole of HA, and per carboxylate for the different ways of carrying out the reaction, are given in Tables 6 and 7 below: Table 6. Relative ratios of coupling agents, catalysts, and linkers in the methods. [Table 6] Table 7. Ratio of coupling agent, catalyst, and linker per polymer carboxylate in the method. [Table 7]

[0237] The solution was mixed by gentle pipetting between each reagent addition, and the final reaction volume was brought up to 1 mL with buffer. The final mixture was allowed to react at room temperature for 45 min to 2 h using a nutation mixer, depending on the method. After reaction, the thiol-reactive biopolymer was purified using a 7 kDa MWCO 5-10 mL Zeba desalting spin column equilibrated with 10% v / v glycerol (optional), pH 6.5 DPBS, and 0.01% v / v polysorbate 20 (optional) and loaded with the crude reactants at 20% of the resin volume. The desired intermediate was eluted into a clean conical tube using a centrifuge at room temperature with an elution time of ~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 measured using UV absorbance, NMR, or a modified Ellman reaction assay.

[0238] Alternatively, the reaction pH or the equivalents of hydrazide linker, catalyst, and coupling agent (EDC) were varied higher or lower to increase or decrease the number of thiol-reactive small molecule linkers covalently linked to the biopolymer (valency) unit.

[0239] Alternative coupling reagents such as DMTMM or oxyma can be used in place of EDC or HOBt. The activated biopolymer intermediate can also be purified from the reaction using size exclusion chromatography, other desalting columns, tangential flow filtration, ion exchange chromatography, dialysis, or alcohol / acetone precipitation.

[0240] After purification, UV spectra (200-324 nm) were acquired for intermediates prepared using different methods on a BioTek Synergy plate reader using a Take3 microspot plate. Maleimide concentrations can be determined by absorbance at 230 nm or by comparing the spectra with authentic standard intermediates.

[0241] NMR analysis of the conjugates was performed on a Bruker Advance III spectrometer equipped with a 5 mm cryoprobe at 25 °C ( 1 H, 600.13 MHz) at the Complex Carbohydrate Research Center (CCRC) of the University of Georgia. Following standard preparation of intermediates using methods 1 and 5 on a 6 mL scale, the intermediates were purified in HPLC grade water using desalting resin and shipped to the CCRC on melting ice. Samples were left at 4°C for several weeks and the resulting partial maleimide hydrolysis was observed by NMR spectroscopy. For NMR sample preparation, 0.7 ml of intermediate stock solution (2.9 mg / ml) was pipetted into 7 ml screw-cap tubes. 1.3 ml of deuterium oxide (99.9%) was added to each sample and mixed thoroughly by vortexing. The samples were then dried using a SpeedVac vacuum concentrator at room temperature. The dried samples were then redissolved in 700 μL of deuterium oxide (99.98%) for NMR analysis.

[0242] The analytical chemistry of the example reaction products is set forth in Table 8 below. Table 8. Exemplary intermediates prepared using the methods described above. [Table 8]

[0243] A tabular representation of the intermediates synthesized using the three different methods, along with the resulting maleimide concentrations, valencies, and reaction efficiencies based on the HA monomer, are shown below in Tables 9, 10, and 11. Table 9. Intermediates for Method 1 [Table 9] Table 10. Intermediates for Method 2 [Table 10] Table 11. Intermediates for Method 5 [Table 11]

[0244] Alternatively, sodium hyaluronate (HA, 830 kDa) was suspended at 4 mg / mL in water or 10 mg / mL or 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 5.7 by gentle rotation or mixing with nutation overnight at room temperature. Prior to the reaction, a 4 mg / mL HA stock in 0.1 M MES was made using water and ~1 M MES pH 5.7 and mixed with nutation at room temperature. To 3 mg (3.6 nmol, amount varies based on polymer composition and MW) of HA in solution was added hydroxybenzotriazole (HOBt) hydrate as a stock solution of 5-100 mg / mL in DMSO, a thiol-reactive linker agent (e.g., hydrazide-X-thiol reactive group such as MP2H) in 1-10% DMSO (stock solution of 10-100 mg / mL), and a coupling agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) in 0.1 M MES buffer pH 5.7. The molar equivalents of each reactant per mole of HA, and per carboxylate for the different ways of carrying out the reaction, are listed in Table 12 below: Table 12. Preparation of intermediates [Table 12]

[0245] The solution was mixed with gentle pipetting between each reagent addition, and the final reaction volume was brought up to 1 mL with buffer. The final mixture was allowed to react for 45 min to 2 h at room temperature using a nutation mixer, depending on the method. Following reaction, the thiol-reactive biopolymer was purified using a 7 kDa MWCO 5-10 mL Zeba desalting spin column equilibrated with 10% v / v glycerol (optional) pH 6.5 DPBS and loaded with the crude reactants at 20% of the resin volume. For NMR samples, the Zeba column was equilibrated and the intermediate was eluted with deuterium oxide and not frozen. The desired intermediate was eluted into a clean conical tube using a centrifuge at room temperature with an elution time of ~25-60 min. The intermediate was either used immediately for reaction with thiols or aliquoted and flash frozen on dry ice or at -80°C. The maleimide concentration and number of modifications per polymer were measured using UV absorbance, NMR or a modified indirect Ellman reaction assay.

[0246] The maleimide concentration / valency for each reaction method is provided in Table 13 below. Table 13. Indirect Ellman Maleimide Substitution Measurements [Table 13]

[0247] NMR analysis of the conjugates was performed on a Bruker Advance III spectrometer equipped with a 5 mm cryoprobe at 25 °C ( 1 H, 600.13 MHz) at the Complex Carbohydrate Research Center (CCRC) at the University of Georgia. After standard preparation of intermediates using methods A-E on a 3-6 mL scale, the intermediates were purified in deuterium oxide using a desalting resin and shipped to the CCRC on melting ice.

[0248] 1Based on H NMR spectroscopy, all samples contained signals corresponding to HA, MP2H, free ethyl dimethylaminopropyl urea (EDU), and by-products of EDC hydrolysis that were not removed during purification. The abundance of MP2H, conjugated n-acylurea adducts, and free EDU were measured relative to HA (repeating polymer unit) by calculating the integrals of the signals from characteristic peaks. Based on the integrals, the raw abundances of HA, MP2H, free EDU, and conjugated N-acylurea (conjug. EDU) were calculated. The abundances of MP2H, conjugated N-acylurea adducts, and free EDU relative to HA are shown in Table 14. Table 14. Relative molar abundance of MP2H, free EDU, and N-acylurea adducts relative to HA (100%) for methods A–E. [Table 14] Example 6. Preparation of purified peptide-polymer conjugates

[0249] To obtain purified peptide-polymer conjugates, 1.1-2 equivalents of peptide per maleimide were combined with the intermediate prepared by the method of Example 1 and reacted at least 2 hours to overnight at either 4° C. or ambient temperature with mixing by rotation or nutation (most reactions were carried out at room temperature to improve solubility). Optionally, 1 M HEPES, pH 7, was added to a final concentration of 0.1 M to adjust the reaction pH. In some cases, prior to the conjugation reaction, a reducing agent such as DTT or TCEP HCl, 10-100 equivalents per protein equivalent, is added to reduce disulfides formed between peptides. This is either removed from the protein solution prior to conjugation by desalting columns or buffer exchange, or added directly to the conjugation in the form of TCEP immobilized on polymer beads. During conjugation, one or more of the following were added to improve reaction efficiency: 0.5-10 mM EDTA to minimize free thiol oxidation, tween 20 to help stabilize the protein and / or reduce non-specific interactions, carbohydrates, additional buffer, or glycerol to help stabilize the protein and / or reduce non-specific interactions between the protein and the activated biopolymer, and increased or decreased salt concentration to stabilize the protein and / or 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 (1:100-1:1000) at least twice for 4 hours each and once for at least 4 hours at 4 °C to room temperature using a 50-1000 kDa MWCO against an appropriate buffer (pH should be >1 unit higher or lower than the pI of the peptide). Depending on the peptide, tangential flow filtration against citrate buffer, DPBS pH 6-8, or 50 mM tris 150 mM NaCl pH 8-8.5 with EDTA and other additives such as tween or trehalose, FPLC advanced purification using size exclusion columns, FPLC advanced purification using affinity chromatography columns designed to bind the polymeric component of the conjugate, or selective precipitation of the conjugate can also be used to purify the conjugate from unreacted peptide.If the reaction efficiency is high enough (i.e., there is <4% unreacted protein), no purification is necessary.

[0250] Alternatively, peptides were added to each solution of the intermediates from Example 1 at the appropriate peptide:polymer molar feed ratio and final concentration of up to 0.03% (optional) in Tween-20. The solutions were allowed to react for 2 hours to overnight with rotational (~5 RPM) or nutational stirring at ambient temperature. Unreacted peptide was removed by dialysis using 50-1000 kDa MWCO membranes against each of the following buffers: first, phosphate buffered saline or equivalent citrate or succinate buffered saline (pH and buffer salts used vary by peptide) with 0.01% Tween-20 (optional) for at least 4 hours, second phosphate buffered saline with 0.01% Tween-20 overnight, and phosphate buffered saline with 0.01% Tween-20 for 4 hours at 4°C or room temperature, with an optional fourth dialysis step. Optionally, additives such as tween 20, EDTA, carbohydrates, etc. are added to enhance protein stability.

[0251] After MVP purification, the conjugates were analyzed for protein concentration, protein valency, MVP radius, and binding affinity using the methods described in the stability study section. A tabular representation of the MVPs synthesized using various intermediates is shown below with their resulting protein concentration, valency, biolayer interferometry (BLI) dissociation constant, and radius.

[0252] MVPs synthesized using Method 1 and Method 2 had similar or improved characteristics (final protein concentration, protein valency, radius, binding kinetics) for MVP therapeutics compared to MVPs synthesized using Method 5 (Table 15). An example of a hydrodynamic distance comparison of DARPin MVPs synthesized using Method 1 or Method 5 intermediates is shown in Figure 12. An example of a VEGF binding curve BLI data comparison for anti-VEGF MVPs synthesized using Method 1 or Method 5 intermediates is shown in Figure 13. Table 15. Conjugates [Table 15]

[0253] Alternatively, to obtain purified peptide-polymer conjugates, 1.1-2 equivalents of peptide per maleimide were combined with HA conjugation substrate prepared by the method shown in Table 16. The conjugation reaction was allowed to react for at least 2 hours to overnight at ambient temperature with mixing by rotation or nutation. 1 M HEPES, pH 7, was added to a final concentration of 0.1 M to adjust the reaction pH. In some cases, unreacted peptide was removed from the peptide-polymer conjugates by at least three dialysis (1:400-1:1000) for 4 hours each at 4° C. to room temperature against an appropriate buffer (pH should be >1 unit higher or lower than the pI of the peptide) using a 50-1000 kDa MWCO. Table 16. Reaction conditions for peptide-polymer conjugates [Table 16]

[0254] To confirm successful conjugation, the products of the conjugation reaction were analyzed by SDS-PAGE and DLS. SDS-PAGE was used to measure the percentage of unreacted peptide separated by migration into the gel consistent with its molecular weight. After the conjugation reaction, a substantial percentage of the peptide appeared as high molecular weight conjugates at the top of the stacking gel, which could not migrate into the gel due to their size (>300 kDa). DLS was used to measure the hydrodynamic distance present in the reaction products. After the conjugation reaction, the highest intensity peak was identified as R, consistent with the conjugation substrate. hand indicated that the peptide was bound to the hyaluronic acid substrate. The conjugate data are shown in Table 17. The percentage of unreacted protein was determined by densiometric analysis of the SDS-PAGE bands of unbound protein relative to BSA standards of known mass, then divided by the total mass loaded into each well. The hydrodynamic radius was measured using dynamic light scattering (DLS) with a Wyatt DynaPro plate reader III (25°C, 5-10 acquisitions, 5 seconds, n=3 samples per conjugate). Data analysis was performed using a Jupyter notebook data analysis program to extract data of appropriate quality and analyzed based on the most intense peak. Table 17. Properties of each peptide-polymer conjugate. [Table 17]

[0255] To achieve therapeutic threshold, maximize therapeutic life, and / or minimize dosage volume, it is often required to achieve high concentration of pharmaceutical formulation. However, at higher concentrations, therapeutic peptides may aggregate. For polymer-peptide conjugation, there is also an additional concern that interactions with the polymer substrate may contribute to aggregation or result in aggregation at lower peptide concentrations that would occur without the attached polymer. The HA conjugation substrate made using method B provided polymer-peptide conjugates with no measured aggregation.

[0256] To synthesize purified conjugate number 28 using Method B: 174 μL of purified conjugation intermediate from Method B was mixed with 3 μL of 2% v / v Tween 20 and 26 μL of 80 mg / mL N42 anti-VEGF VHH (SEQ ID NO: 145) for 1.1 equivalents of peptide per maleimide in a 2 mL v-bottom microcentrifuge tube. The reaction pH was adjusted to pH 7 by the addition of 20 μL of 1 M HEPES, pH 7, for a final concentration of 0.1 M. The reaction was allowed to proceed overnight at room temperature for 16 hours with mixing using nutation. Unreacted peptide was removed from the peptide-polymer conjugate by dialysis (1:1000 based on initial reaction volume) using a 100 kDa MWCO 200 μL microFloat-A-Lyzer dialysis cassette (Repligen) against 25 mM citrate, pH 5.5, 100 mM NaCl, 0.03% tween 20 at room temperature with stirring. A total of four dialysis steps were performed, with three buffer changes every 4 hours and one after 16 hours of overnight dialysis.

[0257] To synthesize purified conjugate number 30 using method B: 186 μL of purified conjugation intermediate from method B was mixed with 3 μL of 2% v / v Tween 20 and 14.4 μL of 80 mg / mL N42 anti-TNFα VHH (SEQ ID NO: 102) for 1.1 equivalents of peptide per maleimide in a 2 mL v-bottom microcentrifuge tube. The reaction pH was adjusted to pH 7 by the addition of 20 μL of 1 M HEPES, pH 7, for a final concentration of 0.1 M. The reaction was allowed to proceed overnight at room temperature for 16 hours with mixing using nutation. Unreacted peptide was removed from the peptide-polymer conjugate by dialysis (1:1000 based on initial reaction volume) using a 100 kDa MWCO 200 μL microFloat-A-Lyzer dialysis cassette (Repligen) against 25 mM citrate, pH 5.5, 100 mM NaCl, 0.03% tween 20 at room temperature with stirring. A total of four dialysis steps were performed, with three buffer changes every 4 hours and one after 16 hours of overnight dialysis.

[0258] After the fourth dialysis step was completed, the purified conjugate was removed from the dialysis cassette and stored at 4° C. The reaction products were analyzed by visual inspection, UV vis absorbance to measure purified protein conjugation, R as described in the Examples herein. h The antibodies were characterized by DLS to measure binding affinity, SDS-PAGE to determine the percentage of unbound protein, and biolayer interferometry to measure binding affinity. Table 18. Characterization of conjugates No. 28 and No. 30 prepared from intermediates of Method B. [Table 18] Example 7. Stability analysis of purified peptide-polymer conjugates

[0259] MVP stability was assessed by setting up long-term accelerated in vivo stability by maintaining MVP in vitreous mimetic buffer pH 7.3 (Table 19) or PBS 0.01% tween 20 pH 7.4 at 5-10x therapeutic concentration at 37° C. MVP stability was assessed using SEC MALS or SEC, DLS, and / or BLI analysis of samples removed after various time periods. Table 19. Composition of vitreous mimetic buffer [Table 19]

[0260] A long-term 37°C stability study was set up to evaluate the effect of the composition on MVP stability. MVP was synthesized under sterile conditions and diluted to approximately 0.4mg / mL in sterile-filtered human vitreous mimicking buffer. This concentration is 5-fold higher than the intravitreal therapeutic concentration of our predicted clinical dose. Samples were either filtered using a sterile 0.2 or 5μm spin filter prior to use or mixed with 0.01% sodium azide as an antimicrobial agent. Several 100μL aliquots of each sample were then added to wells of a sterile 96-well plate at 4°C, where one day 0 aliquot had been prepared. The remaining wells were filled with sterile-filtered human vitreous buffer + 0.01% sodium azide to minimize evaporation. The plates were incubated in a standard tissue culture incubator at 37°C with 5% CO2. At distinct time points, one aliquot from 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 steps to observe any dramatic changes in sample composition. Next, protein concentrations were measured to adjust for any possible volume differences. Binding affinities were measured using BLI. K over time on (binding constant) or K D The change in radius is used to assess relative stability. To observe the change in radius over time, the samples are centrifuged at 5000 g for 5 minutes to remove any large aggregates or dust particles, and the R h is measured using DLS without any sample dilution.

[0261] Stability study samples were evaluated using HPLC size exclusion chromatography (SEC). This method was also used to analyze MVP formation and the percentage of unreacted protein after purification. To evaluate stability by SEC, MVP was filtered to remove particulates and analyzed using a Shodex 1MDa Ohpak LB-804, Shodex KW-404 or 405, or Phenomenex PolySep6000 column with DPBS or appropriate solvent as the mobile phase to obtain baseline traces at 280 nm, 230 nm, etc. After various time points, samples were removed and analyzed using the same SEC method. An increase in retention time and peak width compared to the baseline sample indicates degradation. Additionally, a decrease in MVP peak area and / or an increase in peak area of ​​monomeric and dimeric protein species also indicates MVP degradation. The percentage loss of the conjugate was quantified by comparing the difference in peak area with time. In the future, SEC stability analysis will be combined with MALS to quantify the change in molecular weight and valency of the conjugate with degradation at various temperatures. Representative SEC data for Method 5 or Method 1 intermediate DARPin MVP samples aged at 37° C. for up to 71 days are shown in Figure 14. Due to its large size, conjugate 2 could not be analyzed by the same column used to analyze conjugate 1 in this data.

[0262] Stability study samples also showed that the MVP radius of gyration (R g,zThe conjugates were analyzed by combining SEC with MALS analysis for the determination of the molecular weight and the molecular weight. For this, conjugate stability samples were placed in glass vial inserts (250 μL volume) mounted in 2 mL HPLC vials and sealed. For HPLC analysis, 5-20 μg of MVP (based on protein) was injected into a 1260 Infinity Agilent HPLC system equipped with an isocratic pump, an autosampler, a thermostatically controlled column compartment, and a variable wavelength detector (or equivalent) set for observation at 1280 nm, using a Shodex KW-405-4F (4.6×300 mm, 0.35 mL / min flow rate) or LB-804 or 806 (8×300 mm, 0.4 mL / min flow rate, for the analysis of the unbound VHH peak) with their respective guard columns. For analysis, the column compartment was held at 30°C using an isocratic method with mobile phase made up of HPLC grade water, 0.1 μm filtered pH 7.4 DPBS, 200 mM KCl, 100 mM urea, 50 mM sodium phosphate pH 6 with 0.025% sodium azide, or 0.1 μm filtered 300 mM NaCl, 10 mM sodium phosphate, 0.025% SDS, 0.025% sodium azide pH 6.0, and eluted with at least 2 column volumes of mobile phase after sample injection or after a total run time of 60 minutes. A Dawn Heleos II MALS instrument and Optilab T-rEX refractive index detector (Wyatt Technology) or equivalent was interfaced with the HPLC downstream of the UV detector. The MALS and dRI detector parameters for protein-polymer conjugate analysis using Astra software (Wyatt Technology) are listed in Table 20. System specific calibration numbers, normalization factors, delay volumes, and band broadening periods were measured for the system prior to analysis. Representative SEC traces of MVP stability samples across the EDC range are shown in Figure 14. Increased retention time indicated size reduction / shrinkage / decomposition of MVP due to degradation.In Figure 14, the high EDC conjugate (Conjugate 1) demonstrated a much larger increase in retention time and radius shrinkage compared to the low EDC MVP (Conjugate 2). Peak broadening also suggested an increase in polydispersity of the aged samples, which may indicate sample degradation. The radius decrease was further verified by MALS of the samples in the accelerated aging study shown in Figure 15. Table 20. MALS and dRI instrument parameters for SEC MALS analysis [Table 20]

[0263] Stability can also be assessed by measuring macromolecular size (e.g., R h) for stability analysis using DLS degradation at 37°C. Samples were removed from the 37°C stability study conditions at various time points for analysis. All samples and buffers were at room temperature. Solutions were diluted (typically 1:10 dilution) with sterile 0.1 μm filtered formulation buffer without polysorbate 20 to a final concentration of 100 nM in 100 μL and mixed by gentle trituration in a 1.5 mL centrifuge tube. 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 placed into a Wyatt Technology disposable microcuvette (Wyatt Cat# WNDMC) with a cap, tapped to remove air bubbles, and placed in the analytical instrument for analysis. For multiple measurements using a plate reader, 25-35 μL of sample was added to a clear bottom black well 384 well plate (Corning Cat# P8802-384 or similar). Air bubbles in the sample were removed by a brief spin in a centrifuge with a plate adapter, followed by a gentle puff of 70% EtOH vapor from a squirt bottle using a pipette tip. The instrument settings for this and other sample analysis by DLS in this document are shown in Table 21. All peaks greater than 1000 nm should have an intensity of <6%. DLS collection parameters are shown below in Table 21. Table 21. DLS acquisition parameters [Table 21]

[0264] Table 22 below shows the MVP radius change with accelerated 37° C. degradation for MVP samples synthesized using various methods. The hydrodynamic radius or radius of gyration was measured by DLS or MALS at t=0 and various time points after degradation at 37° C. In these examples, MVP synthesized using low EDC (method 1) had improved 37° C. stability based on the slight shrinkage / change in radius upon degradation. This suggested that the presence of N-acylurea adducts destabilized the conjugates prepared using methods using higher amounts of EDC. Table 22. MVP radius over time [Table 22]

[0265] Changes in binding affinity over time were also measured using biolayer interferometry (BLI). To perform BLI experiments, samples were removed from the 37°C stability study conditions for analysis at various time points. All reagents were equilibrated to room temperature for at least 30 minutes prior to use. Two probes per sample were equilibrated in 250 μL of BLI buffer (0.2 μm filtered PBS pH 7.4, 0.2% Tween and 0.2% BSA) for a minimum of 10 minutes in a Gator Bio Max plate (one for the kinetic assay and one for the no ligand control). Ligands were diluted in BLI buffer to a constant concentration of 25-100 nM based on performance in pilot reactions. Analytes were prepared in BLI buffer at the highest concentration measured in pilot reactions and serially diluted 1:3 2-5 times using BLI buffer. A black flat bottom uncoated 96 well plate (Greiner Bio One Cat# 655209 or similar) was loaded column wise with 200 μL of ligand, analyte dilutions, and BLI buffer for each row of ligand and analyte. One well in each row of analyte should be in BLI buffer to be used as a blank for subtracting the standards. No air bubbles were present in the wells as they were removed with a pipette tip or by gently blowing 70% EtOH vapor from a squirt bottle. The plate was placed in a Gator on a tilting platform set at 25°C. The loading and kinetic steps for the Gator K assay were set to use the dual reference and step times shown in Table 24. The ligand was loaded until the signal reached 0.4-0.6 nm, then returned to buffer and the column was allowed to return to baseline measurements for 60 seconds. The kinetic readout was initiated using the step parameters in Table 24. When the kinetic readout with the ligand-injected probe was completed, a no-ligand control was run using a new probe with no ligand injected. The same kinetic assay timing and same sample wells were used as in the analysis with the ligand-injected probe. This data was used to correct for any non-specific interactions between the sample and the probe.Representative BLI data for high (Method 5) and low (Method 1) EDC MVP samples before and after accelerated aging at 37° C. are shown in FIG. 16(K). D ) and Fig. 17(K on ) as shown in

[0266] When the kinetic assays were completed, the data was analyzed in the Results and Analysis section of the Gator software. The raw data was corrected to include binding times from 1 s to 180 s onwards. The Y-axis was aligned to the beginning of the binding step and inter-step correction was enabled. Savitzky-Goaly Filtering of the data was used. Samples were set up for double referencing by indicating the probe and wells as buffer reference in the software. A reference subtraction equation was then compiled for each assay, which was double referenced using the equation (kinetic assay wells - no ligand assay wells) - (kinetic assay buffer reference wells - no ligand assay buffer wells). All titers of the same MVP were sorted by color and the parameters were adjusted to a 1:1 binding model that included both binding and dissociation using a global, Rmax uncoupled fit. The window of interest was moved to include only the 100 s of dissociation. Binding curves were fitted and a perfect Rmax was used to determine the fit, with the remainder not differing from the actual curve by more than 10%. 2 is >0.98 and perfect X 2 It was confirmed that K was <3.0. Kinetics were calculated and K D , K on , and responses were noted. Different samples had the same K D When we have the result, the binding constant K on was used to differentiate the binding affinities between different constructs (ie, FIG. 17), where higher binding constants indicated faster binding kinetics. Table 23. BLI Ligand and Analyte Pairings [Table 23] Table 24. BLI method parameters and result specification for kinetic quantification [Table 24]

[0267] A tabular representation of the binding kinetics upon accelerated 37° C. degradation of MVP samples synthesized using various methods is shown below. The dissociation constants of the samples were measured by BLI at various time points after degradation at 37° C. In these examples, MVPs synthesized using method 1 and method 2 had similar or improved 37° C. stability based on therapeutic target binding ability, and similar or smaller changes in dissociation constants upon degradation. The anti-VEGF VHH peptide MVP synthesized using intermediate method 5 lost all binding ability after a short period of degradation, whereas the examples synthesized using methods 1 or 2 demonstrated target binding ability throughout the study, suggesting that the presence of the N-acylurea adduct destabilized the therapeutic. Table 25. Conjugate binding over time. [Table 25] Example 8. In vivo half-life of purified peptide-polymer conjugates

[0268] The extended intravitreal retention time of the conjugate was demonstrated with a well-established pharmacokinetic model. New Zealand White rabbits (n=9) were randomized into three groups according to body weight. All animals received a 50 μL ITV injection of hu_anti-TNFα_aH MVP (SEQ ID NO: 102)+HyA (850 kDa) ("anti-TNFα MVP") in the left eye and unconjugated VHH (SEQ ID NO: 102) ("anti-TNFα") in the right eye using a 31G insulin disposable syringe. Both eyes received equimolar doses of antibody. At 1 hour, 5 days, and 10 days after injection, a group of three rabbits was sacrificed and their eyes were enucleated for analysis of intravitreal VHH. Both eyes were snap frozen and the vitreous, retina, and aqueous humor were isolated from the frozen eyes. Each tissue sample was then homogenized using a bead beater. After homogenization, VHH concentrations were quantified either by using ELISA or by digesting the peptides with trypsin and subjecting the samples to LC / mass spectrometry or similar. Representative results for extended intravitreal half-life in rabbit eyes after bioconjugation are shown in Figure 18.

[0269] The method for fluorescent tagging of peptides for this study is as follows. A mouse tumor model to assess the rate of clearance of proteins from solid tumors was used to measure the intratumoral (IT) half-life of MVP to maximize parameters related to tumor retention. We used antibodies tagged with amine-reactive Sulfo-Cy7 NHS ester (Broadpharm Cat# BP-22541) or Alexa Fluor 750 near-infrared fluorophore by the following method (performed under sterile conditions). First, the dye was dissolved in DMSO at a concentration of 10 mg / mL. Next, proteins at concentrations of 5.0-10.0 mg / mL were mixed with 0.1 M sodium bicarbonate at a 3:2 vol:vol ratio. Finally, the fluorophore was added at a 1:2 protein:fluorophore molar ratio, mixed thoroughly, and incubated for 1 hour at room temperature in a nutator protected from light by covering with foil. The NHS ester was quenched by adding 1.5M Tris buffer pH 8.5 at 10% of the reaction volume and mixed on a nutator for another 10 minutes. The tagged protein was purified from unreacted fluorophore using a NAP-10 desalting column (illustra Cat# 17-0854-01) equilibrated with PBS pH 7.0 + 0.01% Tween-20 according to the manufacturer's instructions. Protein concentration and degree of Cy7 labeling were measured by absorbance at 280 and 750 nm. Proteins stored on ice and within 3 hours of Sulfo-Cy7 labeling were used for MVP synthesis according to the protocol described above. Each time the final product was then sterile filtered and stored at 4°C protected from light until it was used in animal studies.

[0270] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate that certain changes and modifications may be practiced within 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 were individually incorporated by reference.In the event of any inconsistency between this application and the references provided herein, this application shall control. Table 26. Arrays [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] [Table 26-6] [Table 26-7] [Table 26-8] [Table 26-9]

Claims

1. The following compound III has a molecular weight of approximately 0.1 MDa to approximately 3 MDa: 【Chemistry 1】 {During the ceremony, Each X is independently a peptide having a molecular weight of approximately 5 kDa to approximately 200 kDa; Each Y is an organic linker; Each X-Y-Z １ The structure of the part is as follows: 【Chemistry 2】 Having; Each Z ２ The structure is as follows: 【Transformation 3】 Having; Each Z ３ It has the following structure independently: 【Chemistry 4】 Having; Each R １ and R ２ is, independently, C １ -C ６ alkyl, -(C １ -C ６ alkyl)-NR ３ R ４ or C ５ -C ８ cycloalkyl; Each R ３ and R ４ H or C １ -C ６ It is alkyl; Each Z ３ａ These are independently OH or Y'; 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 from 0 to 1000 and is less than approximately 10% of the sum of the subscripts n, p, and q; and The subscript q is a conjugate, which is a random polymer of integers between 100 and 10000.

2. The aforementioned conjugate is given by the following formula IIIa: 【Transformation 5】 {During the ceremony, each X １ This is given by the following equation (I): 【Transformation 6】 It contains a peptide having CDR1, CDR2, and CDR3 are each independently complementary determination regions; FR1 is as follows: X １０ VQLX １１ EX １２ GGGX １３ X １４ QX １５ GX １６ SLLSCX １７ X １８ SG (Sequence No. 1) {During the ceremony, X １０ is Q, E, or D, X １１ is V, Q, A, or E, X １２ It is either S or T. X １３ is L, S, or V, X １４ is either V or A, X １５ is P, A, or T, X １６ is G, D, or R, X １７ is A, V, T, or E, and X １８ is either A or V. It has an amino acid sequence containing; FR2 is as follows: X ２０ X ２１ WX ２２ RQX ２３ PGKX ２４ X ２５ EX ２６ VX ２７ X ２８ I (Sequence ID 2) {During the ceremony, X ２０ It is M, I, V, or L, X ２１ is G, S, or A, X ２２ is F, Y, or V, X ２３ is A, V, P, or T, X ２４ is E, G, A, or Q, X ２５ is either R or L, X ２６ These are F, G, W, or L. X ２７ is A, G, or S, and X ２８ is A, S, or G. It has an amino acid sequence containing; FR3 is as follows: YX ３０ DSVKGRFTIS ３１ DX ３２ X ３３ KX ３４ X ３５ VX ３６ LQMX ３７ X ３８ LRX ３９ａ EDTAX ３９ｂ YYCAA (Sequence ID 3) {During the ceremony, X ３０ is A, G, S, or T, X ３１ is R or Q, X ３２ It is N, S, or D. X ３３ It is S, A, or D. X ３４ is N or K, X ３５ is either T or M, X ３６ is Y, D, or S, X ３７ is N or D, X ３８ is either S or N, X ３９ａ is P or A, and X ３９ｂ is V, M, L, or I. It has an amino acid sequence containing; and FR4 is as follows: YWGX ４０ GTX ４１ VTVSS (Sequence No. 4) {During the ceremony, X ４０ is either Q or K, X ４１ is either L or Q. Having an amino acid sequence including; and each X ２ This is a peptide linker containing an α-helix. The conjugate according to claim 1, having the structure described above.

3. (a) CDR1 has an amino acid sequence containing FAYSTYS (SEQ ID NO: 9), CDR2 has an amino acid sequence containing NSGTFRLW (SEQ ID NO: 10), and CDR3 has an amino acid sequence containing RAWSPYSSTVDAGDFR (SEQ ID NO: 11); or (b) CDR1 has an amino acid sequence containing RRFSIEA (SEQ ID NO: 12), CDR2 has an amino acid sequence containing DSGGSTD (SEQ ID NO: 13), and CDR3 has an amino acid sequence containing IGGSWYGRGLD (SEQ ID NO: 14); or (c) CDR1 has an amino acid sequence including GTFSSII (SEQ ID NO: 15), CDR2 has an amino acid sequence including SWSGGTTV (SEQ ID NO: 16), and CDR3 has an amino acid sequence including RPYQKYNWASASYNV (SEQ ID NO: 17); or (d) CDR1 has an amino acid sequence containing GGSDAGT (SEQ ID NO: 18), CDR2 has an amino acid sequence containing SWAGTAWR (SEQ ID NO: 19), and CDR3 has an amino acid sequence containing LGSYEMDHH (SEQ ID NO: 20). The conjugate according to claim 2.

4. Each of the aforementioned X １ The conjugate according to claim 2, wherein the peptide has an amino acid sequence containing any one of sequence numbers 51-58, 61-73, 81-85, 91-95, 101-106, and 111-118.

5. Each of the aforementioned X ２ However, the following: AEAAAAKEAAAAKAGC (Sequence ID 21), AEEEKRKAEEEKRKAEEEAGC (Sequence ID 22), AEEEKRKAEEEEKRKAEEEEKRKAEEEAGC (Sequence ID 23), AEEEEKKKKEEEEKKKAKAGC (Sequence ID 24), AEAAAAKEAAAAKAGC (Sequence ID 25), PSRLEEELRRRRLTEGC (SEQ ID NO: 26), or AEEEEKKKQQEEEEEAERLRRIQEEMEKERKRREEDEEERRRRKEEEEERRMKLEMEAKRKQEEEEERKKREDDEKRKKKAGC (Sequence ID 27), The conjugate according to claim 2, which is a peptide linker having an amino acid sequence containing the above.

6. The aforementioned organic linker has the following structure: 【Transformation 7】 {In the formula, the subscript m is an integer between 1 and 300.} The conjugate according to claim 1, having the following characteristics.

7. The conjugate according to claim 1, wherein the random polymer of formula III has a molecular weight of about 0.8 MDa.

8. Each of the above Rs １ and R ２ is independently C １ -C ３ alkyl or -(C １ -C ３ alkyl)-NR ３ R ４ The conjugate according to claim 1, wherein the conjugate is as defined above.

9. Each of the aforementioned R ３ and R ４ However, independently, C １ -C ３ The conjugate according to claim 1, wherein it is alkyl.

10. The aforementioned 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 1 and 1000, and is less than approximately 10% of the sum of the subscripts n, p, and q; and The conjugate according to claim 1, wherein the subscript q is an integer between 100 and 10000.

11. The aforementioned subscript n is an integer between 1 and 1000, 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 800, and is less than approximately 8% of the sum of the subscripts n, p, and q; and The conjugate according to claim 1, wherein the subscript q is an integer between 100 and 10000.

12. The aforementioned subscript n is an integer between 10 and 450, and is less than approximately 15% of the sum of the subscripts n, p, and q; The subscript p is an integer between 1 and 300, and is less than approximately 10% of the sum of the subscripts n, p, and q; and The conjugate according to claim 1, wherein the subscript q is an integer between 1000 and 3000.

13. The aforementioned 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 240, and is less than approximately 8% of the sum of the subscripts n, p, and q; and The conjugate according to claim 1, wherein the subscript q is an integer between 1000 and 3000.

14. The aforementioned 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; and The conjugate according to claim 1, wherein the subscript q is an integer between 1000 and 3000.

15. A pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof described in claim 1, and a pharmaceutically acceptable excipient.

16. A pharmaceutical composition for use in a method of treating an eye disease of a subject requiring the same, comprising the conjugate described in claim 1.

17. The pharmaceutical composition according to claim 16, wherein the use comprises administering the conjugate into the vitreous body.

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

19. The pharmaceutical composition according to claim 16, wherein the intravitreous half-life of the conjugate is at least five times longer than the half-life of the unbound peptide.

20. The pharmaceutical composition according to claim 16, wherein the eye disease is uveitis, macular degeneration, choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, or ocular inflammation.

21. A pharmaceutical composition for use in a method of treating a joint disease or disorder of a subject requiring the same, comprising the conjugate described in claim 1.

22. The pharmaceutical composition according to claim 21, wherein the use comprises administering the conjugate intraarticularly.

23. The pharmaceutical composition according to claim 21, wherein the use comprises administering the conjugate monthly, every two months, or every three months.

24. The pharmaceutical composition according to claim 21, wherein the intra-articular half-life of the conjugate is at least five times longer than the half-life of the unbound peptide.

25. The pharmaceutical composition according to claim 21, wherein the disease or disorder is rheumatoid arthritis, wear-related osteoarthritis, age-related osteoarthritis, post-traumatic osteoarthritis, psoriatic arthritis, and sterile implant laxity, joint exudate, ankylosing spondylitis, bursitis, gout, reactive arthritis, synovitis, or ischemic necrosis.

26. A method for preparing the conjugate described in claim 1, the following: (a)A hyaluronic acid polymer having a molecular weight of from about 0.1 MDa to about 3 MDa, from about 0.1 to about 2 equivalents of a coupling agent per hyaluronic acid monomer, and the formula H ２ N-R Ｙ {During the ceremony, R Ｙ The following: 【Chemistry 21】 and A first reaction mixture containing an organolinker of the subscript m is an integer between 1 and 300; thereby forming a plurality of the following formulas IV: 【Chemistry 22】 Forms an intermediate polymer having monomers; and (b) A second reaction mixture is formed containing an intermediate polymer and a peptide having a molecular weight of about 5 kDa to about 200 kDa, wherein the peptide contains one or more -SH; thereby, a conjugate is prepared. The method, including the method described above.

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