Humanized multivalent protein conjugates

Abstract

The present invention relates to peptide sequences that have a high degree of humanization and / or are capable of being expressed at a considerable level in a culture medium (e.g., E. coli). The peptide may be covalently linked to a polymer backbone via an alpha-helix peptide linker. The invention also relates to a method for preparing the peptide, polymer conjugates comprising the peptide, and pharmaceutical compositions thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 331,534, filed on April 15, 2022, the entire contents of which are incorporated herein for all purposes.

[0003] Sequence Listing

[0004] The material in the accompanying sequence listing is hereby incorporated by reference in its entirety. The name of the accompanying file is 2023-04-11Sequence_Listing_ST26 052566-506001WO.xml, created on April 11, 2023, and is 187,778 bytes in size. Background of the Invention

[0006] The use of biopolymers to modify the properties of bioactive agents is a recurring theme in a wide range of medical and biological applications. A variety of chemical linkers can be used to attach bioactive peptides or proteins to biopolymers to alter the pharmacological properties of the resulting conjugates for use as drugs that provide optimal treatment for specific diseases. Peptide-polymer conjugates comprising multiple copies of one or more peptides conjugated to a single biopolymer chain have been used to impart specific improvements in the pharmacological properties of peptides, including: (1) higher binding affinity for biological targets, (2) slower diffusion through target tissues, and (3) inhibition of proteases that may inactivate the biological activity of the peptide or protein.

[0007] These improved pharmacological properties of peptide-polymer conjugates are particularly useful for the delivery of effective drugs delivered directly to diseased tissues. The dose delivered directly to the tissue can be lower than the dose required to achieve the same therapeutic effect after systemic administration because the drug has been locally administered to the target tissue. Drugs can also be administered to tissues that otherwise have poor blood transport properties. Specific examples of tissues where direct drug administration is common include the posterior chamber of the eye by intravitreal injection and the joints by intra-articular injection.

[0008] However, local tissue delivery requires specialized personnel to safely deliver the required injections, making them more burdensome and costly to administer than systemic delivery. When peptide drugs are administered as part of a peptide-polymer conjugate, the frequency of dosing can be significantly reduced, thereby reducing the burden on patients to receive effective treatment. In addition, the reduction in the number of local injections reduces the risk of local tissue damage or adverse reactions to injections. Finally, the lack of the need for frequent dosing can reduce the amount of time that drug concentrations in target tissues are below therapeutic concentrations, thereby improving the overall efficacy of the drug. Based on these advantages, there is a strong impetus to develop protein-polymer drug products for a variety of diseases.

[0009] Many humanized monoclonal antibodies exhibit poor biophysical properties, such as low stability and susceptibility to aggregation. These unfavorable tendencies may be more pronounced for humanized antibody fragments, which usually require considerable modification.

[0010] In order to properly formulate a peptide-polymer conjugate into a drug product, sufficiently high drug concentrations must be achieved to enable appropriate dosing of the patient. It is also desirable to prepare purified peptide-polymer conjugates with high biological activity and shelf life, for example, the ability 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 negatively impact the ability to accomplish any of these drug-enabling properties.

[0011] The degree of humanization of the peptide and the secondary structure of the peptide linker used to connect to the polymer can have a significant impact on the pharmacological properties of the conjugate, intra-conjugate interactions, and conjugate-conjugate interactions. Therefore, there is a need to develop humanized peptide-polymer conjugates with specific peptide linkers so that they can achieve preferred pharmacological properties for specific diseases and be successfully formulated into drug products. The present invention meets this need and other needs.

[0012] Brief description of the invention

[0013] In some embodiments, the peptide of the present invention is a peptide represented by formula (I):

[0014] CDR1, CDR2 and CDR3 are each independently a complementarity determining region;

[0015] FR1 has an amino acid sequence comprising:

[0016] X 10 VxD 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1),

[0017] in

[0018] X 10 is Q, E or D,

[0019] X 11 is V, Q, A or E,

[0020] X 12 is S or T,

[0021] X 13 is L, S or V,

[0022] X 14 is V or A,

[0023] X 15 is P, A or T,

[0024] X 16 is G, D or R,

[0025] X 17 is A, V, T, or E, and

[0026] X 18 A or V;

[0027] FR2 has an amino acid sequence comprising:

[0028] X 20 X 21 WX 22 R 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2),

[0029] in

[0030] X 20 is M, I, V or L,

[0031] X 21 is G, S or A,

[0032] X 22 is F, Y or V,

[0033] X 23 is A, V, P or T,

[0034] X 24 is E, G, A or Q,

[0035] X 25 is R or L,

[0036] X 26 is F, G, W or L,

[0037] X 27 is A, G, or S, and

[0038] X 28 is A, S or G;

[0039] FR3 has an amino acid sequence comprising:

[0040] YX 30 DSVKGRFTISX31 DX 32 X 33 KX 34 X 35 VX 36 LqCy 37 X 38 LRX 39a EDTAX 39b YYCAA (SEQ ID NO: 3),

[0041] in

[0042] X 30 is A, G, S or T,

[0043] X 31 is R or Q,

[0044] X 32 is N, S or D,

[0045] X 33 is S, A or D,

[0046] X 34 is N or K,

[0047] X 35 is T or M,

[0048] X 36 is Y, D or S,

[0049] X 37 is N or D,

[0050] X 38 is S or N,

[0051] X 39a is P or A, and

[0052] X 39b V, M, L or I;

[0053] and

[0054] FR4 has an amino acid sequence comprising:

[0055] YW 40 GTX 41 VTVSS (SEQ ID NO: 4),

[0056] in

[0057] X 40 is Q or K, and

[0058] X 41 It is L or Q.

[0059] In some embodiments, the method for preparing the peptide of the present invention includes (a) translating a gene sequence encoding the peptide in bacteria in a first reaction mixture; and (b) removing endotoxin in the first reaction mixture by forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA); thereby preparing the peptide.

[0060] In some embodiments, the conjugate of the present invention is a conjugate represented by Formula IIa:

[0061] (X 1 -X 2 -Y) n -Z(IIa),

[0062] in

[0063] Each X 1 independently a peptide as described herein;

[0064] Each X 2 are independently peptide linkers of 3 to 100 amino acids in length;

[0065] Each Y is independently an organic linker;

[0066] Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; and

[0067] The subscript n is an integer from 1 to 1500.

[0068] In some embodiments, the conjugate of the present invention is a conjugate represented by formula IIb:

[0069] (X 1 -X 2A -Y) n -Z(IIb),

[0070] in

[0071] Each X 1 are independently a peptide having a molecular weight of about 5 kDa to about 200 kDa;

[0072] Each X 2A independently a peptide linker comprising an alpha helix;

[0073] Each Y is independently an organic linker;

[0074] Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; and

[0075] The subscript n is an integer from 1 to 1500.

[0076] In some embodiments, the conjugate of the present invention is a conjugate represented by Formula IIa:

[0077] (X 1 -X 2 -Y) n -Z(IIa),

[0078] in

[0079] Each X 1 independently a peptide as described herein;

[0080] Each X 2 is a peptide linker having an amino acid sequence comprising:

[0081] AEAAAKEAAAKEAAAKAGC(SEQ ID NO:21);;

[0082] Each Y is an organic linker having the following structure;

[0083]

[0084]

[0085] Z is a biocompatible polymer which is hyaluronic acid having a molecular weight of about 0.1 MDa to about 3 MDa;

[0086] Subscript m is an integer from 1 to 300; and

[0087] The subscript n is an integer from 1 to 1500.

[0088] In some embodiments, the conjugate of the present invention is a conjugate of a random polymer represented by formula IIIa:

[0089] (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa),

[0090] Its molecular weight is about 0.8 MDa;

[0091] in

[0092] Each X 1 is a peptide having an anti-VEGF amino acid sequence comprising: SEQ ID NO: 67;

[0093] Each X 2 is a peptide linker having an amino acid sequence comprising:

[0094] AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21);

[0095] Each Y is an organic linker having the following structure:

[0096]

[0097] Each X 1 -X 2 -YZ 1 The section has the following structure:

[0098]

[0099] Each Z 2 Has the following structure:

[0100]

[0101] Each Z 3 Independently has the following structure:

[0102]

[0103] Each Z 3a are independently OH or Y';

[0104] Each Y' has the following structure:

[0105]

[0106] Each R 1 and R 2 is ethyl or -(CH2)3-NMe2;

[0107] Subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q;

[0108] Subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and

[0109] The subscript q is an integer from 1000 to 3000.

[0110] In some embodiments, a pharmaceutical composition of the invention comprises a conjugate as described herein and a pharmaceutically acceptable excipient.

[0111] In some embodiments, the method of the invention is a method of treating an ocular disease in a subject in need thereof, comprising administering to the subject a conjugate as described herein.

[0112] In some embodiments, the method of the invention is a method of treating a disease or disorder in a joint of a subject in need thereof, comprising administering to the subject a conjugate as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1A The expressed open reading frames (ORFs) are shown. Figure 1B Soluble expression (mAU*mL) of Hu2H10 or 2H10 containing different peptide linkers is shown. Figure 1C SDS-PAGE protein expression of Hu2H10_5MUT (SEQ ID NO:55), Hu2H10_5MUT_CYS (SEQ ID NO:141) or Hu2H10_5MUT_aH_CYS (SEQ ID NO:142) is shown. Figure 1D Soluble expression (production yield fold) of HuNb42_A88P (SEQ ID NO: 67) or HuNb42_A88PaH_CYS (SEQ ID NO: 145) is shown.

[0115] Figure 2 The amino acid sequences of 2H10 and point mutants are shown.

[0116] Figure 3 The protein expression of Hu2H10 mutants is shown. The Y-axis shows the immobilized metal affinity chromatography (IMAC) peak area (mAU*mL). The X-axis shows the 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), and Hu2H10_5MUT_R86K_A87P_L115Q ("R86K_A87P_L115Q") (SEQ ID NO: 58).

[0117] Figure 4 The amino acid sequences of Nb42 and point mutants are shown.

[0118] Figure 5 Coomassie Brilliant Blue (CBB) staining of E. coli cell extracts expressing HuNb42 and point mutants is shown.

[0119] Figure 6 Relative E. coli cell culture yields of HuNb42 (SEQ ID NO:61) and HuNb42 A88P (SEQ ID NO:67) point mutants in the cytoplasm and periplasm are shown.

[0120] Figure 7 Shown are the relative degrees of humanization of Nb42 (SEQ ID NO:61), HuNb42 (SEQ ID NO:62) and HuNb42_A88P (SEQ ID NO:67) compared to caplacizumab, bevacizumab and ranibizumab.

[0121] Figure 8 Shown is 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.

[0122] Fig. 9 Relative protein expression of E1-1 (SEQ ID NO:81) and point mutants E1-1F11L (SEQ ID NO:82), E1-1S49A (SEQ ID NO:83), E1-1 F11L / S49A (SEQ ID NO:84), and E1-1 CDR (SEQ ID NO:85) are shown.

[0123] Fig. 10A Shown is Coomassie Brilliant Blue (CBB) staining of purified HuNb42 protein (SEQ ID NO: 62) induced at 18°C ​​and 37°C. Fig. 10B Shown are purified Hu2H10_5MUT proteins (SEQ ID NO: 55) induced at 16°C, 27°C, 30°C and 37°C, respectively.

[0124] Fig.11A Two preparations of HuNb42A88P (SEQ ID NO: 67) are shown without ("220119") and with ("220204") EDTA treatment. Fig. 11B Shown are the recovery of TNFα3 MUT (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), and the endotoxin levels before ("Input") and after filtration through a 50 kDa ("50 kDa FT") or 100 kDa ("100 kDa FT") polyethersulfone membrane (right). Fig. 11C SDS-PAGE gel showing purified aAng2_D4_aH_CYS (SEQ ID NO: 120).

[0125] Fig.12The R values ​​of DARPin multivalent proteins (MVP) prepared by method 1 (conjugate 5, conjugate 2) and method 5 (conjugate 3, conjugate 6, conjugate 7) are shown. h The MVP prepared using method 5 exhibits a smaller MVP radius.

[0126] Figures 13A-13B Shows the activity of MVP. Fig.13A Shown are the biolayer interferometry (BLI) VEGF binding and dissociation curves of anti-VEGF MVP prepared using different intermediates and anti-VEGF E1-1 peptide. Top: Method 2 Conjugate 10 (K D =0.123 nM). D =0.184 nM), showing the baseline, binding and dissociation curves (x-axis: time (seconds), y-axis: BLI signal) of different concentrations of conjugate 8. Both conjugates showed similar binding kinetics curves and calculated dissociation constants. Fig. 13B The VEGF binding affinity ("KD", nM) of the two peptides compared to the corresponding MVP is shown. First column: unconjugated 2H10_5MUT_aH_CYS (SEQ ID NO: 142); second column: unconjugated HuNb42_A88P_aH_CYS (SEQ ID NO: 145); third column: MVP containing 2H10_5MUT_aH_CYS (SEQ ID NO: 142); fourth column: MVP containing HuNb42_A88P_aH_CYS (SEQ ID NO: 145).

[0127] Fig.14 SEC traces of MVP stability samples over time are shown (x-axis: retention time, y-axis: absorbance at 280 nm). Example of SEC retention time changes for anti-VEGF DARPin MVP prepared using intermediates synthesized using Method 5 (Conjugate 1, top) or Method 1 (Conjugate 2, bottom), stored at 37°C for up to 71 days. MVP synthesized using Method 5 (top) showed greater radius loss with aging.

[0128] Fig.15 Shown are the changes in the radius of gyration of DARPin MVP made using intermediate Method 1 (Conjugate 2, upper line) or Method 5 (Conjugate 3, lower line) after aging under accelerated conditions at 37°C for 28 and 32 days.

[0129] Fig.16 Shown are the VEGF binding constants (K) of BI VHH anti-VEGF MVPs prepared using Intermediate Method 1, Method 2, or Method 5 before and after aging at 37°C. D). By day 4 of the stability study, MVP synthesized using the intermediate of method 5 no longer bound VEGF. The BLI limit of detection (LOD) was 0.001 nM. The samples listed in the figure with this value were below K D BLI LOD.

[0130] Fig.17 Shown are the binding constants (K) of DARPin MVP after aging at 37 °C for 28 and 32 days using intermediate method 1 (conjugate 2, squares, upper line) or method 5 (conjugate 1, triangles, lower line). on ). Lower K values ​​were observed in Conjugate 1 both initially and during aging. on and slower binding, indicating that the low-impurity polymer of conjugate 2 is stabilizing and enhancing K on .

[0131] Fig.18 VHH and MVP synthesized with Method 1 intermediates show increased in vivo half-life after intravitreal injection in rabbits. Conjugate 12 anti-TNFα VHH MVP (Method 1 intermediate) rabbit intravitreal pharmacokinetic (IVT PK) study, half-life increased by >2X compared to unconjugated. n=3 eyes per time point. All eyes received 50 μg VHH. DETAILED DESCRIPTION OF THE INVENTION

[0133] I. Overview

[0134] The present invention provides multivalent peptide-hyaluronic acid polymer conjugates and preparation methods thereof, wherein the conjugate uses a peptide linker to covalently link each bioactive peptide to a polymer. In some embodiments, the peptide linker is an alpha-helix. The peptide has been modified to increase the degree of humanization while maintaining stability and the ability to be expressed at an acceptable level in a bacterial system (e.g., E. coli). The corresponding conjugate is expected to have an immunogenicity comparable to other humanized antibodies.

[0135] II. Definitions

[0136] Unless otherwise specifically stated, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. In addition, any method or material similar or equivalent to the method or material described herein can be used in the practice of the present invention. For the purposes of the present invention, the following terms are defined.

[0137] "About" when referring to a value includes + / - 10% of the stated value. For example, about 50% includes a range of 45% to 55%, and about 20 molar equivalents includes a range of 18 to 22 molar equivalents. Thus, when referring to a range, "about" refers to each stated value of + / - 10% of the stated value at each end of the range. For example, a ratio (weight / weight) of about 1 to about 3 includes a range of 0.9 to 3.3.

[0138] "Alkyl" is a straight or branched saturated monovalent or divalent hydrocarbon. For example, an alkyl group can have 1 to 10 carbon atoms (i.e., C 1-10 alkyl) 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 1-4 Examples 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, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl 1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)C H2CH3), 2-methyl-2-propyl (t-Bu, tert-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 (-CH2CH 2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3 )CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3).

[0139] "Cycloalkyl" refers to a single saturated or partially unsaturated all-carbon ring (i.e., C 3-20 Cycloalkyl) such as 3 to 12 ring atoms, such as 3 to 10 ring atoms, or 3 to 8 ring atoms, or 3 to 6 ring atoms, or 3 to 5 ring atoms, or 3 to 4 ring atoms. The term "cycloalkyl" also includes multiple fused, saturated and partially unsaturated full carbocyclic ring systems (for example, ring systems comprising 2, 3 or 4 carbocyclic rings). Therefore, cycloalkyl includes polycyclic carbocyclic rings, such as bicyclic carbocyclic rings (for example, bicyclic carbocyclic rings with about 6 to 12 ring carbon atoms, such as bicyclic [3.1.0] hexane and bicyclic [2.1.1] hexane) and polycyclic carbocyclic rings (for example, tricyclic and tetracyclic carbocyclic rings with up to about 20 ring carbon atoms). When the valence requirement allows, the rings of multiple fused ring systems can be interconnected by fused bonds, spiral bonds and bridge bonds. 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.

[0140] 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 can be about 100 Da to 500 Da. The types of organic linkers of the present invention include, but are not limited to, imides, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. It will be appreciated by those skilled in the art that other types of organic linkers may also be used in the present invention.

[0141] "Thiol" refers to a -SH functional group.

[0142] "Thiol-reactive groups" refer to groups that can react with thiols to form covalent bonds with sulfur atoms. Representative thiol-reactive groups include, but are not limited to, thiols, TNB-thiols, haloacetyl groups, aziridines, acryloyl groups, vinyl sulfones, APN (3-arylpropionitrile), maleimides, and pyridyl disulfides. The reaction of thiol-reactive groups with thiols can form disulfides or thioethers.

[0143] As used herein, "conjugating agent" refers to a reagent that reacts a carboxylic acid (-(C=O)-OH) with an amine (-NH2) group to form an amide (-(C=O)-NH-).

[0144] "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 the naturally occurring amino acids. The term "peptide" includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, fusions with or without N-terminal methionine residues; immunolabeled proteins; and the like. Peptides also include post-translationally modified peptides.

[0145] As used herein, "VHH" refers to a single domain heavy chain antibody.

[0146] “DARPin” refers to designed ankyrin repeat protein, which is a genetically engineered antibody-mimicking protein that can exhibit highly specific and high-affinity binding to target proteins.

[0147] The "alpha-helix" or "α-helix" is a common motif in protein secondary structure, a right-handed helical conformation in which each backbone NH group is hydrogen-bonded to the backbone C=O group of the amino acid located in the first four residues of the protein sequence. The alpha helix is ​​also called the classic Pauling-Corey-Branson alpha-helix or 3.6 13 Helix, which represents the average number of residues per helical turn (3.6), wherein 13 atoms participate in the ring formed by hydrogen bonds. Peptides containing alpha-helices are referred to as alpha-helices. Such peptides may be partially or entirely alpha-helices. As understood in the art, an alpha-helix has at least four amino acid residues. In some embodiments, an alpha-helix has 4 to 40 amino acids.

[0148] Also provided are pharmaceutically acceptable salts of the peptides or conjugates described herein."Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms and other materials that can be used to prepare pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0149] As used herein, "pharmaceutical composition" refers to a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. A pharmaceutical composition is generally safe for biological use.

[0150] As used herein, "pharmaceutically acceptable excipient" refers to a substance that helps the active agent to be absorbed by the 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 pigments. Those skilled in the art will appreciate that other pharmaceutically acceptable excipients may also be used in the present invention.

[0151] The conjugates described herein can be prepared and / or formulated as pharmaceutically acceptable salts, or as free bases where appropriate. Pharmaceutically acceptable salts are non-toxic salts of free base forms of compounds having the pharmacological activity required for free bases. These salts can be derived from inorganic or organic acids or bases. For example, conjugates containing basic nitrogen can be prepared into pharmaceutically acceptable salts by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butynes The pharmaceutically acceptable salts of the present invention are hydroxybenzoic acid, ...

[0152] Examples of "pharmaceutically acceptable salts" of the conjugates disclosed herein also include salts derived from appropriate bases, such as alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., magnesium), ammonium and NR4 + (wherein R is C1-C4 alkyl). Also included are base addition salts such as sodium or potassium salts.

[0153] As used herein, "therapeutically effective amount" refers to the dosage that produces a therapeutic effect. The exact dosage will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 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, Gennaro, ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose may be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0154] As used herein, "Inhibition," "inhibits," and "inhibitors" refer to a compound or method that prevents a specific effect or function.

[0155] As used herein, "treatment" or "treat" or "treating" refers to a method of obtaining a beneficial or desired result. For the purposes of this disclosure, a beneficial or desired result includes, but is not limited to, relieving symptoms and / or reducing the extent 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 the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition, and / or reducing the extent of a disease or condition); b) slowing or preventing the development of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, delaying worsening or progression of a disease or condition); c) alleviating a disease or condition, for example, resulting in regression of clinical symptoms, improving the disease state, slowing disease progression, improving quality of life, and / or prolonging survival.

[0156] "Prevention" refers to preventing or delaying clinical progression of a disease in a patient suffering from the disease.

[0157] A "subject" of the present invention is a mammal, which can be a human or a non-human mammal, for example a companion animal, such as a dog, cat, rat, etc., or a farm animal, such as a horse, donkey, mule, goat, sheep, pig, or cow, etc. In some embodiments, the subject is a human.

[0158] As used herein, "joint" refers to a fibrous or cartilaginous joint, which is an area of ​​fibrous or cartilaginous where two or more bones are interconnected.

[0159] As used herein, "diffusion half-life" refers to the time required for the initial concentration of a conjugate to decrease by half within a given volume or space, where the decrease in concentration is a function of a concentration gradient.

[0160] As used herein, "intra-articular half-life" refers to the time required for the initial concentration of a conjugate to decrease by half within a particular joint, where transport out of the joint is by convection. Convective transport is transport by a combination of diffusion and advection, where advective transport is the transport of a substance by bulk motion.

[0161] III. Peptides

[0162] In some embodiments, the peptides of the present invention have advantages over similar peptides in the art, such as higher degree of humanization, higher solubility, higher stability, lower tendency to aggregate in solution, and / or higher expression levels in convenient systems such as E. coli.

[0163] In some embodiments, the peptide is a peptide represented by formula (I):

[0164] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I), CDR1, CDR2 and CDR3 are each independently a complementarity determining region;

[0165] FR1 has an amino acid sequence comprising:

[0166] X 10 VxD 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1),

[0167] in

[0168] X 10 is Q, E or D,

[0169] X 11 is V, Q, A or E,

[0170] X 12 is S or T,

[0171] X 13 is L, S or V,

[0172] X14 is V or A,

[0173] X 15 is P, A or T,

[0174] X 16 is G, D or R,

[0175] X 17 is A, V, T, or E, and

[0176] X 18 A or V;

[0177] FR2 has an amino acid sequence comprising: X 20 X 21 WX 22 R 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2),

[0178] in

[0179] X 20 is M, I, V or L,

[0180] X 21 is G, S or A,

[0181] X 22 is F, Y or V,

[0182] X 23 is A, V, P or T,

[0183] X 24 is E, G, A or Q,

[0184] X 25 is R or L,

[0185] X 26 is F, G, W or L,

[0186] X 27 is A, G, or S, and

[0187] X 28 is A, S or G;

[0188] FR3 has an amino acid sequence comprising:

[0189] YX 30 DSVKGRFTISX 31 DX 32 X 33 KX34 X 35 VX 36 LqCy 37 X 38 LRX 39a EDTAX 39b YYCAA (SEQ ID NO: 3),

[0190] in

[0191] X 30 is A, G, S or T,

[0192] X 31 is R or Q,

[0193] X 32 is N, S or D,

[0194] X 33 is S, A or D,

[0195] X 34 is N or K,

[0196] X 35 is T or M,

[0197] X 36 is Y, D or S,

[0198] X 37 is N or D,

[0199] X 38 is S or N,

[0200] X 39a is P or A, and

[0201] X 39b is V, M, L or I; and

[0202] FR4 has an amino acid sequence comprising: YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4),

[0203] in

[0204] X 40 is Q or K, and

[0205] X 41 It is L or Q.

[0206] In some embodiments, X 13 For L.

[0207] In some embodiments, X 27 is A.

[0208] In some embodiments, X 30 is A.

[0209] In some embodiments, X 39a For P.

[0210] In some embodiments, X 40 For Q.

[0211] In some embodiments, FR1 has an amino acid sequence comprising:

[0212] QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 5).

[0213] In some embodiments, FR2 has an amino acid sequence comprising:

[0214] MGWFRQAPGKEREFVAAI (SEQ ID NO:6).

[0215] In some embodiments, FR3 has an amino acid sequence comprising:

[0216] YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7).

[0217] In some embodiments, FR4 has an amino acid sequence comprising: YWGQGTLVTVSS (SEQ ID NO: 8).

[0218] In some embodiments, FR1 has an amino acid sequence comprising:

[0219] QVQLVESGGGLVQPGGSLRLSCAASG(SEQ ID NO:5);

[0220] FR2 has an amino acid sequence comprising: MGWFRQAPGKEREFVAAI (SEQ ID NO: 6);

[0221] FR3 has an amino acid sequence comprising:

[0222] YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7); and

[0223] FR4 has an amino acid sequence comprising: YWGQGTLVTVSS (SEQ ID NO: 8).

[0224] In some embodiments, CDR1, CDR2 and CDR3 are each complementary determining regions from an antibody or a cytokine. 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, each of the complementarity determining regions is specific for vascular endothelial growth factor (VEGF), tumor necrosis factor-α (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-associated protein (GITR), V-domain immunoglobulin inhibitor of T cell activation (VISTA), T-cell immunoglobulin mucin 3 (TIM-3), lymphocyte activation gene-3 (LAG3), interleukin-1-β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin 12 (IL-12), or interleukin 15 (IL-15). In some embodiments, each of the complementarity determining regions is specific for vascular endothelial growth factor (VEGF).

[0225] In some embodiments, the peptide consists of Formula I.

[0226] In some embodiments, the peptide has one or more of: (a) a CDR1 of 7 amino acids in length; (b) a CDR2 of 7 or 8 amino acids in length; and / or (c) a CDR3 of 9 to 16 amino acids in length.

[0227] 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

[0228] (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

[0229] (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

[0230] (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).

[0231] In some embodiments, the amino acid sequence comprises any of the following: 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.

[0232] In some embodiments, the amino acid sequence comprises any of the following: 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 of the following: SEQ ID NOS: 73, 81, 91, and 92. In some embodiments, the peptide has an amino acid sequence comprising any of the following: SEQ ID NOS: 101-106. In some embodiments, the peptide has an amino acid sequence comprising: SEQ ID NO: 67.

[0233] IV. Conjugates

[0234] In some embodiments, the conjugate is a conjugate of formula IIa:

[0235] (X 1 -X 2 -Y) n -Z(IIa),

[0236] in

[0237] Each X 1 independently a peptide as described herein;

[0238] Each X 2 are independently peptide linkers of 3 to 100 amino acids in length;

[0239] Each Y is independently an organic linker;

[0240] Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; and

[0241] The subscript n is an integer from 1 to 1500.

[0242] In some embodiments, the conjugate is a conjugate of formula IIb:

[0243] (X 1 -X 2A -Y) n -Z(IIb),

[0244] in

[0245] Each X 1 are independently a peptide having a molecular weight of about 5 kDa to about 200 kDa;

[0246] Each X 2A independently a peptide linker comprising an alpha helix;

[0247] Each Y is independently an organic linker;

[0248] Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; and

[0249] The subscript n is an integer from 1 to 1500.

[0250] In some embodiments, each X 1 Independently a peptide as described in the present invention.

[0251] In some embodiments, each peptide linker is independently 7 to 100 amino acids in length. In some embodiments, each peptide linker is independently 10 to 30 amino acids in length.

[0252] In some embodiments, each peptide linker independently has an amino acid sequence comprising:

[0253] AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21),

[0254] AEEEKRKAEEEEKRKAEEEAGC(SEQ ID NO:22),

[0255] AEEEKRKAEEEKRKAEEEKRKAEEEAGC(SEQ ID NO:23),

[0256] AEEEEKKKKEEEEKKKKAGC(SEQ ID NO:24),

[0257] AEAAAKEAAAKAGC(SEQ ID NO:25),

[0258] PSRLEEELRRRLTEGC (SEQ ID NO: 26), or

[0259] AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEERKKREDDEKRKKKAGC (SEQ ID NO: 27).

[0260] In some embodiments, each peptide linker has an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

[0261] Each peptide can be linked to a biocompatible polymer via a variety of organic linkers commonly used in the art to form antibody-drug conjugates, such as those provided by Conju-Probe or BroadPharm in San Diego, California, or Creative Biolabs in Shirley, New York. Methods for forming bioconjugate bonds are described in Bioconjugation Technology, 3rd Edition, Greg T. Hermanson. Organic linkers can react with amines, carbonyls, carboxyls, and active esters, can react by click chemistry (with or without copper), or react with thiols.

[0262] Representative organic linkers include amides or disulfides, or are formed by reactive groups, such as succinic anhydride, succinimide, N-hydroxysuccinimide, N-chlorosuccinimide, N-bromosuccinimide, maleic anhydride, maleimide, hydantoin, phthalimide, etc. The organic linkers that can be used for the present invention are relatively small, and the molecular weight is generally about 100Da to about 500Da, comprising two functional groups consisting of maleimide and amine or hydrazide. In some embodiments, the peptide is covalently attached to the polymer by a sulfide bond and an organic linker with a molecular weight of about 100Da to about 500Da. In some embodiments, the molecular weight of the organic linker is about 100Da to about 300Da. In some embodiments, the organic linker includes succinimide. In some embodiments, the organic linker is formed using N-β-maleimidopropionic acid hydrazide (BMPH), N-ε-maleimidohexanoic acid hydrazide (EMCH), N-aminoethylmaleimide, N-κ-maleimidoundecanoic acid hydrazide (KUMH), hydrazide-PEG2-maleimide, amine-PEG2-maleimide, hydrazide-PEG3-maleimide or amine-PEG3-maleimide.

[0263] Representative organic linkers include, but are not limited to:

[0264]

[0265] In some embodiments, the organic linker can be N-ε-maleimidocaproic acid hydrazide (EMCH):

[0266]

[0267] In some embodiments, the organic linker has the following structure:

[0268]

[0269] The subscript m is an integer from 1 to 300.

[0270] In some embodiments, the organic linker has the following structure:

[0271]

[0272] In some embodiments, preparing the conjugate of the invention comprises covalently linking an organic linker to a biocompatible polymer and then covalently linking the peptide to the organic linker. In some embodiments, after preparing the conjugate of the invention, unreacted organic linkers are present on the biocompatible polymer. The structure of the unreacted organic linker depends on the organic linker and will be understood by those skilled in the art.

[0273] Representative unreacted organic linkers include, but are not limited to,

[0274]

[0275] In some embodiments, the unreacted organic linker has the following structure:

[0276]

[0277] In some embodiments, the unreacted organic linker has the following structure:

[0278]

[0279] wherein the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

[0280] In some embodiments, the unreacted organic linker has the following structure:

[0281]

[0282] In some embodiments, the biocompatible polymer is a polysaccharide.

[0283] In some embodiments, the biocompatible polymer is a glycosaminoglycan.

[0284] In some embodiments, the biocompatible polymer is hyaluronic acid.

[0285] 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.

[0286] In some embodiments, subscript n is an integer from 1 to 1500. In some embodiments, subscript n is an integer from 5 to 1000. In some embodiments, subscript n is an integer from 10 to 400. In some embodiments, subscript n is an integer from 10 to 100.

[0287] In some embodiments, the conjugate is a conjugate of formula IIa:

[0288] (X 1 -X 2 -Y) n -Z(IIa),

[0289] in

[0290] Each X1 independently a peptide as described herein;

[0291] Each X 2 is a peptide linker having an amino acid sequence comprising:

[0292] AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21);

[0293] Each Y is an organic linker having the following structure:

[0294]

[0295] Z is a biocompatible polymer which is hyaluronic acid having a molecular weight of about 0.1 MDa to about 3 MDa;

[0296] Subscript m is an integer from 1 to 300; and

[0297] The subscript n is an integer from 1 to 1500.

[0298] In some embodiments, the conjugate of the present invention is a conjugate of a random polymer represented by Formula III:

[0299] (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (III)

[0300] Its molecular weight is from about 0.1 MDa to about 3 MDa;

[0301] in

[0302] Each X is independently a peptide having a molecular weight of about 5 kDa to about 200 kDa;

[0303] Each Y is an organic linker;

[0304] Each XYZ 1 The section has the following structure:

[0305]

[0306] Each Z 2 Has the following structure:

[0307]

[0308] Each Z 3 Independently has the following structure:

[0309]

[0310] Each R 1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl;

[0311] Each R 3 and R 4 are independently H or C1-C6 alkyl;

[0312] Each Z 3a are independently OH or Y';

[0313] Each Y' is an unreacted organic linker;

[0314] Subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q;

[0315] Subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; and

[0316] Subscript q is an integer from 100 to 10,000.

[0317] In some embodiments, each X is a peptide having an amino acid sequence comprising any 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.

[0318] In some embodiments, each X is a peptide having an amino acid sequence comprising any of SEQ ID NOs: 51-58, 61-73, 81-85, 91-95, 101-106, and 111-118.

[0319] In some embodiments, the conjugate has the structure shown in Formula IIIa:

[0320] (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 )q (IIIa),

[0321] in

[0322] Each X 1 are independently a peptide having a molecular weight of about 5 kDa to about 200 kDa; and

[0323] Each X 2 It is a peptide linker containing an alpha helix.

[0324] In some embodiments, each X 1 Contains a peptide represented by formula I:

[0325] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I), CDR1, CDR2 and CDR3 are each independently a complementarity determining region;

[0326] FR1 has an amino acid sequence comprising:

[0327] X 10 VxD 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1),

[0328] in

[0329] X 10 is Q, E or D,

[0330] X 11 is V, Q, A or E,

[0331] X 12 is S or T,

[0332] X 13 is L, S or V,

[0333] X 14 is V or A,

[0334] X 15 is P, A or T,

[0335] X 16 is G, D or R,

[0336] X 17 is A, V, T, or E, and

[0337] X 18 A or V;

[0338] FR2 has an amino acid sequence comprising:

[0339] X 20 X 21 WX 22 R 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2),

[0340] in

[0341] X 20 is M, I, V or L,

[0342] X 21 is G, S or A,

[0343] X 22 is F, Y or V,

[0344] X 23 is A, V, P or T,

[0345] X 24 is E, G, A or Q,

[0346] X 25 is R or L,

[0347] X 26 is F, G, W or L,

[0348] X 27 is A, G, or S, and

[0349] X 28 is A, S or G;

[0350] FR3 has an amino acid sequence comprising:

[0351] YX 30 DSVKGRFTISX 31 DX 32 X 33 KX 34 X 35 VX 36 LqCy 37 X 38 LRX 39a EDTAX 39b YYCA

[0352] (SEQ ID NO:3),

[0353] in

[0354] X 30 is A, G, S or T,

[0355] X 31 is R or Q,

[0356] X 32 is N, S or D,

[0357] X 33 is S, A or D,

[0358] X 34 is N or K,

[0359] X 35 is T or M,

[0360] X 36 is Y, D or S,

[0361] X 37 is N or D,

[0362] X 38 is S or N,

[0363] X 39a is P or A, and

[0364] X 39b is V, M, L or I; and

[0365] FR4 has an amino acid sequence comprising: YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4),

[0366] in

[0367] X 40 is Q or K, and

[0368] X 41 It is L or Q.

[0369] In some embodiments of the peptide of Formula I, X 13 For L.

[0370] In some embodiments of the peptide of Formula I, X 27 is A.

[0371] In some embodiments of the peptide of Formula I, X 30 is A.

[0372] In some embodiments of the peptide of Formula I, X 39a For P.

[0373] In some embodiments of the peptide of Formula I, X 40For Q.

[0374] In some embodiments of the peptide of Formula I, FR1 has an amino acid sequence comprising: QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 5).

[0375] In some embodiments of the peptide of Formula I, FR2 has an amino acid sequence comprising: MGWFRQAPGKEREFVAAI (SEQ ID NO: 6).

[0376] In some embodiments of the peptide of Formula I, FR3 has an amino acid sequence comprising: YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO: 7).

[0377] In some embodiments of the peptide of Formula I, FR4 has an amino acid sequence comprising: YWGQGTLVTVSS (SEQ ID NO: 8).

[0378] In some embodiments of the peptide of Formula I, FR1 has an amino acid sequence comprising: QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 5);

[0379] FR2 has an amino acid sequence comprising: MGWFRQAPGKEREFVAAI (SEQ ID NO: 6);

[0380] FR3 has an amino acid sequence comprising:

[0381] YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7); and

[0382] FR4 has an amino acid sequence comprising: YWGQGTLVTVSS (SEQ ID NO: 8).

[0383] In some embodiments of the peptides of Formula I, CDR1, CDR2, and CDR3 are each complementarity determining regions from an antibody or a cytokine.

[0384] 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.

[0385] In some embodiments, each of the complementary determining regions is specific to vascular endothelial growth factor (VEGF), tumor necrosis factor-α (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-associated protein (GITR), V-domain immunoglobulin inhibitor of T cell activation (VISTA), T cell immunoglobulin mucin 3 (TIM-3), lymphocyte activation gene-3 (LAG3), interleukin-1-β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin 12 (IL-12) or interleukin 15 (IL-15). In some embodiments, each of the complementary determining regions is specific to vascular endothelial growth factor (VEGF). In some embodiments, each of the complementarity determining regions is specific for tumor necrosis factor-α (TNF-α).In some embodiments, each of the complementarity determining regions is specific for interleukin-1-β (IL-1β).

[0386] In some embodiments, the peptide consists of Formula I.

[0387] In some embodiments, the peptide has one or more of: (a) a CDR1 of 7 amino acids in length; (b) a CDR2 of 7 or 8 amino acids in length; and / or (c) a CDR3 of 9 to 16 amino acids in length.

[0388] In some embodiments of the peptide,

[0389] (a) CDR1 has an amino acid sequence comprising: FAYSTYS (SEQ ID NO: 9),

[0390] CDR2 has an amino acid sequence comprising: NSGTFRLW (SEQ ID NO: 10), and

[0391] CDR3 has an amino acid sequence comprising: RAWSPYSSTVDAGDFR (SEQ ID NO: 11); or

[0392] (b) CDR1 has an amino acid sequence comprising: RRFSIEA (SEQ ID NO: 12),

[0393] CDR2 has an amino acid sequence comprising: DSGGSTD (SEQ ID NO: 13), and

[0394] CDR3 has an amino acid sequence comprising: IGGSWYGRGLD (SEQ ID NO: 14); or

[0395] (c) CDR1 has an amino acid sequence comprising: GTFSSII (SEQ ID NO: 15),

[0396] CDR2 has an amino acid sequence comprising: SWSGGTTV (SEQ ID NO: 16), and

[0397] CDR3 has an amino acid sequence comprising: RPYQKYNWASASYNV (SEQ ID NO: 17); or

[0398] (d) CDR1 has an amino acid sequence comprising: GGSDAGT (SEQ ID NO: 18),

[0399] CDR2 has an amino acid sequence comprising: SWAGTAWR (SEQ ID NO: 19), and

[0400] CDR3 has an amino acid sequence comprising: LGSYEMDHH (SEQ ID NO: 20).

[0401] In some embodiments, each X 1 is a peptide having an amino acid sequence comprising any of the following: SEQ ID NOS: 51-58, 61-73, 81-85, 91-95, 101-106 and 111-118. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising the following: SEQ ID NO: 55. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising the following: SEQ ID NO: 67. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising the following: SEQ ID NO: 73. In some embodiments, each X 1 A peptide having the following amino acid sequence: SEQ ID NO:91.

[0402] In some embodiments, each X 2 A peptide linker having the following amino acid sequence:

[0403] AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21),

[0404] AEEEKRKAEEEEKRKAEEEAGC(SEQ ID NO:22),

[0405] AEEEKRKAEEEKRKAEEEKRKAEEEAGC(SEQ ID NO:23),

[0406] AEEEEKKKKEEEEKKKKAGC(SEQ ID NO:24),

[0407] AEAAAKEAAAKAGC(SEQ ID NO:25),

[0408] PSRLEEELRRRLTEGC (SEQ ID NO: 26), or

[0409] AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEERKKREDDEKRKKKAGC(SEQ ID NO:27)

[0410] In some embodiments, each X 2 is a peptide linker having the following amino acid sequence: AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

[0411] In some embodiments, the organic linker has the following structure:

[0412]

[0413] In some embodiments, the organic linker can be N-ε-maleimidocaproic acid hydrazide (EMCH):

[0414]

[0415] In some embodiments, the organic linker has the following structure:

[0416]

[0417] and wherein subscript m is an integer from 1 to 300. In some embodiments, subscript m is an integer from 1 to 100.

[0418] In some embodiments, the organic linker has the following structure:

[0419]

[0420] The organic linker having the above structure is called MP2H.

[0421] In some embodiments, the molecular weight of the random polymer shown in formula III is about 0.4 MDa to about 2 MDa. In some embodiments, the molecular weight of the random polymer shown in formula III is about 0.7 MDa to about 1.5 MDa. In some embodiments, the molecular weight of the random polymer shown in formula III is about 0.8 MDa.

[0422] 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 -(CH2)3-NMe2. 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 It is ethyl.

[0423] In some embodiments, each R 3 and R 4 is independently C1-C3 alkyl. 3 and R 4 It is methyl.

[0424] In some embodiments, subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; subscript q is an integer from 100 to 10000. In some embodiments, subscript n is an integer from 1 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 800 and is less than about 8% of the sum of subscripts n, p, and q; subscript q is an integer from 100 to 10000. In some embodiments, subscript n is an integer from 10 to 450 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript q is an integer from 1000 to 3000. In some embodiments, subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 240 and is less than about 8% of the sum of subscripts n, p, and q; subscript q is an integer from 1000 to 3000. In some embodiments, subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 60 and is less than about 2% of the sum of subscripts n, p, and q; subscript q is an integer from 1000 to 3000. In some embodiments, subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 30 and is less than about 1% of the sum of subscripts n, p, and q; subscript q is an integer from 1000 to 3000. In some embodiments, subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer from 1000 to 3000.

[0425] In some embodiments, the conjugate of the present invention is a conjugate of a random polymer represented by Formula III:

[0426] (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (III)

[0427] Its molecular weight is from about 0.1 MDa to about 3 MDa;

[0428] in

[0429] Each X is independently a peptide having a molecular weight of about 5 kDa to about 200 kDa;

[0430] Each Y is an organic linker;

[0431] Each XYZ1 The section has the following structure:

[0432]

[0433] Each Z 2 Has the following structure:

[0434]

[0435] Each Z 3 Independently has the following structure:

[0436]

[0437] Each R 1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl;

[0438] Each R 3 and R 4 are independently H or C1-C6 alkyl;

[0439] Each Z 3a are independently OH or Y';

[0440] Each Y' is an unreacted organic linker;

[0441] Subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q;

[0442] Subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; and

[0443] Subscript q is an integer from 100 to 10,000.

[0444] In some embodiments, each X is a peptide having an amino acid sequence comprising any 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.

[0445] In some embodiments, the conjugate of the present invention is a conjugate of a random polymer represented by Formula III:

[0446] (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (III)

[0447] Its molecular weight is from about 0.1 MDa to about 3 MDa;

[0448] in

[0449] Each X is independently an anti-TNF-α or anti-IL-1β peptide comprising:

[0450] QVQLQES GGGLVQPGGS LRLSCAASGR TFSDHSGYTY TIGWFRQAPG KEREFVARIYWSSGNTYYAD SVKGRFAISR DIAKNTVDLT MNNLEPEDTA VYYCAARDGIPTSRSVESYN YWGQGTQVTVSSPSTPPTPS PSTPPGGCDD DDK(SEQ ID NO:101),

[0451] QVQLQES GGGLVQPGGS LRLSCAASGR TFSDHSGYTY TIGWFRQAPG KEREFVARIYWSSGNTYYAD SVKGRFAISR DIAKNTVDLT MNNLEPEDTA VYYCAARDGIPTSRSVESYN YWGQGTQVTVSSAEAAAAKEA AAKEAAAKAG C (SEQ ID NO: 102),

[0452] QVQLQDS GGGLVQAGGS LRLSCAASGG TFSSIIMAWF RQAPGKEREF VGAVSWSGGTTVYADSVLGR FEISRDSARK SVYLQMNSLK PEDTAVYYCA ARPYQKYNWA SASYNVWGQG TQVTVSSAEAAAKEAAAKEA AAKAGC (SEQ ID NO: 103),

[0453] QVQLQES GGGLVQAGGS LRLSCAASGG TFSSIIMAWF RQAPGKEREF VGAVSWSGGTTVYADSVKGR FTISRDSARK SVYLQMNSLK PEDTAVYYCA ARPYQKYNWA SASYNVWGQG TQVTVSSAEAAAKEAAAKEA AAKAGC (SEQ ID NO: 104),

[0454] CGGGVDNKFN KEVGWAFGEI GALPNLNALQ FRAFIISLWD DPSQSANLLA EAKKLNDAQA PK(SEQ ID NO:105), or

[0455] EIVMTQS PSTLSASVGD RVIITCQASQ SIDNWLSWYQ QKPGKAPKLL IYRASTLASGVPSRFSGSGS GAEFTLTISS LQPDDFATYY CQNTGGGVSI AFGQGTKLTV LGGGGGSGGG GSGGGGSGGGGSEVQLVESG GGLVQPGGSL RLSCTASGFS LSSAAMAWVR QAPGKGLEWV GIIYDSASTY YASWAKGRFTISRDTSKNTV YLQMNSLRAE DTAVYYCARE RAIFSGDFVL WGQGTLVTVS SSPSTPPTPS PSTPPGGC (SEQ ID NO: 106);

[0456] Each Y is an organic linker having the following structure:

[0457]

[0458] Each XYZ 1 The section has the following structure:

[0459]

[0460] Each Z 2 Has the following structure:

[0461]

[0462] Each Z 3 Independently has the following structure:

[0463]

[0464] Each R1 and R 2 are independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl;

[0465] Each R 3 and R 4 are independently H or C1-C6 alkyl;

[0466] Each Z 3a are independently OH or Y';

[0467] Each Y' has the following structure:

[0468]

[0469] Subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q;

[0470] Subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; and

[0471] Subscript q is an integer from 100 to 10,000.

[0472] In some embodiments, the conjugate is a conjugate of a random polymer represented by Formula IIIa:

[0473] (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa),

[0474] Its molecular weight is about 0.8 MDa;

[0475] in

[0476] Each X 1 A peptide having an amino acid sequence comprising the following: SEQ ID NO: 55;

[0477] Each X 2 A peptide linker having the following amino acid sequence: AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21);

[0478] Each Y is an organic linker having the following structure:

[0479]

[0480] Each X1 -X 2 -YZ 1 The section has the following structure:

[0481]

[0482] Each Z 2 Has the following structure:

[0483]

[0484] Each Z 3 Independently has the following structure:

[0485]

[0486] Each Z 3a are independently OH or Y';

[0487] Each Y' has the following structure:

[0488]

[0489] Each R 1 and R 2 is ethyl or (CH2)3-NMe2;

[0490] Subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q;

[0491] Subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and

[0492] The subscript q is an integer from 1000 to 3000.

[0493] In some embodiments, the conjugate is a conjugate of a random polymer represented by Formula IIIa:

[0494] (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa),

[0495] Its molecular weight is about 0.8 MDa;

[0496] in

[0497] Each X 1 is a peptide having an anti-VEGF amino acid sequence comprising: SEQ ID NO: 67;

[0498] Each X 2 is a peptide linker having an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21);

[0499] Each Y is an organic linker having the following structure:

[0500]

[0501] Each X 1 -X 2 -YZ 1 The section has the following structure:

[0502]

[0503] Each Z 2 Has the following structure:

[0504]

[0505] Each Z 3 Independently has the following structure:

[0506]

[0507] Each Z 3a are independently OH or Y';

[0508] Each Y' has the following structure:

[0509]

[0510] Each R 1 and R 2 is ethyl or -(CH2)3-NMe2;

[0511] Subscript n is an integer from 10 to 300 and is less than about 10% of the sum of subscripts n, p, and q;

[0512] Subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and

[0513] The subscript q is an integer from 1000 to 3000.

[0514] In some embodiments, the conjugates of the invention exhibit an in vivo half-life of about 12 hours to about 24 hours, about 1 day to about 3 days, about 3 days to about 7 days, 1 week to about 2 weeks, about 2 weeks to about 4 weeks, or about 1 month to about 6 months.

[0515] 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, 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.

[0516] The biological activity of the conjugate is enhanced relative to the activity of the corresponding peptide in a soluble form (e.g., compared to the activity of a peptide not conjugated to a polymer). In some embodiments, the biological activity of the conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times, or greater than 1000 times, compared to the biological activity of the peptide in a soluble (unconjugated) form.

[0517] V. Composition

[0518] In some embodiments, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable excipient.

[0519] A. Preparation

[0520] To prepare pharmaceutical compositions from the conjugates of the invention, pharmaceutically acceptable carriers may be solid or liquid. Solid form preparations include powders, cachets, and dispersible granules. A solid carrier may be one or more substances that may also serve as a diluent, binder, preservative, disintegrant, or encapsulating material. Details of formulations and administration techniques are described in detail in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Company, Easton, Pennsylvania ("Remington's").

[0521] 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 compacted into the desired shape and size. Powders and tablets preferably contain from 5% or 10% to 70% of the conjugate of the invention.

[0522] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. For parenteral injection, liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

[0523] Aqueous solutions suitable for oral use can be prepared by dissolving the conjugate of the present invention in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as needed. Aqueous suspensions suitable for oral use can be prepared by dispersing finely divided active ingredients with viscous substances (e.g., natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth gum and gum arabic), and dispersants or wetting agents (e.g., naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain fatty alcohols (e.g., heptadecaethyleneoxyhexadecanoate), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (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)) in water. The aqueous suspension may also contain one or more preservatives (eg, 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 formulation may be adjusted according to osmotic pressure.

[0524] Also included are solid form preparations that are intended to be converted into liquid form preparations for oral administration shortly before use. Such liquid forms include solutions, suspensions, and emulsions. These preparations may also contain colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc. in addition to the active ingredient.

[0525] Oily suspensions can be prepared by suspending the conjugate of the present invention in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (e.g., liquid paraffin); or a mixture of these. Oily suspensions may contain thickeners, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners may be added to provide a palatable oral preparation, such as glycerol, sorbitol, or sucrose. These preparations may be preserved by adding antioxidants, such as ascorbic acid. As an example of an injectable oil carrier, see Minto, J. Pharmacol. Exp. Ther. 281: 93-102, 1997. The pharmaceutical preparations of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be the above-mentioned vegetable oil or mineral oil, or a mixture thereof. Suitable emulsifiers include naturally occurring gums, such as gum arabic and gum tragacanth, naturally occurring phospholipids, such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavorings, such as in the preparations of syrups and elixirs. Such preparations may also contain a demulcent, a preservative, or a coloring agent.

[0526] The compositions of the present invention can also be delivered as microspheres for slow release in vivo. For example, the microspheres can be formulated to be administered by intradermal injection of drug-containing microspheres, which are slowly released subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7: 623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12: 857-863, 1995); or as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49: 669-674, 1997). Both transdermal and intradermal routes can provide sustained delivery for weeks or months.

[0527] In another embodiment, the composition of the present invention can be formulated for parenteral administration into a body cavity, such as intratumoral administration, intravitreal administration into the eye or intra-articular space. The preparation for administration generally includes a solution of the composition of the present invention dissolved in a pharmaceutically acceptable carrier. Acceptable carriers and solvents that can be used include water and Ringer's solution, isotonic sodium chloride. In addition, sterile fixed oils can generally be used as solvents or suspension media. For this purpose, any light fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used to prepare injections. These solutions are sterile and generally do not contain undesirable substances. These preparations can be sterilized by conventional, well-known sterilization techniques. The preparation can contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the composition of the present invention in these preparations can vary greatly, and will be selected mainly based on fluid volume, viscosity, body weight, etc., according to the selected specific mode of administration and the needs of the patient. For intravenous injection, intratumoral or intravitreal administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. The suspension can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution.

[0528] In another embodiment, the preparation of the composition of the present invention can be delivered by using liposomes that fuse with the cell membrane or are endocytosed, that is, by using ligands attached to liposomes or directly attached to oligonucleotides, which bind to the surface membrane protein receptors of the cell to cause endocytosis. By using liposomes, especially when the surface of the liposomes carries specific ligands for target cells or otherwise preferentially targets specific organs, the composition of the present invention can be delivered to the target cells in the body. (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).

[0529] 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). Specifically, SEDDS and SMEDDS are isotropic mixtures of lipids, surfactants and cosurfactants that can spontaneously disperse in aqueous media and form miniemulsions (SEDDS) or microemulsions (SMEDDS). Lipids that can be used in the formulations of the present invention include any natural or synthetic lipids, including but not limited to sesame oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerides, and

[0530] B. Application

[0531] The conjugates and compositions of the invention can be delivered by any suitable method, including oral, parenteral, and topical methods. 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.

[0532] The pharmaceutical preparation is preferably in unit dosage form. In this form, the preparation is subdivided into unit doses containing appropriate amounts of the conjugates and compositions of the present invention. The unit dosage form can be a packaged preparation containing discrete quantities of the preparation, such as packaged tablets, capsules, and powders in vials or ampoules.

[0533] The conjugates and compositions of the present invention can be co-administered with other agents. Co-administration includes administering the conjugates or compositions of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours of administering other agents. Co-administration also includes simultaneous administration, approximately simultaneous administration (e.g., within about 1, 5, 10, 15, 20 or 30 minutes of each other) or sequential administration in any order. In addition, the conjugates and compositions of the present invention can each be administered once a day, or twice, three times or more a day to provide a preferred daily dosage level.

[0534] In some embodiments, co-administration can be achieved by co-formulation, ie, preparing a single pharmaceutical composition including the conjugates and compositions of the invention and any other agents. Alternatively, the various components can be formulated separately.

[0535] The conjugates and compositions of the present invention and any other agents may be present in any suitable amount and may depend on various factors, including but not limited to the weight and age of the subject, disease state, etc. Suitable dosage ranges include about 0.1 mg to about 10,000 mg, or about 1 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable dosages also include about 1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg. The composition may also include other compatible therapeutic agents. The conjugates described herein may be used in combination with each other, with other active agents known to be useful for regulating glucocorticoid receptors, or with adjuvants that may be ineffective when used alone but may contribute to the efficacy of the active agent.

[0536] VI. Treatment Methods

[0537] In some embodiments, the invention relates to methods and / or uses comprising a conjugate or composition as described herein for treating a disease or disorder in a subject in need thereof.

[0538] In some embodiments, the method comprises administering the conjugate multiple times. In some embodiments, the method comprises administering the conjugate every day, every other day, every three days, or every week. In some embodiments, the method comprises administering the conjugate every week, every 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 twice or three times per year. In some embodiments, the method comprises administering the conjugate annually.

[0539] A. Eye diseases

[0540] In some embodiments, the method of the invention is a method of treating an ocular disease in a subject in need thereof, comprising administering to the subject a conjugate as described herein.

[0541] In some embodiments, the method comprises administering the conjugate intravitreally.

[0542] In some embodiments, the method comprises administering the conjugate once per month, every two months, or every three months.

[0543] In some embodiments, the vitreous 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 vitreous half-life of the conjugate is at least 5 times longer than the half-life of the unconjugated peptide.

[0544] Ocular diseases that can be treated using the methods 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.

[0545] 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, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior part or location of the eye; ocular tumors; retinal diseases, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (P VR), retinal artery occlusive disease, retinal detachment, uveitis retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal diffusion; posterior ocular disease caused by or affected by ocular laser therapy; posterior ocular disease caused by or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disease; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy (cone-rod dystrophy); Stargardt disease (macular fundus); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders, including achromatopsia, protanopia, deuteranopia, and tritanopia.

[0546] In some instances, the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's Congenital Amaurosis, diabetic retinopathy, achromatopsia, or color blindness.

[0547] In some cases, the composition comprising the conjugate is administered via intravitreal, scleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar or intraspinal routes of administration. In some cases, the composition comprising the conjugate is administered via intravitreal. In some cases, the composition is delivered via intravitreal or near the posterior segment of the eye. In some cases, the composition is administered via injection into the vitreous. In some cases, the composition comprising the conjugate is administered via intraocular injection.

[0548] B. Joint diseases

[0549] In some embodiments, the methods of the invention are methods of treating a joint disease or disorder in a subject in need thereof, comprising administering to the subject a conjugate as described herein.

[0550] In some embodiments, the method comprises administering the conjugate intra-articularly.

[0551] In some embodiments, the half-life of the conjugate in the joint is at least 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, or at least 100 times longer than the half-life of the unconjugated peptide. In some embodiments, the half-life of the conjugate in the joint is at least 5 times longer than the half-life of the unconjugated peptide.

[0552] The present invention also provides methods of treating diseases and disorders of joint tissue using the conjugates of the present invention. Examples of diseases and disorders of joint tissue 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.

[0553] Many peptides are used as drugs to attenuate immune cell function and have significant utility in the treatment of many joint diseases. Joint tissue is particularly susceptible to injury and disease because the typical cellular response to these insults, the upregulation of inflammatory mediators, also signals the breakdown of articular cartilage and the resorption of underlying bone tissue. Degeneration of the joint surface promotes worsening of joint tissue damage and further upregulation of inflammatory mediators. Over time, these mechanisms create a feed-forward loop that leads to cumulative damage to joint tissue.

[0554] Any joint of the human or animal body can be treated using the methods and conjugates of the present invention. Representative joints include, but are not limited to, fibrous joints, cartilaginous joints, synovial joints, small joints, immobile joints, micro-movement joints, and diarthrosis. The joint can be a simple joint with two joint surfaces, a compound joint with three or more joint surfaces, or a complex joint with two or more joint surfaces and a joint knee or meniscus. The anatomical joints that can be treated using the conjugates and methods of the present invention include, but are not limited to, hand joints (including fingers), elbow joints, wrist joints, shoulder joints, sternal and clavicular joints, vertebral joints, jaw and skull joints, pelvis and hip joints, knee joints, ankle joints, and foot joints including toes. Joints can also be divided into plane 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 joint tissues, including, but not limited to, connective tissue, cartilage, articular surfaces, synovial cavities, menisci, and the like.

[0555] Examples of drugs intended to attenuate immune cell function include antibodies that can 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 can 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, affinity bodies, anticalins, DARPins, and engineered Kunitz-type inhibitors. Other examples also include receptor decoys for immunomodulatory cytokines, such as tumor necrosis factor-α and IL-1β, IL-6 or interferon-γ.

[0556] A common side effect of using the above anti-inflammatory drugs is a higher risk of infection. Because they weaken the body's immune response, the immune system's ability to fight off bacteria, viruses, and parasites is impaired. Therefore, the benefits of systemic use of these drugs need to be carefully weighed against the risks associated with systemic immunosuppression. For diseases where the whole body is affected by a hyperimmune disorder, such as rheumatoid arthritis, systemic use of immunosuppressive drugs can be justified. However, for diseases that affect only one or a limited number of joints, the risk of systemic infection does not usually justify the use of these drugs systemically.

[0557] As an alternative, 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 space and do not provide sufficient treatment time after IA administration. After IA injection, the half-life of anti-inflammatory proteins in the synovium is short (<1.5 hours). This is evident from clinical studies in which inflammatory inhibitors (including infliximab and etanercept) have been administered to humans via IA injection to treat a variety of joint diseases. Some of these studies reported significant reductions in joint inflammation, but acknowledged that frequent (e.g., weekly) administration was required to achieve successful results. Therefore, IA anti-inflammatory therapy using existing drugs will be limited by the high cost and inconvenience of frequent IA administration. Clearly, methods are needed to prolong the bioactivity of anti-inflammatory drugs in the synovial fluid to enable this therapeutic approach to treat joint diseases.

[0558] The cardinal symptoms associated with joint diseases are pain, effusion, limited range of motion and pathological remodeling of joint anatomical structures. The therapeutic effect of treating joint diseases can include pain relief as measured by general assessment (e.g., visual assessment score). The therapeutic effect can also be measured based on the improved scoring of using a system (e.g., WOMAC score for osteoarthritis, ACR20 for rheumatoid arthritis, psoriatic arthritis quality of life for psoriatic arthritis or SASSS for ankylosing spondylitis) with specificity for specific joint diseases. The therapeutic effect can also be measured using functional output (e.g., an increase in pain-free walking distance or an increase in joint range of motion). The therapeutic effect can also be measured based on the radiological evidence showing the recovery of normal joint anatomical structures.

[0559] The conjugate can be administered at any suitable frequency or amount as described above. In some embodiments, the conjugate is injected into the joint no more than about once a month. In some embodiments, the conjugate is injected into the joint about once a month to once every 6 months. In some embodiments, the conjugate is injected into the joint once every 2 months or once every 3 months.

[0560] 1. Osteoarthritis

[0561] In 2015, an estimated 7.75 million Americans experienced symptoms of osteoarthritis (OA) that may be related to a known joint injury. Post-traumatic OA (PTOA) accounts for at least 15% of all OA cases, although it is thought that many other OA diagnoses may also be related to previous joint trauma. Due to the lack of disease-modifying therapies, joint replacement surgery is often the only treatment option to eliminate the associated discomfort and restore mobility. However, PTOA is often diagnosed in younger patients, for whom joint replacement is not a viable option. Overall, treating these PTOA patients costs the healthcare community more than $4 billion per year.

[0562] Short-term suppression of injury-associated inflammation will limit the long-term symptoms of PTOA. Many types of joint injuries are associated with PTOA, including dislocations, ligament tears, meniscal injuries, and intra-articular fractures. Although the initial injury may be acute, the injury is sufficient to initiate a cascade of inflammatory mediators. The resulting chronic pan-joint inflammation can promote the 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, which they do by downregulating the expression of cartilage matrix components and upregulating the expression of matrix metalloproteinases (MMPs). TNFα also stimulates osteoclast recruitment and induces apoptosis of bone-forming osteoblasts in the inflammatory environment, which leads to erosion of articular cartilage tissue. TNFα and IL-1β are potent targets for alleviating the inflammatory response to joint injury. Inhibition of these key acute inflammatory cytokines in the joint environment has been proposed for early intervention to delay the progression of PTOA.

[0563] 2. Inflammation caused by immune response to particles in the joints

[0564] Wear between joint surfaces can produce micron-sized particles, leading to joint inflammation and osteolysis. Wear particles can be produced by wear between endogenous surfaces, such as ossified cartilage lesions, osteophytes (bone spurs), or exposed subchondral bone lesions. This type of wear particle production often occurs in the later stages of OA, leading to severe joint pain and limited mobility. This additional inflammatory response accelerates the rate of joint tissue degeneration in OA.

[0565] Wear particles can also form between the surfaces of artificial joints. In 2015, more than 7 million Americans were living with artificial joints. Nearly 250,000 of these people will eventually require revision surgery because the bone around the device dissolves, eventually causing the device to loosen and fail.

[0566] Wear-related inflammation originates from a foreign body response to inert particles shed from joint surfaces. Macrophages in the synovial lining readily recognize wear particles as foreign matter, release proinflammatory factors, recruit other active immune cells to the synovium, and stimulate osteoclast expansion while inhibiting bone formation. Thus, continued inflammation triggers a feed-forward loop in which cartilage degradation and bone dissolution lead to more wear between joint surfaces and more motion and physical stress, which in turn generates more particles.

[0567] In some embodiments, the peptide modulates the activity of 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.

[0568] Tumor necrosis factor (TNFα) is a potent target for controlling foreign body responses. TNFα plays a well-known role in mediating joint inflammation. TNFα also stimulates osteoclast recruitment and induces osteoblast apoptosis in the inflammatory environment, leading to osteolysis of subchondral bone. Inhibition of TNFα using a systemically administered receptor antagonist (etanercept) has been shown to reduce wear particle-induced bone resorption in mice, although the risks associated with systemic anti-TNFα are generally considered unacceptable for localized conditions. As an alternative, IA anti-TNFα therapy has been proposed to prevent or inhibit the osteolytic response to wear particles within the joint.

[0569] In some embodiments, the use of the invention is the use of a conjugate as described herein for the preparation of a medicament for use in a method of treating a disease or disorder in a subject.

[0570] In some embodiments, the subject is a human.

[0571] In some embodiments, the use of the invention is use for treating a disease or disorder, comprising a conjugate or pharmaceutical composition as described herein.

[0572] In some embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for treating a disease or disorder, comprising a conjugate as described herein.

[0573] In some embodiments, the conjugates of the invention are conjugates for use in treating a disease or disorder as described herein.

[0574] VII. Preparation Method

[0575] In some embodiments, the method is a method for preparing a peptide of the present invention, comprising (a) translating a gene sequence encoding the peptide in a bacterium in a first reaction mixture; and (b) removing endotoxin in the first reaction mixture by forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA); thereby preparing the peptide.

[0576] In some embodiments, the method is a method for preparing a peptide of the present invention, comprising (a) translating a gene sequence encoding the peptide in a bacterium 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 at pH 5.5.

[0577] In some embodiments, the bacteria is Escherichia coli.

[0578] In some embodiments, the second reaction mixture comprises about 0.1 mM to about 5 mM EDTA. In some embodiments, the second reaction mixture comprises about 0.2 mM to about 1 mM EDTA.

[0579] Filtering the second reaction mixture can be completed by any method known in the art. In some embodiments, filtering the second reaction mixture includes a filter membrane. In some embodiments, the filter membrane includes polyethersulfone (PES) or regenerated cellulose. For example, the filter membrane can include a 50kDa or 100kDa PES membrane.

[0580] In some embodiments, the method for preparing the conjugate of the present invention comprises: (a) forming a first reaction mixture, the first reaction mixture comprising 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 conjugating agent per hyaluronic acid monomer, and a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa, and a hyaluronic acid polymer having a molecular weight of about 0.1 to about 2 equivalents of a conjugating agent per hyaluronic acid monomer. Y The organic linker shown, wherein R Y for

[0581]

[0582] and subscript m is an integer from 1 to 300; thereby forming an intermediate polymer having a plurality of monomers represented by formula IV:

[0583] 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, wherein the peptide comprises one or more -SH; thereby preparing the conjugate.

[0584] In some embodiments, the molecular weight of the hyaluronic acid polymer is about 0.4 MDa to about 2 MDa. In some embodiments, the molecular weight of the hyaluronic acid polymer is about 0.7 MDa to about 1.5 MDa. In some embodiments, the molecular weight of the hyaluronic acid polymer is about 0.8 MDa.

[0585] In some embodiments, the first reaction mixture comprises about 0.2 to about 1.5 equivalents of conjugating agent per hyaluronic acid monomer. In some embodiments, the first reaction mixture comprises about 0.2 to about 1 equivalents of conjugating agent per hyaluronic acid monomer.

[0586] In some embodiments, the conjugating agent comprises a carbodiimide. In some embodiments, the conjugating agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1,3-diisopropylcarbodiimide or dicyclohexylcarbodiimide, or a salt thereof. In some embodiments, the conjugating agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof.

[0587] In some embodiments, R Y yes

[0588]

[0589] In some embodiments, the first reaction mixture comprises from about 0.2 to about 6 equivalents of organic linker per hyaluronic acid monomer.

[0590] In some embodiments, the first reaction mixture comprises 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.

[0591] In some embodiments, the second reaction mixture comprises about 0.5 to about 1.5 equivalents of peptide per organic linker.

[0592] In some embodiments, the method for preparing the conjugate of the present invention comprises: (a) forming a first reaction mixture comprising a hyaluronic acid polymer having a molecular weight of about 0.8 MDa, about 0.2 to about 1 equivalent of a conjugating agent per hyaluronic acid monomer, and a hyaluronic acid having a formula of H2N-R Y An organic linker, wherein R Y for

[0593]

[0594] Thus, an intermediate polymer having multiple monomers of formula IV is formed:

[0595]

[0596] The conjugating agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof; and

[0597] The first reaction mixture comprises from about 0.2 to about 6 equivalents of an organic linker per hyaluronic acid monomer; and

[0598] (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; thereby preparing a conjugate.

[0599] The organic linker has the following structure:

[0600]

[0601] Its name is 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-(3-hydrazino-3-oxopropoxy)ethoxy)ethyl)propionamide, abbreviated as MP2H. "MP2H" mentioned herein as an organic linker or organic linker can be understood by those skilled in the art based on the background of its use. Example

[0602] Certain abbreviations are used in describing the experimental details. Although most of them will be understood by those skilled in the art, the following table contains a list of many of these abbreviations.

[0603] Table 1. List of abbreviations.

[0604]

[0605]

[0606] The general methods used in the examples are as follows.

[0607] Adding peptide linkers to improve protein / conjugate stability

[0608] In-frame fusions between therapeutic proteins and C-terminal peptide linkers were 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 sequenced as a linear gene block (from IDT or analog) with overhangs compatible for direct cloning into protein expression plasmids. Alternatively, oligonucleotide primers complementary to the therapeutic ORF containing the codon-optimized sequence encoding the peptide linker were extended and amplified in a PCR reaction, and the resulting linear amplicons were cloned directly into protein expression plasmids. All isolated plasmids were Sanger sequenced to ensure correct placement and integrity of the ORF containing the therapeutic fused to the peptide linker. All ORFs were expressed in commercially available T7-compatible E. coli strains via an IPTG-inducible T7 promoter.

[0609] E. coli cytoplasmic expression

[0610] To determine the amount of soluble protein expressed by E. coli, the protein was placed under the control of an IPTG-inducible T7 promoter (NEB Shuffle T7 Express) and grown in Terrific Broth to an OD of 600nm The pH of the culture medium is 0.6 and induced with 0.5 mM IPTG for 4 hours at 37° C. The size of the culture medium varies according to needs and purposes, but is between 10 mL and 1 L.

[0611] Expression in the periplasm of E. coli

[0612] For expression in the E. coli periplasm, the periplasmic targeting sequence of E. coli MalE was added to the N-terminus of the ORF. All subsequent expression and downstream purification techniques remained unchanged.

[0613] IMAC and affinity tag removal

[0614] E. coli pellets from 1 L culture were lysed by sonication, supplemented with 25 mM HEPES, 20 mM imidazole, 400 mM NaCl, 0.5 mM EDTA, 5% glycerol, 0.01% Tween 20, pH 7.5, clarified at 20 k*g, and applied to GENi-NTA HisTrap TM on the column. Nonspecific proteins were washed away in the above buffer containing an additional 40 mM imidazole. The target protein was then gradient eluted to 260 mM imidazole by FPLC. Purity was checked by SDS-PAGE, and the elution peak area identified by AKTA Unicorn software was used as a comparative indicator of culture yield. In some cases, proteins were expressed with a TEV-cleavable IMAC affinity tag at the N-terminus, which was removed after IMAC purification.

[0615] Protein A purification

[0616] Without the use of a polyhistidine affinity tag, sdAbs were captured using protein A resin (JSR Life Sciences, Amsphere A3) from clarified E. coli lysate in 20 mM Tris, 25 mM NaCl, 0.5 mM EDTA, pH 8.5. The immobilized sdAbs were then washed in fresh lysis buffer and then eluted with 50 mM sodium citrate, 25 mM NaCl, 1 mM EDTA, pH 5.

[0617] Chromatographic polishing

[0618] In order to further purify protein, the IMAC eluent merged is diluted 5 times with nanopure water, and is applied to the GE HiTrap Q HP post pre-equilibrated with 20mM Tris, 25mM sodium chloride, 0.5mM EDTA, pH 8.5. These conditions are enough to remove the contaminated Escherichia coli proteins from the affinity chromatography eluent pool, and the target protein remains in the column flow-through liquid (column flow-through). The Q column flow-through liquid is further diluted twice with nanopure water, and the pH value is adjusted to 5 with acetic acid, and is applied to the GE HiTrap SP HP post pre-equilibrated with 10mM sodium citrate, 0.25mM EDTA, pH 5.0. The purified protein gradient is eluted to 25mM sodium citrate, 0.5M sodium chloride, 1mM EDTA, pH 5.5. Purity is confirmed by SDS-PAGE, and the elution peak area identified using AKTA Unicorn software is used as a comparative index of protein output.

[0619] Endotoxin removal and protein post-processing

[0620] The pure SP elution fractions were pooled and the material was passed twice through a 100 kDa regenerated cellulose spin concentrator to remove endotoxins. The protein solution that passed through the 100 kDa spin concentrator was then concentrated on a 3 kDa regenerated cellulose spin concentrator to a protein concentration of >175 mg / mL. Sterile glycerol was then added to 10% C F (v / v) and then snap frozen and stored at -80°C.

[0621] Alternatively, after Q anion exchange chromatography, a final concentration / purification step is performed with cation exchange chromatography. The protein binds at pH 5 and is eluted on a gradient from solution A (10 mM sodium citrate pH 5, 0.25 mM EDTA) to solution B (25 mM citrate pH 5.5, 1 M NaCl, 1 mM EDTA), typically eluting between 10%-25% B. The peak fractions are then pooled and these pooled protein solutions are passed through a 100 kDa filter (PES or regenerated cellulose) at 3000*g. The pure protein is then further concentrated on a 10 kDa filter.

[0622] Free cysteine ​​gel analysis

[0623] To ensure that a single free cysteine ​​was available for conjugation to the biopolymer, ~20 equivalents of the 1.2 kDa PEG-maleimide moiety were incubated at 42°C for 45 min and run on a 4-20% SDS-PAGE to confirm the mobility shift due to a single 1.2 kDa gel mobility shift.

[0624] Other protein / conjugate stabilizing mutations

[0625] Protein sequences were retrieved by BLAST query against the PDB database. MSA was performed using the Jalview program, which included sequence alignment using Clustal Omega, manual curation of sequences in the alignment to include only sdAbs with the desired topology, and removal of sequence redundancy to retain only ∼100 sequences in the MSA. Positions with 9 or more conservation scores in the MSA were considered consistent and were included in the sdAb sequence containing a C-terminal α-helical linker peptide between the ORF and the conjugated cysteine.

[0626] Site-directed mutagenesis

[0627] Complementary oligonucleotides containing the desired codon-optimized amino acid substitution mutations were designed according to the guidelines disclosed 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. Sanger sequencing was performed on the freshly isolated plasmids to confirm the correct amino acid substitution.

[0628] Thermal precipitation of soluble lysine proteins

[0629] 25 volumes of lysis buffer 25mM HEPES, 20mM imidazole, 400mM sodium chloride, 0.5mM EDTA, 5% glycerol, 0.01% Tween 20, pH 7.5 were added to the E. coli pellet harvested from 25mL TB culture and sonicated 5 times at 40% output power on ice using a probe sonicator. After lysis, the whole cell extract was clarified at 10K*g, 100uL of the supernatant was aliquoted and incubated at 50℃, 60℃, 70℃ and 80℃ for 15 minutes, then incubated on ice for 10 minutes. Heat precipitated proteins were removed at 10K*g for five minutes, and the soluble extract fraction was directly mixed with Laemmli sample buffer, denatured, and run on 4-20% SDS-PAGE to assess yield and stability.

[0630] Example 1. Peptide Linker Evaluation

[0631] based on Figure 1A The peptide containing the target bioactive peptide attached to the peptide linker was expressed in E. coli prior to attachment to the polymer using an expression open reading frame (ORF) in. Various peptide linkers were evaluated for attachment of the bioactive peptide to the polymer (Table 2).

[0632] Table 2. Peptide linkers

[0633]

[0634] Figure 1B Soluble expression of the exemplary protein 2H10 in E. coli is shown. Improved yield of the soluble fraction was observed using 2H10 variants containing an alpha helical peptide linker. The presence of an alpha helical peptide linker increased expression when a reactive cysteine ​​was present at the C-terminus of the protein.

[0635] Figure 1C The increase in soluble expression is evident in the SDS-PAGE analysis of the same peptide. Cultures containing autoinduction medium and carbenicillin (5 mL) were grown overnight at 37 ° C to saturation. Saturated cultures were collected by centrifugation, washed with 1 mL PBS, and collected by repeated centrifugation. The supernatant was aspirated and the culture was frozen at -80 ° C. Cells were lysed on ice by a probe sonicator, cleared by centrifugation, normalized to protein content, and run on 4-20% SDS-PAGE. The protein expression of Hu2H10_5MUT (SEQ ID NO: 55) and Hu2H10_5MUT_CYS (SEQ ID NO: 141) demonstrated by the band at about 15 kDa was lower than that of Hu2H10_5MUT_aH_CYS (SEQ ID NO: 142) (band at about 17 kDa) containing the alpha helical peptide linker of SEQ ID NO: 21 at the C-terminus.

[0636] The enhanced soluble expression was not limited to the above proteins. The soluble expression of the anti-VEGF protein HuNb42 was also increased with the addition of the C-terminal α-helical peptide. Figure 1D Shown is a comparison of soluble expression of HuNb42_A88P (SEQ ID NO: 67) ("null") and HuNb42_A88P aH_Cys (SEQ ID NO: 145) ("+aH_CYS"). The total process yield per liter of culture medium with the alpha helical linker was approximately four times higher than the corresponding protein without the C-terminal alpha helical peptide.

[0637] Example 2. Effect of humanization on soluble expression

[0638] Therapeutic peptides and proteins require a higher degree of humanization to reduce the risk of immunogenicity. However, certain residues in single-domain antibodies can affect humanization while reducing stability. Therefore, specific point mutations in the framework regions were systematically evaluated for humanization and stability.

[0639] Sequence humanization was performed using the computational resources of the Abysis Antibody Analyzer and LakePharma's T20 score analyzer. For sdAbs targeting human proteins, certain amino acids in the consensus sequence were changed such that the framework-only T20 score was 85 or higher.

[0640] Figure 2The amino acid sequences of 2H10 and point mutation variants tested for expression in E. coli are shown. As shown in Fig. 3, the protein expression of variants Hu2H10 R86K A87P (SEQ ID NO: 56) and Hu2H10 R86K A87P L115Q (SEQ ID NO: 58) was higher than that of Hu2H10-5MUT (SEQ ID NO: 55) or Hu2H10 L115Q (SEQ ID NO: 57).

[0641] Figure 4 The amino acid sequences of Nb42 and point mutation variants tested for expression in E. coli are shown. Figure 5 The HuNb42 A88P variant (SEQ ID NO:67) was shown to exhibit increased soluble expression. Figure 6 It was shown that the higher relative expression was independent of the cellular compartment, as both the cytoplasmic and periplasmic compartments showed higher relative levels of soluble expression.

[0642] Figure 7 Humanization of Nb42 (SEQ ID NO: 61), HuNb42 (SEQ ID NO: 62) and HuNb42 A88P variant (SEQ ID ID: 67) compared to caprolactam, bevacizumab and ranibizumab is shown. HuNb42 (SEQ ID NO: 62) and HuNb42 A88P variant (SEQ ID ID: 67) are comparable to humanized antibodies in the literature in terms of humanization detected by Z score or T20 score.

[0643] Figure 8 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. is shown. The staining shows that three point mutations within aTNFaMu increase protein production and thermal stability up to about 70° C.

[0644] Fig. 9 The effects of specific point mutations in E1-1 on protein expression yields are shown. Compared to E1-1 (SEQ ID NO: 81) or 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) showed about 10 times or higher relative protein expression.

[0645] Figure 10 shows that when protein synthesis is performed at higher temperatures, the gel shift assay is consistent with less reactive cysteines in the final protein preparation (upper graph). The reduction in reactive cysteines indicates that naturally occurring disulfide bonds are formed within the antibody, resulting in a more stable product. A similar gel shift (lower graph) shows a gradient from low to high disulfide bond formation when the protein synthesis temperature is increased.

[0646] Example 3. Endotoxin Removal

[0647] Endotoxin is undesirable in protein pretreatment because it can enter the drug conjugation step and is a source of contamination in animal experiments (endotoxins can cause immune reactions).

[0648] Endotoxin removal depends on the presence of EDTA in the buffer, which causes the endotoxin to aggregate to a certain size and become filterable while minimizing protein loss during the filtration process. Figures 11A-11B The endotoxin removal process showed that more than 99.5% of the endotoxins in the samples were removed.

[0649] Fig. 11B The removal of endotoxin and recovery of anti-TNFα3 MUT VHH (mouse)-aH (SEQ ID NO: 104) content using 50 and 100 kDa filters is shown. Methods: A protein solution of 15 mg / mL in 25 mM sodium citrate, 100 mM NaCl, 1 mM EDTA, pH 5.5 was 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 an A280 spectrophotometer (nanodrop) and endotoxin was measured using the Charles River Endosafe LAL kit.

[0650] method:

[0651] 5 mL culture was grown to saturation at 37°C in TB autoinduction medium + antibiotics. Cells were collected by centrifugation at 4000 RPM for 10' at 4°C. The pellet was washed with 1 mL of PBS and transferred to an Eppendorf tube. Cells were pelleted at 14000 RPM, 2.5'4°C, 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, 5" pulses on, 5" pulses off, for a total of 60", at 40% output power. Cell lysates were normalized to total protein content using a Nanodrop A280 and subjected to 4-20% SDS-PAGE under denaturing and reducing conditions. Gels were stained in InstaBlue protein stain and destained extensively in water. ImageJ software was used to calculate the density signal % normalized across the gel lane. Overexpression was considered when >10% of the total lane protein signal was due to bands of approximately the predicted molecular weight that were absent in the uninduced sample control. Fig. 11C An exemplary SDS-PAGE gel is shown in .

[0652] Table 3 below shows a summary of the expression density determinations of certain peptides of the present invention.

[0653] Table 3. Measured values ​​of expression density

[0654]

[0655]

[0656]

[0657] § Each sequence listed is covalently linked to the C-terminal alpha-spiro peptide of SEQ ID NO:21 unless marked with an asterisk ("*").

[0658] Example 4. Consensus sequence for expression of single domain antibody proteins

[0659] Based on the data provided in the above examples, the following framework sequences allow single domain antibodies to be more stably expressed and / or more human-like. Tables 4 and 5 show exemplary framework regions and allowed amino acid substitutions.

[0660] Table 4. Framework region

[0661]

[0662] Table 5. Allowable substitutions for exemplary peptides

[0663]

[0664]

[0665]

[0666]

[0667] Example 5. Preparation of purified thiol-reactive hyaluronic acid conjugated intermediates

[0668] Sodium hyaluronate (HA, 830 kDa) was suspended in water or 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.7 at a concentration of 4 mg / mL and mixed overnight at room temperature with gentle rotation or nutation. To 3 mg (3.6 nmol, the amount will vary depending on the polymer composition and molecular weight) of HA in solution was added hydroxybenzotriazole (HOBt) hydrate as a ~5-100 mg / mL stock solution in DMSO, a thiol-reactive linker (such as a hydrazine-X thiol reactive group, such as MP2H) in 10-100% DMSO (10-100 mg / mL stock), and a conjugating agent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)) in 0.1 M MES buffer at pH 5.7. The molar equivalents of each reactant per mole of HA and per carboxylate in the different reaction methods and exemplary methods are described in Tables 6 and 7 below:

[0669] Table 6. Relative proportions of conjugating agent, catalyst and linker in the method

[0670] Reactants Method 1 Method 2 Method 5* Conjugating agent (EDC) 500-750 1000-1500 9500-12000 HOB 3000 2000 50 Connector MP2H 3000 1500 500-1000

[0671] The EDC range reaction was set up using molar equivalents of 830 kDa HA per mole.

[0672] *EMCH was used instead of MP2H in the specific intermediates indicated.

[0673] Table 7. Ratios of conjugating agent, catalyst and linker per polymer carboxylate in the process

[0674]

[0675]

[0676] Equivalents of reactants per HA monomer (approximately 2000 carboxylates per 830 kDa HA).

[0677] *EMCH was used instead of MP2H in the specific intermediates indicated.

[0678] The solution was mixed with a gentle pipette between each reagent addition, and the final reaction volume was raised to 1mL with buffer. According to the method, a nutating mixer was used, and the final mixture was reacted at room temperature for 45 minutes to 2 hours. After the reaction, a 7kDa MWCO 5-10mL Zeba desalting spin column was used to purify the thiol-reactive biopolymer, which was balanced with 10% v / v glycerol (optional) pH 6.5 DPBS and 0.01% v / v polysorbate 20 (optional), and the crude reactant of 20% volume resin was loaded. Centrifuge was used at room temperature to elute the required intermediate into a clean conical tube, and the elution time was about 25-60 minutes. The intermediate was immediately used for reacting with thiol or aliquoted and quickly frozen on dry ice. The maleimide concentration and the modification number of each polymer were determined using UV absorbance, NMR or improved Ellman reaction assay.

[0679] Alternatively, the reaction pH or equivalents of the hydrazide linker, catalyst, and conjugating agent (EDC) are adjusted up or down to increase or decrease the number (valency) of thiol-reactive small molecule linkers covalently attached per biopolymer.

[0680] Other conjugating agents can be used instead of EDC and HOBt, such as DMTMM or oxyma. 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.

[0681] After purification, UV spectroscopy (200-324 nm) was performed on the BioTek Synergy plate reader using the Take3 microplate reader for intermediates prepared using different methods. Maleimide concentration can be determined by absorbance at 230 nm or by comparing the spectrum to a reference standard intermediate.

[0682] The data were obtained at the University of Georgia Complex Carbohydrate Research Center (CCRC) using a Bruker Advance III spectrometer equipped with a 5 mm cryoprobe. 1 H, 600.13MHz) at 25°C for NMR analysis of the conjugate. After preparing the intermediate using Method 1 and Method 5 on a 6mL scale standard, the intermediate was purified into HPLC grade water using a desalting resin and shipped to CCRC on wet ice. The sample was placed at 4°C for several weeks, and partial maleimide hydrolysis was observed in the NMR spectrum. For NMR sample preparation, 0.7ml of the intermediate stock solution (2.9mg / ml) was pipetted into a 7ml screw cap tube. 1.3mlD2O (99.9%) was added to each sample and mixed thoroughly with a vortex. The samples were then dried using a SpeedVac vacuum concentrator at room temperature. The dried samples were then redissolved in 700μL of D2O (99.98%) for NMR analysis.

[0683] Chemical analyses of exemplary reaction products are described in Table 8 below.

[0684] Table 8. Exemplary intermediates prepared using the above method

[0685]

[0686] ND = Not Determined, * = Determined on the day the CCRC receives the sample

[0687] Tables 9, 10 and 11 below show in tabular form the intermediates synthesized using three different methods, and the maleimide concentration, valency and reaction efficiency based on the HA monomer.

[0688] Table 9. Method 1 intermediates

[0689]

[0690] Table 10. Method 2 intermediates

[0691]

[0692]

[0693] Table 11. Method 5 intermediates

[0694]

[0695] *Use UV absorbance to quantify maleimide concentration and valency.

[0696] Alternatively, sodium hyaluronate (HA, 830 kDa) was suspended at 10 mg / mL in water or 4 mg / mL in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.7 and mixed overnight at room temperature with gentle rotation or nutation. A 0.1 mg / mL HA stock solution was prepared in water and approximately 1 M MES at pH 5.7 prior to the reaction and mixed with nutation at room temperature. To 3 mg (3.6 nmol, amount will vary depending on polymer composition and molecular weight) of HA in solution was added hydroxybenzotriazole (HOBt) hydrate as a ~5-100 mg / mL stock solution in DMSO, a thiol-reactive linker (such as a hydrazine-X thiol reactive group such as MP2H) in 1-10% DMSO (10-100 mg / mL stock), and a conjugating agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) in 0.1 M MES buffer at pH 5.7. The molar equivalents of each reactant per mole of HA and per carboxylate in the different reaction methods and exemplary methods are as described in Table 12 below:

[0697] Table 12. Intermediate preparation method

[0698]

[0699] Equivalents of reactants per HA monomer (approximately 2000 carboxylates per 830 kDa HA).

[0700] The solution was mixed with a gentle pipette between each reagent addition, and the final reaction volume was raised to 1 mL with buffer. The final mixture was reacted at room temperature for 45 minutes to 1.5 hours using a nutating mixer according to the method. After the reaction, the thiol-reactive biopolymer was purified using a 7kDa MWCO 5-10mL Zeba desalting spin column, which was balanced with pH 6.5 DPBS with 10% v / v glycerol (optional), and the crude reaction of 20% volume resin was loaded. For NMR samples, the Zeba column was balanced and the intermediate was eluted in deuterated water without freezing. The desired intermediate was eluted into a clean conical tube using a centrifuge at room temperature, and the elution time was about 25-60 minutes. The intermediate was immediately used for reaction with thiol or aliquoted and quickly frozen at dry ice or -80°C. The maleimide concentration and modification number of each polymer were determined using UV absorbance, NMR or modified indirect Ellman reaction assay.

[0701] The maleimide concentration / valence for each reaction method is provided in Table 13 below.

[0702] Table 13. Maleimide substitution detection by indirect Ellman method

[0703]

[0704] *Reactive maleimide concentration and approximate valency of intermediates analyzed by NMR were determined using the Ellman reaction assay.

[0705] The data were obtained at the University of Georgia Complex Carbohydrate Research Center (CCRC) using a Bruker Advance III spectrometer equipped with a 5 mm cryoprobe. 1 H, 600.13 MHz) NMR analysis of the conjugate was performed at 25° C. After preparation of the intermediate using standard method AE at 3-6 mL scale, the intermediate was purified into deuterated water using desalting resin and shipped on wet ice to CCRC.

[0706] according to 1H NMR spectroscopy analysis showed that all samples contained signals corresponding to HA, MP2H, and free ethyldimethylaminopropyl urea (EDU), which is a byproduct of EDC hydrolysis and is not removed during the purification process. The abundance of MP2H, conjugated n-acylurea adducts, and free EDU relative to HA (repeating polymer units) was determined by calculating the integral of the characteristic peak signal. Based on the integral value, the original abundance of HA, MP2H, free EDU, and conjugated N-acylurea (conjugated EDU) was calculated. The abundance of MP2H, conjugated N-acylurea adducts, and free EDU relative to HA is shown in Table 14.

[0707] Table 14. Relative molar abundance of MP2H, free EDU and N-acylurea adducts relative to HA in Method AE (100%)

[0708] MP2H Free EDU N-Acylurea Adducts Method A 21.7 11.7 15.2 Method B 23.0 1.6 nd Method C 25.9 6.4 nd Method D 26.8 8.0 nq Method E 20.7 18.2 5.0

[0709] nd: species not detected

[0710] nq: the species is present but cannot be quantified due to low signal

[0711] Example 6. Preparation of purified peptide-polymer conjugates

[0712] To obtain a purified peptide-polymer conjugate, 1.1 to 2 equivalents of peptide per maleimide are combined with the intermediate prepared by the method of Example 1 and reacted with rotation or nutation mixing at 4°C or ambient temperature for at least 2 hours to overnight (most reactions are performed at room temperature to improve solubility). Optionally, 1M pH7 HEPES is added to a final concentration of 0.1M to adjust the reaction pH. In some cases, 10-100 equivalents of a reducing agent, such as DTT or TCEP HCl, is added per protein equivalent prior to the conjugation reaction to reduce any disulfide bridging between peptides. This is removed from the peptide solution by desalting columns or buffer exchange prior to conjugation, or added directly to the conjugation reaction in the form of TCEP immobilized on polymer beads. During the conjugation reaction, one or more of the following are added to improve the efficiency of the reaction: 0.5-10 mM EDTA to minimize oxidation of free thiols, Tween 20, carbohydrates, additional buffer or glycerol to stabilize the protein and / or help reduce non-specific interactions between the protein and the activated biopolymer, increased or decreased salt concentration to stabilize the protein and / or help reduce non-specific interactions between the protein and the activated biopolymer. Unreacted peptide is removed from the peptide-polymer conjugate by one or more of the following methods: dialysis (1:100 to 1:1000) against 50-1000 kDa MWCO in a suitable buffer (pH should be >1 unit above or below the pI of the peptide) at least twice for 4 hours each, and at least once at 4°C to room temperature for 4 hours. The conjugate can be purified from unreacted peptide using citrate buffer containing EDTA and Tween or other additives such as trehalose (depending on the peptide), tangential flow filtration in DPBS pH 6-8 or 50mM tris 150mM NaCl pH 8-8.5, FPLC polishing using a size exclusion column, FPLC polishing using an affinity chromatography column designed to bind the polymer component of the conjugate, or selective precipitation of the conjugate. If the reaction efficiency is high enough (i.e., the unreacted protein content is <5%), no purification is necessary.

[0713] Alternatively, to each solution of the intermediate of Example 1, peptide is added at an appropriate peptide: polymer molar feed ratio and Tween-20 to a final concentration of up to 0.03% (optional). The solution is allowed to react for 2 hours to overnight while stirring by rotation (~5 RPM) or nutation at ambient temperature. Unreacted peptide is removed by dialysis using a 50-1000 kDa MWCO membrane in each of the following buffer solutions in sequence: first, phosphate buffered saline containing 0.01% Tween-20 (optional) or an equal amount of citrate or succinate buffered saline (the pH and buffer salt used depend on the peptide) for at least 4 hours, secondly phosphate buffered saline containing 0.01% Tween-20 overnight, phosphate buffered saline containing 0.01% Tween-20 at 4°C or room temperature for 4 hours, and the fourth dialysis step is optional. Optionally, additives such as Tween 20, EDTA and carbohydrates are added to enhance protein stability.

[0714] 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. The table below shows MVP synthesized using different intermediates, as well as their resulting protein concentration, valency, dissociation constant by biolayer interferometry (BLI), and radius.

[0715] Compared to MVP synthesized using Method 5, MVP synthesized using Method 1 and Method 2 had similar or improved MVP therapeutic properties (final protein concentration, protein valency, radius, binding kinetics) (Table 15). Examples of comparisons of hydrodynamic radii of DARPin MVPs synthesized using Method 1 or Method 5 intermediates are shown in Fig.12 An example of the comparison of VEGF binding curve BLI data of anti-VEGF MVP synthesized using intermediates from Method 1 or Method 5 is shown in Fig.13A shown.

[0716] Table 15. Conjugates

[0717]

[0718]

[0719] *The BLI detection limit is 0.001 nM. The samples listed in the table above with this value are below K D BLI LOD. **Cy7 fluorophore labeled peptide

[0720] Alternatively, to obtain a purified peptide-polymer conjugate, 1.1 to 2 equivalents of peptide per maleimide are combined with a HA conjugated substrate prepared by the method shown in Table 16. The conjugation reaction is allowed to react at ambient temperature with rotation or nutation mixing for at least 2 hours to overnight. 1M pH 7 HEPES is added to a final concentration of 0.1 M to adjust the reaction pH. In some cases, unreacted peptide is removed from the peptide-polymer conjugate by dialysis (1:400 to 1:1000) at least three times for 4 hours each with a 50-1000 kDa MWCO in a suitable buffer (pH should be higher or lower than the pI of the peptide>1 unit) at 4°C to room temperature.

[0721] Table 16. Reaction conditions for peptide-polymer conjugates

[0722]

[0723] To confirm successful conjugation, the products of the conjugation reaction were analyzed using SDS-PAGE and DLS. SDS-PAGE was used to determine the percentage of unreacted peptides that were separated by migration into a gel consistent with their molecular weight. After the conjugation reaction, a significant percentage of the peptides were present at the top of the stacking gel as high molecular weight conjugates that were unable to migrate into the gel due to their size (>300 kDa). DLS was used to determine the hydrodynamic radius present in the reaction products. After the conjugation reaction, the highest intensity peak and R consistent with the conjugated substrate h Aligned, indicating that the peptide is conjugated to the hyaluronic acid substrate. The data of the conjugates are shown in Table 17. The percentage of unreacted protein is determined by density determination analysis of the SDS-PAGE bands of the unconjugated protein, which are referenced to the BSA standard of known mass and then divided by the total mass loaded in each well. The hydrodynamic radius is measured using dynamic light scattering (DLS) using a Wyatt Dyna Pro plate reader III (25°C, 5-10 acquisitions, 5 seconds, n=3 samples per conjugate). Data analysis is performed using the Jupyter notebook data analysis program to extract data of sufficient quality and analyze according to the highest intensity peak.

[0724] Table 17. Properties of various peptide-polymer conjugates

[0725]

[0726] It is often necessary to achieve high concentrations of pharmaceutical formulations to reach therapeutic thresholds, maximize therapeutic durability, and / or minimize dose volume. However, at higher concentrations, therapeutic peptides may aggregate. For polymer-peptide conjugates, there is also concern that interactions with the polymer substrate may promote aggregation or lead to aggregation at lower peptide concentrations, which would occur in the absence of a conjugated polymer. The HA conjugated substrate prepared using method B provided a polymer-peptide conjugate with no measured aggregation.

[0727] Synthesis of purified conjugate #28 obtained using method B: In a 2mL v-bottom microcentrifuge tube, 174μL of the purified conjugated intermediate from method B was mixed with 3μL 2% v / v Tween 20 and 26μL 80mg / mL N42 anti-VEGF VHH (SEQ ID NO: 145) to obtain 1.1 equivalents of peptide per maleimide. The reaction pH was adjusted to pH 7 by adding 20μL 1M pH7 HEPES to a final concentration of 0.1M. The reaction was nutated and mixed overnight at room temperature for 16 hours. Unreacted peptide in the peptide-polymer conjugate was removed by stirring dialysis (1:1000 based on the initial reaction volume) in pH 5.5 25mM citrate, 100mM NaCl, 0.03% Tween 20 at room temperature using a 100kDaMWCO 200μL microFloat-A-Lyzer dialysis cassette (Repligen). A total of four dialysis steps were performed, with the buffer changed three times after 4 hours each time and once after 16 hours of overnight dialysis.

[0728] Synthesis of purified conjugate #30 obtained using method B: In a 2 mL v-bottom microcentrifuge tube, 186 μL of the purified conjugated intermediate from method B was mixed with 3.3 μL of 2% v / v Tween 20 and 14.4 μL of 80 mg / mL anti-TNFα VHH (SEQ ID NO: 102) to give 1.1 equivalents of peptide per maleimide. The reaction pH was adjusted to pH 7 by adding 20 μL of 1M pH 7 HEPES to a final concentration of 0.1 M. The reaction was nutated and mixed overnight at room temperature for 16 hours. Unreacted peptide in the peptide-polymer conjugate was removed by agitation dialysis (1:1000 based on the initial reaction volume) in a 100 kDa MWCO 200 μL microFloat-A-Lyzer dialysis cassette (Repligen) at room temperature in pH 5.5 25 mM citrate, 100 mM NaCl, 0.03% Tween 20. A total of four dialysis steps were performed, with the buffer changed three times after 4 hours each time and once after 16 hours of overnight dialysis.

[0729] After the fourth dialysis step, the purified conjugate was removed from the dialysis cassette and stored at 4°C. The reaction products were characterized by visual inspection, UV-vis absorbance to determine the purified protein conjugation, and DLS to determine R h , SDS-PAGE to determine the percentage of unconjugated protein, and bio-layer interferometry to determine binding affinity, as described in the Examples herein.

[0730] Table 18. Characterization of Conjugates #28 and #30 Prepared from Method B Intermediates

[0731]

[0732] Example 7. Stability analysis of purified peptide-polymer conjugates

[0733] MVP stability was assessed by establishing long-term accelerated in vivo stability by maintaining MVP at 37°C at 5-10 times therapeutic concentrations in pH 7.3 vitreous mimetic buffer (Table 19) or pH 7.4 PBS 0.01% Tween 20. MVP stability was assessed by analyzing samples removed after different times using SEC MALS or SEC, DLS and / or BLI.

[0734] Table 19. Vitreous Simulation Buffer Composition

[0735] Components mg / mL NaCl 7.14 KCl 0.38 <![CDATA[CaCl2 2H2O]]> 0.154 <![CDATA[MgCl2 6H2O]]> 0.2 <![CDATA[Sodium hydrogen phosphate (NaH2PO4)]]> 0.42 <![CDATA[NaHCO3]]> 2.1 glucose 0.92 lactic acid 0.358 <![CDATA[CuSO4]]> <![CDATA[8.28x10 -5 ]]> <![CDATA[ZnSO4 heptahydrate]]> 0.000561 <![CDATA[FeCl2 tetrahydrate]]> 0.000618 Transferrin (2 Fe binding sites) 0.0878 Reduced Glutathione (GSH) 0.0154

[0736] A long-term 37°C stability study was developed to evaluate the effect of composition on MVP stability. MVP was synthesized under sterile conditions and diluted to approximately 0.4 mg / mL in sterile-filtered human vitreous mimetic buffer. This concentration is 5-fold higher than the intravitreal therapeutic concentration we predict for clinical doses. Samples were filtered using sterile 0.2 or 5 μm spin filters or mixed with 0.01% sodium azide (as an antimicrobial agent) before use. Several 100 μL aliquots of each sample were then added to wells of a sterile 96-well plate, with one aliquot from day 0 stored at 4°C. 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 discrete time points, one aliquot of each sample was removed from the plate under sterile conditions and analyzed. First, UV-VIS spectra of the samples were acquired from 200-600 nm in steps of 10 nm to monitor any significant changes in sample composition. The protein concentration was then determined to adjust for any volume differences that may have occurred. Binding affinity was determined using BLI. on (binding constant) or K DChanges over time were used to assess relative stability. To monitor changes in radius over time, samples were spun at 5000 g for 5 min to remove any large aggregates or dust particles, and then the samples were measured undiluted using DLS to measure R. h .

[0737] Stability study samples were analyzed using HPLC size exclusion chromatography (SEC). The method was also used to analyze the MVP formation and unreacted protein percentage after purification. In order to evaluate stability by SEC, MVP was filtered to remove particles and analyzed using Shodex1 MDa OhpakLB-804, Shodex KW-404 or 405 or Phenomenex PolySep6000 columns with DPBS or a suitable solvent as mobile phase to obtain baseline traces at 280nm, 230nm, etc. After different time points, samples were taken out and analyzed using the same SEC method. Increases in retention time and peak width relative to baseline samples indicate degradation. In addition, a decrease in MVP peak area and / or an increase in monomer and dimer protein species peak area also indicate MVP degradation. The percentage loss of the conjugate was quantified by comparing the change in peak area over time. In the future, SEC stability analysis will be combined with MALS to quantify the molecular weight and valence changes of the conjugate when aged at different temperatures. Fig.14 Shown are representative SEC data for Method 5 or Method 1 intermediate DARPin MVP samples aged for up to 71 days at 37°C. Due to its larger size, Conjugate 2 could not be analyzed on the same column used for Conjugate 1 in this data.

[0738] Stability study samples were also analyzed by combining SEC with MALS analysis to determine the MVP radius of gyration (R g，z) and molecular weight. To this end, the conjugate stability samples were loaded into glass vial inserts (250 μL capacity) nested in 2 mL HPLC vials and capped. For HPLC analysis, 5-20 μg of MVP (based on protein) was injected into a 1260 Infinity Agilent HPLC system equipped with a constant pump, autosampler, thermostat column box, and a variable wavelength detector (or equivalent instrument) set to monitor at 280 nm, using Shodex KW-405-4F (4.6x300mm, 0.35mL / min flow rate) or LB-804 or 806 (8x300mm, 0.4mL / min flow rate for analysis of unconjugated VHH peaks) with respective guard columns. For analysis, the column chamber was maintained at 30°C and an isocratic method was used using 0.1 μm filtered pH 7.4 DPBS, 200 mM KCl, 100 mM urea, 50 mM sodium phosphate pH 6 and 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 mobile phase, made with HPLC grade water, so that there were at least 2 column volumes of mobile phase to elute after sample injection, or a total run time of 60 minutes. A DawnHeleos II MALS instrument and an Optilab T-rEX differential refractive index detector (Wyatt Technologies) or equivalent instrument were coupled to the HPLC, located downstream of the UV detector. The MALS and dRI detector parameters for protein-polymer conjugate analysis using Astra software (Wyatt Technologies) are shown in Table 20. Prior to analysis, system-specific calibration numbers, normalization factors, delay volumes, and band broadening terms were determined. A representative SEC trace of a MVP stability sample within the EDC range is shown in Figure 2. Fig.14 The increase in retention time indicates that MVP will decrease / shrink / degrade with aging. Fig.14 In the Figure 1, the retention time and radius of the high EDC conjugate (Conjugate 1) shrink much more than the low EDC MVP (Conjugate 2). Peak broadening also indicates that the polydispersity of the samples increases with aging, which may indicate sample decomposition. Fig.15 The radius loss was further verified by MALS of samples from the accelerated aging study shown in .

[0739] Table 20. MALS and dRI instrument parameters for SEC MALS analysis

[0740]

[0741]

[0742] Stability is also determined using DLS based on changes in macromolecular size (e.g., Rh ) for evaluation. For stability analysis based on radius change upon aging at 37°C using DLS, samples were removed from the 37°C stability study conditions at various time points for analysis. All samples and buffers were at room temperature. The solution was diluted to a final concentration of 100 nM in 100 μL (typically diluted 1:10) in sterile 0.1 μm filtered formulation buffer without polysorbate 20 and mixed by gentle grinding in a 1.5 mL centrifuge tube. Large aggregates and dust particles can be removed by spinning the tube at 5000 g for 5 minutes in a centrifuge. For single cuvette measurements in the NanoStar, a 40 μL sample of the sample solution was loaded into a Wyatt Technologies disposable microcuvette with a lid (Wyatt Cat# WNDMC), tapped to remove bubbles, and then placed in the instrument for analysis. For multiple readings 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). Remove air bubbles from the sample by briefly spinning in a centrifuge with a plate adapter and then gently blowing with a pipette tip or with 70% ethanol vapor from a squirt bottle. The instrument settings for this and other samples analyzed by DLS in this article are shown in Table 21. The intensity of any peak greater than 1000 nm should be <6%. The DLS acquisition parameters are shown in Table 21.

[0743] Table 21. DLS acquisition parameters

[0744] instrument Laser wavelength 664nm Laser power 100％ Automatic decay Open temperature 25℃ Collection time(s) 5-10 collection# 5 Fixed parameters Correlation function low cutoff 1.5μs Correlation function high cutoff value (μs) 103000μs Peak radius low cutoff 0nm Peak radius high cutoff <![CDATA[1x10 6 nm]]> Analysis Type dynamic Determining time constraints 5 Automatic decay time limit 60 sample Mw-R model Globular protein Solvents PBS dn / dc 0.185 Rg Model ball

[0745] Table 22 below shows the change in radius of MVP during accelerated 37°C aging for MVP samples synthesized using different methods. The hydrodynamic radius or radius of gyration was measured by DLS or MALS at t=0 and at different time points after aging at 37°C. In these examples, the MVP synthesized with low EDC (method 1) has better improved 37°C stability because of less shrinkage / change in radius with aging. This suggests that the presence of N-acylurea adducts destabilizes the conjugates prepared using methods with higher amounts of EDC.

[0746] Table 22. Changes in MVP radius over time

[0747]

[0748] The changes in binding affinity over time were also determined using biolayer interferometry (BLI). For BLI experiments, samples were removed from 37°C stability study conditions at different time points for analysis. All reagents were equilibrated to room temperature for at least 30 minutes before use. In a Gator Bio Max plate, two probes for each sample (one for kinetic assays and one for ligand-free control) were equilibrated in 250 μL BLI buffer (PBS pH 7.4, 0.2% Tween and 0.2% BSA filtered at 0.2 μm) for at least 10 minutes. Depending on the performance in the pilot reaction in BLI buffer, the ligand was diluted to a fixed concentration of 25-100 nM. The analyte was prepared in BLI buffer at the highest concentration determined in the pilot reaction and diluted two to five times in a 1:3 series using BLI buffer. A black flat-bottom, non-coated 96-well plate (Greiner Bio-One Cat#655209 or similar) is loaded column by column with 200 μL of ligand, analyte dilution, and one column of BLI buffer for each column of ligand and analyte. One well in each column of analyte should be BLI buffer to serve as a blank for reference subtraction. There are no bubbles in the wells, which can be removed by gently blowing with a pipette tip or with 70% ethanol vapor from a squirt bottle. The plate is placed on a tilting platform in a Gator set to 25°C. The Gator K assay loading and kinetic steps are set up using the dual reference and step times shown in Table 24. Load the ligand until the signal reaches between 0.4 and 0.6 nm, then return to the buffer column for a 60 second baseline measurement. Start the kinetic read using the step parameters in Table 24. When the kinetic read is completed using the probe with the ligand loaded, a no-ligand control is performed using a new probe without the ligand loaded. The analysis is performed using the same kinetic assay time and the same sample wells as the probe with the ligand loaded. This data is used to correct for any nonspecific interactions between the sample and the probe. Representative BLI data of high (method 5) and low (method 1) EDC MVP samples before and after accelerated aging at 37°C are shown in Figure 2. Fig.16 (K D )and Fig.17 (K on ) as shown.

[0749] Once the kinetic assay is complete, analyze the data in the Results and Analysis section of the Gator software. Correct the raw data to include binding times after 1 second to 180 seconds. Align the Y axis to the start of the binding step and turn on inter-step correction. Use Savitzky-Goaly data filtering. Set up samples to be dual-referenced by indicating the probes and wells for buffer reference in the software. Then, for each assay, edit the reference subtraction formula for each assay to be dual-referenced with the equation (kinetic assay wells - no ligand assay wells) - (kinetic assay buffer reference wells - no ligand assay buffer wells). Group all titrations of the same MVP by color and adjust the parameters to a 1:1 binding model that includes both association and dissociation with a global, Rmax unconnected fit. Move the window of interest to include only 100 seconds of dissociation. Fit the binding curves and check that the residuals do not differ from the actual curve by more than 10% and that the full R 2 >0.98, all X 2 <3.0. Calculate kinetics and record K D , K on and response. When different samples have the same K D As a result, the binding constant K on To distinguish the binding affinity between different constructs (i.e. Fig.17 ), where a higher binding constant indicates faster binding kinetics.

[0750] Table 23. BLI ligand and analyte pairs

[0751]

[0752] Table 24. BLI method parameters and result description for kinetic quantification

[0753]

[0754] A tabular representation of the binding kinetics of MVP samples synthesized using different methods after accelerated 37°C aging is shown below. The dissociation constants of the samples were determined by BLI at different time points after aging 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 the change in dissociation constant was similar or smaller with aging. The anti-VEGF VHH peptide MVP synthesized using Intermediate Method 5 lost all binding ability after brief aging, while the examples synthesized using Methods 1 or 2 exhibited target binding ability throughout the study, indicating that the presence of the N-acylurea adduct destabilizes the therapeutic.

[0755] Table 25. Binding of conjugates over time

[0756]

[0757] Example 8. In vivo half-life of purified peptide-polymer conjugates

[0758] The extended intravitreal retention time of the conjugate was shown in the established pharmacokinetic model. New Zealand white rabbits (n=9) were randomly divided into 3 groups according to weight. All animals received a 50 μL ITV injection of hu_anti-TNFα_aHMVP (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 syringe. Both eyes received equimolar doses of antibodies. One hour, five days and ten days after injection, a group of three rabbits were sacrificed and their eyeballs were removed for analysis of intravitreal VHH. Both eyes were snap frozen, and the vitreous, retina and aqueous humor were separated from the frozen eyes. Each tissue sample was then homogenized using a bead mill. After homogenization, the VHH concentration was quantified using ELISA or by digesting the peptides with trypsin and performing LC / mass spectrometry analysis or similar methods on the samples. Representative results of the extended half-life in rabbit vitreous after bioconjugation are shown in Fig.18 shown.

[0759] The fluorescent labeling method for peptides in this study is as follows. The intratumoral (IT) half-life of MVP was determined using a mouse tumor model used to evaluate protein clearance from solid tumors to maximize the parameters of tumor retention. We used antibodies labeled with amine-reactive Sulfo-Cy7 NHS ester (Broadpharm Cat# BP-22541) or Alexa Fluor 750 near-infrared fluorophores as follows, performed under sterile conditions. First, the fluorophore was dissolved in DMSO at a concentration of 10 mg / mL. Then, the protein at a concentration of 5.0-10.0 mg / mL was mixed with 0.1 M sodium bicarbonate in a volume ratio of 3:2. Finally, the fluorophore was added at a protein:fluorophore molar ratio of 1:2, mixed thoroughly, and incubated for one hour at room temperature on a nutator covered with foil to avoid light. The NHS ester was quenched by adding 1.5 M Tris buffer pH 8.5, which accounted for 10% of the reaction volume, and then mixed on the nutator for another 10 minutes. The labeled 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 the extent of Cy7 labeling were determined by absorbance at 280 and 750 nm. Following the above protocol, proteins stored on ice and 3 hours after Sulfo-Cy7 labeling were used for MVP synthesis. In each case, the final product was sterile filtered and stored at 4°C in the dark until used in animal studies.

[0760] Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it is understood by those skilled in the art that certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was incorporated by reference individually. In the event of a conflict between the present application and the references provided herein, the present application shall prevail.

[0761] Table 26. Sequence

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

Claims

1. A peptide represented by formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I), CDR1, CDR2 and CDR3 are each independently a complementarity determining region; FR1 has an amino acid sequence comprising: X 10 VQLX 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG(SEQ ID NO:1), in 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 A or V; FR2 has an amino acid sequence comprising: X 20 X 21 WX 22 RQX 23 PGKX 24 X 25 EX 26 VX 27 X 28 I(SEQ ID NO:2), in 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; FR3 has an amino acid sequence comprising: YX 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 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 V, M, L or I; and FR4 has an amino acid sequence comprising: YWGX 40 GTX 41 VTVSS(SEQ ID NO:4), in X 40 is Q or K, and X 41 It is L or Q.

2. The peptide according to claim 1, wherein X 27 is A.

3. The peptide according to any one of claims 1 to 2, wherein X 39a For P.

4. The peptide according to any one of claims 1 to 3, wherein FR1 has an amino acid sequence comprising: QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 5).

5. The peptide according to any one of claims 1 to 4, wherein FR2 has an amino acid sequence comprising: MGWFRQAPGKEREFVAAI (SEQ ID NO:6).

6. The peptide according to any one of claims 1 to 5, wherein FR3 has an amino acid sequence comprising: YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7).

7. The peptide according to any one of claims 1 to 6, wherein FR4 has an amino acid sequence comprising: YWGQGTLVTVSS (SEQ ID NO: 8).

8. The peptide of any one of claims 1 to 7, wherein CDR1, CDR2 and CDR3 are each a complementarity determining region from an antibody or a cytokine.

9. The peptide of claim 8, wherein 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.

10. A peptide as described in claim 8 or 9, wherein each of the complementary determining regions is specific for: vascular endothelial growth factor (VEGF), tumor necrosis factor-α (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-associated protein (GITR), V-domain immunoglobulin inhibitor of T cell activation (VISTA), T cell immunoglobulin mucin 3 (TIM-3), lymphocyte activation gene-3 (LAG3), interleukin-1-β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin 12 (IL-12) or interleukin 15 (IL-15).

11. The peptide of claim 8 or 9, wherein each of the complementarity determining regions is specific for vascular endothelial growth factor (VEGF).

12. The peptide according to any one of claims 1 to 11, wherein the peptide consists of formula I.

13. The peptide of any one of claims 1 to 12, which has one or more of the following: (a) CDR1 is 7 amino acids long; (b) a CDR2 of 7 or 8 amino acids in length; and / or (c) CDR3 of 9 to 16 amino acids in length.

14. The peptide according to any one of claims 1 to 13, wherein: (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).

15. The peptide of any one of claims 1 to 14, 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.

16. A method for preparing a peptide as claimed in any one of claims 1 to 15, comprising: (a) translating a gene sequence encoding the peptide in bacteria in the 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; Thus, the peptide was prepared.

17. The conjugate represented by formula IIa: (X 1 -X 2 -Y) n -Z (IIa), in Each X 1 Independently a peptide according to any one of claims 1 to 15; Each X 2 are independently peptide linkers of 3 to 100 amino acids in length; 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.

18. The conjugate represented by formula IIb: (X 1 -X 2A -Y) n -Z (IIb), in Each X 1 are independently a peptide having a molecular weight of about 5 kDa to about 200 kDa; Each X 2A is independently a peptide linker comprising an alpha 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.

19. The conjugate of claim 18, wherein each X 1 Independently a peptide as claimed in any one of claims 1 to 15.

20. The conjugate of any one of claims 17 to 19, wherein each peptide linker is independently 7 to 100 amino acids in length.

21. The conjugate of any one of claims 17 to 20, wherein each peptide linker is independently 10 to 30 amino acids in length.

22. The conjugate of any one of claims 17 to 21, wherein each peptide linker independently has an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21), AEEEKRKAEEEEKRKAEEEAGC(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 AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEERKKREDDEKRKKKAGC (SEQ ID NO: 27).

23. The conjugate of any one of claims 17 to 22, wherein each peptide linker has an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

24. The conjugate of any one of claims 17 to 23, wherein the organic linker has the following structure: The subscript m is an integer from 1 to 300.

25. The conjugate of any one of claims 17 to 24, wherein the organic linker has the following structure:

26. The conjugate of any one of claims 17 to 25, wherein the biocompatible polymer is a polysaccharide.

27. The conjugate of any one of claims 17 to 25, wherein the biocompatible polymer is a glycosaminoglycan.

28. The conjugate of any one of claims 17 to 25, wherein the biocompatible polymer is hyaluronic acid.

29. The conjugate of any one of claims 17 to 28, wherein the biocompatible polymer has a molecular weight of about 0.4 MDa to about 2 MDa.

30. The conjugate of any one of claims 17 to 29, wherein the biocompatible polymer has a molecular weight of about 0.7 MDa to about 1.5 MDa.

31. The conjugate of any one of claims 17 to 30, wherein the biocompatible polymer has a molecular weight of about 0.8 MDa.

32. The conjugate of any one of claims 17 to 31, wherein subscript n is an integer from 10 to 400.

33. The conjugate of any one of claims 17 to 32, wherein subscript n is an integer from 10 to 100.

34. The conjugate represented by formula IIa: (X 1 -X 2 -Y) n -Z (IIa), in Each X 1 Independently a peptide according to any one of claims 1 to 15; Each X 2 is a peptide linker having an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC(SEQ ID NO:21); Each Y is an organic linker having the following structure: Z is a biocompatible polymer which is hyaluronic acid having a molecular weight of about 0.1 MDa to about 3 MDa; Subscript m is an integer from 1 to 300; and The subscript n is an integer from 1 to 1500.

35. A conjugate, wherein the conjugate is a random polymer represented by formula IIIa: (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa), Its molecular weight is about 0.8 MDa; in Each X 1 A peptide having an amino acid sequence comprising the following: SEQ ID NO: 55; Each X 2 is a peptide linker having the following amino acid sequence: AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21); Each Y is an organic linker having the following structure: Each X 1 -X 2 -YZ 1 The section has the following structure: Each Z 2 Has the following structure: Each Z 3 Independently has the following structure: Each Z 3a are independently OH or Y'; Each Y' has the following structure: 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; Subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and The subscript q is an integer from 1000 to 3000.

36. A conjugate, wherein the conjugate is a random polymer represented by formula IIIa: (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa), Its molecular weight is about 0.8 MDa; in 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 is an organic linker having the following structure: Each X 1 -X 2 -YZ 1 The section has the following structure: Each Z 2 Has the following structure: Each Z 3 Independently has the following structure: Each Z 3a are independently OH or Y'; Each Y' has the following structure: 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; Subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and The subscript q is an integer from 1000 to 3000.

37. A pharmaceutical composition comprising the conjugate of any one of claims 17 to 36 and a pharmaceutically acceptable excipient.

38. A method of treating an ocular disease in a subject in need thereof, comprising administering to the subject a conjugate as claimed in any one of claims 17 to 36.

39. The method of claim 38, comprising administering the conjugate intravitreally.

40. The method of claim 38 or 39, comprising administering the conjugate monthly, every two months, or every three months.

41. The method of any one of claims 38 to 40, wherein the conjugate has a vitreous half-life that is at least 5-fold longer than the half-life of the unconjugated peptide.

42. The method of any one of claims 38 to 41, wherein the ocular disease is uveitis, macular degeneration, choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, or ocular inflammation.

43. A method of treating a disease or condition in a joint of a subject in need thereof, comprising administering to the subject a conjugate of any one of claims 17 to 36.

44. The method of claim 43, comprising administering the conjugate intra-articularly.

45. The method of claim 43 or 44, comprising administering the conjugate monthly, every two months, or every three months.

46. ​​The method of any one of claims 43 to 45, wherein the intra-articular half-life of the conjugate is at least 5-fold longer than the half-life of the unconjugated peptide.

47. The method of any one of claims 43 to 46, wherein the disease or condition 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.

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