Humanized Multivalent Protein Conjugates

Abstract

The present invention relates to peptide sequences that have a high degree of humanization and / or the ability to be expressed at significant levels in culture media, such as E. coli. The peptides can be covalently attached to a polymer backbone via an alpha-helical peptide linker. The present invention further relates to methods for preparing the peptides, polymer conjugates containing the peptides, and pharmaceutical compositions thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 331,534, filed April 15, 2022, which is incorporated herein in its entirety for all purposes.

[0002] Sequence Listing The attached Sequence Listing material is incorporated herein by reference in its entirety. 2023-04-11 The attachment named Sequence_Listing_ST26 052566-506001WO.xml was created on April 11, 2023 and is 187,778 bytes in size. [Background technology]

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

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

[0005] However, local tissue administration requires a specialist to safely perform the required injections, which is burdensome and costly to administer compared to systemic administration. When peptide drugs are administered as part of a peptide-polymer conjugate, the frequency of drug administration can be substantially reduced, thereby reducing the burden on the patient to receive effective treatment. Furthermore, reducing the number of local injections reduces the risk of local tissue damage or adverse effects from the injection. Finally, the need to reduce the frequency of administration can shorten the time that the drug concentration in the target tissue falls below the therapeutic concentration, thereby improving the overall efficacy of the drug. Given these advantages, there is a strong motivation to develop protein-polymer drug products for various diseases.

[0006] Many humanized monoclonal antibodies have poor biophysical properties, including poor stability and a tendency to aggregate. These unfavorable tendencies can be even more pronounced in humanized antibody fragments, which often require a significant degree of modification.

[0007] In order to properly formulate a peptide-polymer conjugate as a pharmaceutical product, it is necessary to achieve a sufficiently high drug concentration to allow for proper dosing in patients. It is also necessary to prepare purified peptide-polymer conjugates that exhibit high biological activity and storage stability, e.g., that can be kept in solution for up to two years from the date of manufacture to the date of clinical use. Interactions between peptide-polymer conjugates can adversely affect the ability to achieve any of these drug-enabling properties.

[0008] The degree of humanization of the peptide and the secondary structure of the peptide linker used to attach it to the polymer can have a substantial effect on the pharmacological properties, intra-conjugate interactions, and inter-conjugate interactions of the conjugate.Therefore, there is a need to develop humanized peptide-polymer conjugates with specific peptide linkers that achieve favorable pharmacological properties for a given disease and allow successful formulation into pharmaceutical products.The present invention meets these and other needs. Summary of the Invention

[0009] In some embodiments, the peptide of the present invention is a peptide having the formula (I): wherein CDR1, CDR2, and CDR3 are each independently a complementarity determining region; FR1, X 10 VQLX 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1) (In the formula, X 10 is Q, E, or D; X 11 is V, Q, A, or E; X 12 is S or T, X 13 is L, S, or V; X 14 is V or A, X 15 is P, A, or T, X 16 is G, D, or R; X 17 is A, V, T, or E; X 18 is A or V, FR2, X 20 X 21 WX 22 RQX 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2) (In the formula, X 20 is M, I, V, or L; X 21 is G, S, or A; X 22 is F, Y, or V; X 23 is A, V, P, or T; X 24 is E, G, A, or Q; X 25 is R or L, X 26 is F, G, W, or L; X 27 is A, G, or S; X 28 is A, S, or G, FR3, 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 the formula, X30 is A, G, S, or T, X 31 is R or Q, X 32 is N, S, or D, X 33 is S, A, or D, X 34 is N or K, X 35 is T or M, X 36 is Y, D, or S; X 37 is N or D, X 38 is S or N, X 39a is P or A, X 39b is V, M, L, or I, FR4, YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4) (In the formula, X 40 is Q or K, X 41 is L or Q).

[0010] In some embodiments, a method of preparing a peptide of the present invention comprises: (a) translating in bacteria in a first reaction mixture a genetic sequence encoding the peptide; and (b) removing endotoxin from the first reaction mixture by forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA), thereby preparing the peptide.

[0011] In some embodiments, the conjugate of the invention has formula IIa: (X 1 -X 2 -Y) n -Z(IIa) is a conjugate of During the ceremony, each X 1 is independently a peptide described herein, each X 2 are independently a peptide linker having a length of 3 to 100 amino acids, each Y is independently an organic linker; Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1500.

[0012] In some embodiments, the conjugate of the invention has formula IIb: (X 1 -X 2A -Y) n -Z(IIb) is a conjugate of During the ceremony, each X 1 are independently peptides having a molecular weight of about 5 kDa to about 200 kDa, each X 2A is independently a peptide linker comprising an α-helix; each Y is independently an organic linker; Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1500.

[0013] In some embodiments, the conjugate has formula IIa: (X 1 -X 2 -Y) n -Z(IIa) is a conjugate of During the ceremony, each X 1 is independently a peptide described herein, each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21), Each Y has the structure: [ka] is an organic linker having the formula Z is a biocompatible polymer that is hyaluronic acid having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript m is an integer from 1 to 300; The subscript n is an integer from 1 to 1500.

[0014] In some embodiments, the conjugate of the present invention has a molecular weight of about 0.8 MDa and formula IIIa: (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa) is a conjugate which is a random polymer of During the ceremony, each X 1 is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO: 67; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21), Each Y has the structure: [ka] is an organic linker having the formula each X 1 -X 2 -YZ 1 The part has the structure: [ka] having each Z 2 The structure: [ka] having each Z 3 Independently, the structure: [ka] having each Z 3a is independently OH or Y′; Each Y' has the structure: [ka] having Each R 1 and R 2 is ethyl or -(CH 2 ) 3 -NMe 2 and the subscript n is an integer from 10 to 300 and is less than about 10% of the sum of the subscripts n, p, and q; the subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of the subscripts n, p, and q; The subscript q is an integer from 1000 to 3000.

[0015] In some embodiments, a pharmaceutical composition of the invention comprises a conjugate described herein and a pharma- ceutically acceptable excipient.

[0016] In some embodiments, the method of the present invention is a method of treating an ocular disorder in a subject in need of such treatment, comprising administering to the subject a conjugate described herein.

[0017] In some embodiments, the method of the present invention is a method of treating a joint disease or disorder in a subject in need of such treatment, comprising administering to the subject a conjugate described herein. [Brief description of the drawings]

[0018] [Figure 1A] The expressed open reading frames (ORFs) are shown. [Figure 1B] shows the soluble expression (mAU*mL) of Hu2H10 or 2H10 containing different peptide linkers. [Figure 1C] FIG. 1 shows protein expression by SDS-PAGE of Hu2H10_5MUT (SEQ ID NO: 55), Hu2H10_5MUT_CYS (SEQ ID NO: 141), or Hu2H10_5MUT_aH_CYS (SEQ ID NO: 142). [Figure 1D] Figure 1 shows soluble expression (fold process yield) of HuNb42_A88P (SEQ ID NO: 67) or HuNb42_A88P aH_CYS (SEQ ID NO: 145).

[0019] [Diagram 2] The amino acid sequences of 2H10 and point mutation variants are shown.

[0020] [Diagram 3] Figure 1 shows protein expression of Hu2H10 mutants. Y-axis shows immobilized metal affinity chromatography (IMAC) peak area (mAU*mL). X-axis shows protein expression of Hu2H10_5MUT ("WT(5MUT)") (SEQ ID NO: 55), Hu2H10_5MUT_R86K_A87P ("R86K_A87P") (SEQ ID NO: 56), Hu2H10_5MUT_L115Q ("L115Q") (SEQ ID NO: 57), Hu2H10_5MUT_R86K_A87P_L115Q ("R86K_A87P_L115Q") (SEQ ID NO: 58).

[0021] [Figure 4] The amino acid sequences of Nb42 and point mutation variants are shown.

[0022] [Diagram 5] Coomassie brilliant blue (CBB) staining of E. coli cell extracts expressing HuNb42 and point mutation variants is shown.

[0023] [Figure 6] FIG. 1 shows the relative E. coli cell culture yields of HuNb42 (SEQ ID NO: 61) and HuNb42 A88P (SEQ ID NO: 67) point mutation variants in the cytoplasm and periplasm.

[0024] [Figure 7] 1 shows the relative degree 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.

[0025] [Figure 8] 1 shows Coomassie Brilliant Blue (CBB) staining of E. coli cell extracts expressing aTNFaMu (SEQ ID NO: 71) or aTNFaMu_3MUT (SEQ ID NO: 72) at room temperature, 50°C, 60°C, 70°C, and 80°C.

[0026] [Figure 9] Relative protein expression of E1-1 (SEQ ID NO: 81), and point mutants E1-1 F11L (SEQ ID NO: 82), E1-1 S49A (SEQ ID NO: 83), E1-1 F11L / S49A (SEQ ID NO: 84) and E1-1 CDR (SEQ ID NO: 85) is shown.

[0027] [Figure 10A] Shown is Coomassie Brilliant Blue (CBB) staining of purified HuNb42 protein (SEQ ID NO: 62) induced at 18°C ​​and 37°C. [Figure 10B] Purified Hu2H10_5MUT protein (SEQ ID NO:55) induced at 16°C, 27°C, 30°C and 37°C is shown.

[0028] [Figure 11A] Two preparations of HuNb42 A88P (SEQ ID NO: 67) are shown, without EDTA treatment ("220119") and with EDTA treatment ("220204"). [Figure 11B]Shown are the recovery of TNFα3MUT (SEQ ID NO: 104) before treatment with EDTA ("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 endotoxin levels before filtration ("Input") and after filtration through a 50 kDa ("50 kDa FT") or 100 kDa ("100 kDa FT") polyethersulfone membrane (right). [Figure 11C] Shown is an SDS-PAGE gel of purified aAng2_D4_aH_CYS (sequence number 120).

[0029] [Figure 12] Figure 1 shows the Rh distribution of DARPin multivalent proteins (MVPs) prepared by method 1 (conjugate 5, conjugate 2) and method 5 (conjugate 3, conjugate 6, conjugate 7). MVPs prepared using method 5 showed a smaller MVP radius.

[0030] [Figure 13A] The activity of MVP is shown. Figure 13A shows the VEGF association and dissociation curves of Biolayer Interferometry (BLI) of anti-VEGF MVPs made using different intermediates and anti-VEGF E1-1 peptide. Top: Association and dissociation curves of conjugate 10 (KD=0.123 nM) from method 2. Bottom: Conjugate 8 (KD=0.184 nM) from method 5, showing baseline, association, and dissociation curves of conjugate 8 at different concentrations (x-axis: time in seconds, y-axis: BLI signal). Both conjugates show similar binding kinetics curves and calculated dissociation constants. Figure 13B shows the VEGF binding affinity ("KD", nM) of the two peptides compared to the corresponding MVPs. First bar: unconjugated 2H10_5MUT_aH_CYS (sequence number 142); second bar: unconjugated HuNb42_A88P_aH_CYS (sequence number 145); third bar: MVP containing 2H10_5MUT_aH_CYS (sequence number 142); fourth bar: MVP containing HuNb42_A88P_aH_CYS (sequence number 145). [Figure 13B] The activity of MVP is shown. Figure 13A shows the VEGF association and dissociation curves of Biolayer Interferometry (BLI) of anti-VEGF MVPs made using different intermediates and anti-VEGF E1-1 peptide. Top: Association and dissociation curves of conjugate 10 (KD=0.123 nM) from method 2. Bottom: Conjugate 8 (KD=0.184 nM) from method 5, showing baseline, association, and dissociation curves of conjugate 8 at different concentrations (x-axis: time in seconds, y-axis: BLI signal). Both conjugates show similar binding kinetics curves and calculated dissociation constants. Figure 13B shows the VEGF binding affinity ("KD", nM) of the two peptides compared to the corresponding MVPs. First bar: unconjugated 2H10_5MUT_aH_CYS (sequence number 142); second bar: unconjugated HuNb42_A88P_aH_CYS (sequence number 145); third bar: MVP containing 2H10_5MUT_aH_CYS (sequence number 142); fourth bar: MVP containing HuNb42_A88P_aH_CYS (sequence number 145).

[0031] [Figure 14] SEC traces of stability samples of MVP with various aging are shown (x-axis: retention time, y-axis: absorbance at 280 nm). Example of change in SEC retention time of anti-VEGF DARPin MVPs made with intermediates synthesized using Method 5 (conjugate 1, top) or Method 1 (conjugate 2, bottom) and stored at 37° C. for up to 71 days. MVPs synthesized by Method 5 (top) showed a greater radius loss with aging.

[0032] [Figure 15] FIG. 1 shows the change in radius of gyration of DARPin MVPs made using intermediates of Method 1 (conjugate 2, top line) or Method 5 (conjugate 3, bottom line) after aging under accelerated conditions at 37° C. for 28 and 32 days.

[0033] [Figure 16]Figure 1 shows the VEGF binding constant (KD) of BI VHH anti-VEGF MVPs made using Method 1, Method 2, or Method 5 intermediates before and after aging at 37°C. By day 4 of the stability study, MVPs synthesized using Method 5 intermediates no longer bound VEGF. The BLI limit of detection (LOD) is 0.001 nM. Samples with this value listed on the figure were below the BLI LOD of the KD.

[0034] [Figure 17] Figure 1 shows the change in association constant (Kon) of DARPin MVP made using intermediates of Method 1 (conjugate 2, squares, upper line) or Method 5 (conjugate 1, triangles, lower line) after aging under accelerated conditions for 28 and 32 days at 37° C. A lower Kon and slower association was observed for conjugate 1 initially and upon aging, suggesting that the less impure polymer of conjugate 2 stabilized and enhanced Kon.

[0035] [Figure 18]

[0036] Figure 1 shows the in vivo half-life extension of MVP synthesized using VHH and method 1 intermediate following intravitreal injection in rabbits. Rabbit intravitreal pharmacokinetic (IVT PK) study of conjugate 12 anti-TNFα VHH MVP (method 1 intermediate), more than 2-fold half-life extension versus unconjugated. n=3 eyes per time point. All eyes received 50 μg of VHH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] I. General The present invention provides a multivalent peptide-hyaluronic acid polymer conjugate, and a method for preparing the same, which uses a peptide linker to covalently link each biologically active peptide to the polymer. In some embodiments, the peptide linker is an alpha helix. The peptides are modified to increase the degree of humanization while retaining stability and the ability to express at acceptable levels in bacterial systems (e.g., E. coli). The corresponding conjugates are expected to have immunogenicity comparable to other humanized antibodies.

[0037] II. Definition Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In addition, any methods or materials similar or equivalent to those described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined:

[0038] "About" when referring to a value includes the stated value + / - 10% of the stated value. For example, about 50% includes the range of 45% to 55%, and about 20 molar equivalents includes the range of 18 to 22 molar equivalents. Thus, when referring to a range, "about" refers to the stated value + / - 10% of each stated value at either end of the range. For example, a ratio of about 1 to about 3 (weight / weight) includes the range of 0.9 to 3.3.

[0039] "Alkyl" refers to a straight or branched chain saturated monovalent or divalent hydrocarbon. For example, alkyl refers to an alkyl group having 1 to 10 carbon atoms (i.e., C 1~10 alkyl) or 1 to 8 carbon atoms (i.e., C 1~8 alkyl) or 1 to 6 carbon atoms (i.e., C 1~6 alkyl) or 1 to 4 carbon atoms (i.e., (C 1~4 Examples of alkyl groups include methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-Butyl (n-Bu, n-Butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 ) 2 ), 2-Butyl (s-Bu, s-Butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 ), 1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2-methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH 3 ), 3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 ), 2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3 , and octyl (-(CH 2 ) 7 CH 3 ), but are not limited to these.

[0040] "Cycloalkyl" refers to an alkyl group having 3 to 20 cyclic carbon atoms (i.e., C 3~20Cycloalkyl) refers to a single saturated or partially unsaturated all-carbocyclic ring, e.g., having 3 to 12 ring atoms, e.g., 3 to 10 ring atoms, or 3 to 8 ring atoms, or 3 to 6 ring atoms, or 3 to 5 ring atoms, or 3 to 4 ring atoms. The term "cycloalkyl" also includes multiple fused, saturated and partially unsaturated all-carbocyclic ring systems (e.g., ring systems containing 2, 3, or 4 carbocyclic rings). Thus, cycloalkyl includes polycyclic carbocycles, such as bicyclic carbocycles (e.g., bicyclic carbocycles having about 6 to 12 annular carbon atoms, such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbocycles (e.g., tricyclic and tetracyclic carbocycles having up to about 20 annular carbon atoms). Rings of multiple fused ring systems may be linked together through fused, spiro, and bridged bonds, if permitted by valence requirements. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl.

[0041] As used herein, "organic linker" refers to a chemical moiety that directly or indirectly covalently attaches a peptide to a polymer. Organic linkers useful in the present invention can be about 100 Da to 500 Da. Types of organic linkers of the present invention include, but are not limited to, imides, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. One of skill in the art will appreciate that other types of organic linkers are useful in the present invention.

[0042] "Thiol" refers to the -SH functional group.

[0043] "Thiol-reactive group" refers to a group that can react with a thiol to form a covalent bond to a sulfur atom. Representative thiol-reactive groups include, but are not limited to, thiol, TNB-thiol, haloacetyl, aziridine, acryloyl, vinylsulfone, APN (3-arylpropiolonitrile), maleimide, and pyridyl disulfide. The reaction of a thiol-reactive group with a thiol can form a disulfide or a thioether.

[0044] As used herein, a "coupling agent" refers to a compound that reacts a carboxylic acid (-(C=O)-OH) with an amine (-NH 2 ) group to form an amide (-(C=O)-NH-).

[0045] "Peptide", "polypeptide", and "protein" are used interchangeably herein to refer to naturally occurring and synthetic amino acids of any length, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The term "peptide" includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue), immunologically tagged proteins, and the like. Peptides further include post-translationally modified peptides.

[0046] As used herein, "VHH" refers to a single domain heavy chain antibody.

[0047] "DARPin" refers to designed ankyrin repeat proteins, which are engineered antibody-mimetic proteins that can exhibit highly specific and high affinity target protein binding.

[0048] An "alpha helix" or "α-helix" is a common motif in protein secondary structure, a right-handed structure in which all backbone NH groups are hydrogen bonded to the backbone C=O group of the amino acid located four residues earlier in the protein sequence. An α-helix can be the classical Pauling-Corey-Branson α-helix, or the 3.6 13 -Also known as the helix. 3.6 13 -helix represents the average number of residues per helix turn (3.6) and 13 atoms in the ring formed by hydrogen bonds. Peptides that contain an α-helix are said to be α-helical. Such peptides may be partially or completely α-helical. As understood in the art, an α-helix has at least four amino acid residues. In some embodiments, an α-helix has between 4 and 40 amino acids.

[0049] Pharmaceutically acceptable salts of the peptides or conjugates described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms and other materials that are useful in the preparation of pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0050] As used herein, a "pharmaceutical composition" refers to a product containing specified ingredients in specified amounts, and any product that results directly or indirectly from the combination of specified ingredients in specified amounts. Pharmaceutical compositions are generally safe for biological use.

[0051] As used herein, "pharmaceutically acceptable excipient" refers to a substance that aids in the administration and absorption of an active agent by a subject. Pharmaceutically acceptable excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, and colorings. Those skilled in the art will recognize that other pharmaceutically acceptable excipients are useful in the present invention.

[0052] The conjugates described herein can be prepared and / or formulated as pharmaceutically acceptable salts, or as free bases, if appropriate. Pharmaceutically acceptable salts are non-toxic salts of the free base form of the compound that retain the desired pharmacological activity of the free base. These salts can be derived from inorganic or organic acids or bases. For example, conjugates containing basic nitrogen can be prepared as pharmaceutically acceptable salts by contacting the compound with inorganic or organic acids. Non-limiting examples of pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne, hexyne-1,4-dioate ... Examples of suitable pharma- ceutically acceptable salts include 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, besylate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, and mandelate. A list of other suitable pharma- ceutically acceptable salts can be found in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa., 2006.

[0053] Examples of "pharmaceutically acceptable salts" of the conjugates disclosed herein also include salts of suitable bases, such as alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., magnesium), ammonium and NR 4 + (Wherein, R is C 1~C 4 Also included are salts derived from alkyl groups such as alkyl groups (alkyl groups). Base addition salts, such as sodium or potassium salts, are also included.

[0054] As used herein, "therapeutically effective amount" refers to a dose that produces the therapeutic effect for which it is administered. The exact dose varies depending on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0055] As used herein, "inhibit," "inhibit" and "inhibitor" refer to a compound or method that prevents a particular action or function.

[0056] As used herein, "treatment" or "treat" or "treating" refers to an approach to obtain a beneficial or desired result. For purposes of this disclosure, beneficial or desired results include, but are not limited to, alleviating symptoms and / or reducing the severity of symptoms and / or preventing the worsening of symptoms associated with a disease or condition. In one embodiment, "treatment" or "treating" includes one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition and / or reducing the severity of the disease or condition), b) delaying or halting the onset of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, slowing the worsening or progression of a disease or condition), and c) relieving a disease or condition, e.g., regressing clinical symptoms, improving a disease state, slowing disease progression, improving quality of life, and / or prolonging survival.

[0057] "Prophylaxis" refers to preventing or slowing the progression of clinical disease in a patient suffering from a disease.

[0058] A "subject" of the present invention is a mammal, which may be a human or a non-human mammal, such as a companion animal, such as a dog, cat, rat, 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.

[0059] "Joint" as used herein refers to a fibrous or cartilaginous joint, which is a fibrous or cartilaginous area where two or more bones connect to one another.

[0060] As used herein, "diffusion half-life" refers to the time it takes for the initial concentration of a conjugate in a given volume or space to decrease by half, where the decrease in concentration is a function of the concentration gradient.

[0061] As used herein, "intra-articular half-life" refers to the time it takes for the initial concentration of a conjugate in a particular joint to decrease by half, where transport out of the joint is by convection. Convective transport is a combination of transport by diffusion and advection, where advection transport is the transport of a substance by bulk motion.

[0062] III. Peptides In some embodiments, the peptides of the present invention offer advantages over comparable peptides in the art, such as, for example, improved degree of humanization, improved solubility, improved stability, reduced tendency to aggregate in solution, and / or improved expression levels in favorable systems such as E. coli.

[0063] In some embodiments, the peptide has formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(I) A peptide having the formula: wherein CDR1, CDR2, and CDR3 are each independently a complementarity determining region; FR1, X 10 VQLX 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1) (In the formula, X 10 is Q, E, or D; X 11 is V, Q, A, or E; X 12 is S or T, X 13 is L, S, or V; X 14 is V or A, X 15 is P, A, or T, X 16 is G, D, or R; X17 is A, V, T, or E; X 18 is A or V, FR2, X 20 X 21 WX 22 RQX 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2) (In the formula, X 20 is M, I, V, or L; X 21 is G, S, or A; X 22 is F, Y, or V; X 23 is A, V, P, or T; X 24 is E, G, A, or Q; X 25 is R or L, X 26 is F, G, W, or L; X 27 is A, G, or S; X 28 is A, S, or G, FR3, 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 the formula, X 30 is A, G, S, or T, X 31 is R or Q, X 32 is N, S, or D, X 33is 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, X 39b is V, M, L, or I, FR4, YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4) (In the formula, X 40 is Q or K, X 41 is L or Q).

[0064] In some embodiments, X 13 is L.

[0065] In some embodiments, X 27 is A.

[0066] In some embodiments, X 30 is A.

[0067] In some embodiments, X 39a is P.

[0068] In some embodiments, X 40 is Q.

[0069] In some embodiments, FR1 is It has an amino acid sequence comprising: QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5).

[0070] In some embodiments, FR2 is It has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6).

[0071] In some embodiments, FR3 has an amino acid sequence comprising YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7).

[0072] In some embodiments, FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0073] In some embodiments, FR1 is QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5), FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6); FR3 has an amino acid sequence comprising: YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO: 7); FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0074] In some embodiments, CDR1, CDR2, and CDR3 are each a complementarity determining region from an antibody or a cytokine, hi some embodiments, the antibody is a monoclonal IgG, an IgG fragment, a single chain scFv, a single domain heavy chain VHH, an adnectin, an affibody, an anticalin, a DARPin, or an engineered Kunitz-type inhibitor. In some embodiments, the complementarity determining regions are each specific for vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), programmed cell death protein 1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), cluster of differentiation 40 (CD40), cluster of differentiation 134 (CD134), cluster of differentiation 137 (CD137), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), V-domain immunoglobulin suppressor of T-cell activation (VISTA), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), lymphocyte activation 3 (LAG3), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), or interleukin-15 (IL-15). In some embodiments, the complementarity determining regions are each specific for vascular endothelial growth factor (VEGF).

[0075] In some embodiments, the peptide consists of Formula I.

[0076] In some embodiments, the peptide has one or more of: (a) a CDR1 that is 7 amino acids in length, (b) a CDR2 that is 7 or 8 amino acids in length, and / or (c) a CDR3 that is 9-16 amino acids in length.

[0077] In some embodiments of the peptide, (a) CDR1 has an amino acid sequence comprising FAYSTYS (SEQ ID NO:9), CDR2 has an amino acid sequence comprising NSGTFRLW (SEQ ID NO:10), and CDR3 has an amino acid sequence comprising RAWSPYSSTVDAGDFR (SEQ ID NO:11); or (b) CDR1 has an amino acid sequence comprising RRFSIEA (SEQ ID NO: 12), CDR2 has an amino acid sequence comprising DSGGSTD (SEQ ID NO: 13), and CDR3 has an amino acid sequence comprising IGGSWYGRGLD (SEQ ID NO: 14); or (c) CDR1 has an amino acid sequence comprising GTFSSII (SEQ ID NO: 15), CDR2 has an amino acid sequence comprising SWSGGTTV (SEQ ID NO: 16), and CDR3 has an amino acid sequence comprising RPYQKYNWASASYNV (SEQ ID NO: 17); or (d) CDR1 has an amino acid sequence comprising GGSDAGT (SEQ ID NO: 18), CDR2 has an amino acid sequence comprising SWAGTAWR (SEQ ID NO: 19), and CDR3 has an amino acid sequence comprising LGSYEMDHH (SEQ ID NO: 20).

[0078] In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-98, 101-109, 111-131, and 141-170. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 142. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 145.

[0079] In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-95, 101-106, and 111-118. In some embodiments, the peptide has an amino acid sequence comprising any one of SEQ ID NOs: 73, 81, 91, and 92. In some embodiments, the peptide has an amino acid sequence comprising any one of SEQ ID NOs: 101-106. In some embodiments, the peptide has an amino acid sequence comprising SEQ ID NO: 67.

[0080] IV. Conjugates In some embodiments, the conjugate has formula IIa: (X1 -X 2 -Y) n -Z(IIa) is a conjugate of During the ceremony, each X 1 is independently a peptide described herein, each X 2 are independently a peptide linker having a length of 3 to 100 amino acids, each Y is independently an organic linker; Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1500.

[0081] In some embodiments, the conjugate has formula IIb: (X 1 -X 2A -Y) n -Z(IIb) is a conjugate of During the ceremony, each X 1 are independently peptides having a molecular weight of about 5 kDa to about 200 kDa, each X 2A is independently a peptide linker comprising an α-helix; each Y is independently an organic linker; Z is a biocompatible polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1500.

[0082] In some embodiments, each X 1 are independently peptides of the invention.

[0083] In some embodiments, each peptide vector is independently between 7 and 100 amino acids in length. In some embodiments, each peptide vector is independently between 10 and 30 amino acids in length.

[0084] In some embodiments, each peptide vector independently comprises: AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21), AEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:22), AEEEKRKAEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:23), AEEEEKKKKEEEEKKKKAGC (SEQ ID NO:24), AEAAAKEAAAKAGC (SEQ ID NO:25), PSRLEEELRRRLTEGC (SEQ ID NO:26), or It has an amino acid sequence comprising: AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEERKKREDDEKRKKKAGC (SEQ ID NO:27).

[0085] In some embodiments, each peptide linker has an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21).

[0086] Each peptide can be linked to a biocompatible polymer by various organic linkers commonly known in the art for forming antibody-drug conjugates, such as those provided by Conju-Probe or BroadPharm (San Diego, CA) or Creative Biolabs (Shirley, NY). Methods for forming bioconjugate bonds are described in Bioconjugate Techniques, 3 rd Edition, Greg T. Hermanson. The organic linkers may be reactive with amines, carbonyls, carboxyls, and activated esters, may react via click chemistry (with or without copper), or may be reactive with thiols.

[0087] Representative organic linkers include amides or disulfides or are formed from reactive groups such as succinic anhydride, succinimide, N-hydroxysuccinimide, N-chlorosuccinimide, N-bromosuccinimide, maleic anhydride, maleimide, hydantoin, phthalimide, and the like. Organic linkers useful in the present invention are small, generally having a molecular weight of about 100 Da to about 500 Da, and containing two functional groups consisting of a maleimide and either an amine or a hydrazide. In some embodiments, the peptide is covalently attached to the polymer via a sulfide bond and an organic linker having a molecular weight of about 100 Da to about 500 Da. In some embodiments, the organic linker has a molecular weight of about 100 Da to about 300 Da. In some embodiments, the organic linker comprises a succinimide. In some embodiments, the organic linker is formed using N-β-maleimidopropionic acid hydrazide (BMPH), N-ε-maleimidocaproic acid hydrazide (EMCH), N-aminoethylmaleimide, N-κ-maleimidoundecanoic acid hydrazide (KUMH), hydrazide-PEG2-maleimide, amine-PEG2-maleimide, hydrazide-PEG3-maleimide, or amine-PEG3-maleimide.

[0088] Representative organic linkers include: [ka] These include, but are not limited to:

[0089] In some embodiments, the organic linker is N-ε-maleimidocaproic acid hydrazide (EMCH): [ka] It could be.

[0090] In some embodiments, the organic linker has the structure: [ka] having In the formula, the subscript m is an integer from 1 to 300.

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

[0092] In some embodiments, preparing the conjugate of the present invention comprises covalently binding an organic linker to a biocompatible polymer, and then covalently binding a peptide to the organic linker. In some embodiments, after preparing the conjugate of the present invention, unreacted organic linkers are present on the biocompatible polymer. The structure of the unreacted organic linker varies depending on the organic linker, and will be understood by those skilled in the art.

[0093] Representative unreacted organic linkers include: [ka] These include, but are not limited to:

[0094] In some embodiments, the unreacted organic linker has the structure: [ka] has.

[0095] In some embodiments, the unreacted organic linker has the structure: [ka] having wherein the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

[0096] In some embodiments, the unreacted organic linker has the structure: [ka] has.

[0097] In some embodiments, the biocompatible polymer is a polysaccharide.

[0098] In some embodiments, the biocompatible polymer is a glycosaminoglycan.

[0099] In some embodiments, the biocompatible polymer is hyaluronic acid.

[0100] 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.

[0101] 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.

[0102] In some embodiments, the conjugate has formula IIa: (X 1 -X 2 -Y) n -Z(IIa) is a conjugate of During the ceremony, each X 1 is independently a peptide described herein, each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21), Each Y has the structure: [ka] is an organic linker having the formula Z is a biocompatible polymer that is hyaluronic acid having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript m is an integer from 1 to 300; The subscript n is an integer from 1 to 1500.

[0103] In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa of Formula III: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (III) is a conjugate which is a random polymer of During the ceremony, each X is independently a peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; each XYZ 1 The part has the structure: [ka] having each Z 2 The structure: [ka] having each Z 3 Independently, the structure: [ka] having Each R 1 and R 2 is independently 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-NR 3 R 4 , or C 5 ~C 8 is cycloalkyl, Each R 3 and R4 are independently H or C 1 ~C 6 is alkyl, each Z 3a is independently OH or Y′; each Y' is an unreacted organic linker; the subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of the subscripts n, p, and q; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; The subscript q is an integer from 100 to 10,000.

[0104] In some embodiments, each X is a peptide having an amino acid sequence comprising any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-98, 101-109, 111-131, and 141-170. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 142. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 145.

[0105] In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-95, 101-106, and 111-118.

[0106] In some embodiments, the conjugate has formula IIIa: (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa) The structure is During the ceremony, each X 1are independently peptides having a molecular weight of about 5 kDa to about 200 kDa, each X 2 is a peptide linker that contains an α-helix.

[0107] In some embodiments, each X 1 is represented by formula I: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(I) A peptide of the formula: wherein CDR1, CDR2, and CDR3 are each independently a complementarity determining region; FR1, X 10 VQLX 11 EX 12 GGGX 13 X 14 QX 15 GX 16 SLRLSCX 17 X 18 SG (SEQ ID NO: 1) (In the formula, X 10 is Q, E, or D; X 11 is V, Q, A, or E; X 12 is S or T, X 13 is L, S, or V; X 14 is V or A, X 15 is P, A, or T, X 16 is G, D, or R; X 17 is A, V, T, or E; X 18 is A or V, FR2, X 20 X 21 WX 22 RQX 23 PGKX 24 X 25 EX 26 VX 27 X 28 I (SEQ ID NO:2) (In the formula, X 20 is M, I, V, or L; X 21 is G, S, or A; X 22 is F, Y, or V; X 23 is A, V, P, or T; X 24 is E, G, A, or Q; X 25 is R or L, X 26 is F, G, W, or L; X 27 is A, G, or S; X 28 is A, S, or G, FR3, 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 the formula, X 30 is A, G, S, or T, X 31 is R or Q, X 32 is N, S, or D, X 33 is S, A, or D, X 34 is N or K, X 35 is T or M, X 36 is Y, D, or S; X 37 is N or D, X 38 is S or N, X 39a is P or A, X 39b is V, M, L, or I, FR4, YWGX 40 GTX 41 VTVSS (SEQ ID NO: 4) (In the formula, X 40 is Q or K, X 41 is L or Q).

[0108] In some embodiments of the peptide of formula I, X 13 is L.

[0109] In some embodiments of the peptide of formula I, X 27 is A.

[0110] In some embodiments of the peptide of formula I, X 30 is A.

[0111] In some embodiments of the peptide of formula I, X 39a is P.

[0112] In some embodiments of the peptide of formula I, X 40 is Q.

[0113] In some embodiments of the peptide of Formula I, FR1 has an amino acid sequence comprising QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5).

[0114] In some embodiments of the peptide of Formula I, FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6).

[0115] In some embodiments of the peptide of Formula I, FR3 has an amino acid sequence comprising YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO:7).

[0116] In some embodiments of the peptide of Formula I, FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0117] In some embodiments of the peptide of Formula I, FR1 has an amino acid sequence comprising QVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO:5); FR2 has an amino acid sequence comprising MGWFRQAPGKEREFVAAI (SEQ ID NO:6); FR3 has an amino acid sequence comprising: YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA (SEQ ID NO: 7); FR4 has an amino acid sequence comprising YWGQGTLVTVSS (SEQ ID NO:8).

[0118] In some embodiments of the peptides of Formula I, CDR1, CDR2, and CDR3 are each a complementarity determining region from an antibody or a cytokine.

[0119] In some embodiments of the peptide of formula I, 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.

[0120] In some embodiments of the peptide of Formula I, the complementarity determining regions are selected from the group consisting of vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), programmed cell death protein 1 (PD-1), programmed cell 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 (GFR), and / or IL-1. In some embodiments, the complementarity determining regions are each specific for vascular endothelial growth factor (VEGF). In some embodiments, the complementarity determining regions are each specific for tumor necrosis factor-alpha (TNF-α). In some embodiments, the complementarity determining regions are each specific for interleukin-1-beta (IL-1β).

[0121] In some embodiments, the peptide consists of Formula I.

[0122] In some embodiments, the peptide has one or more of: (a) a CDR1 that is 7 amino acids in length, (b) a CDR2 that is 7 or 8 amino acids in length, and / or (c) a CDR3 that is 9-16 amino acids in length.

[0123] 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), 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), 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), 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), CDR3 has an amino acid sequence comprising LGSYEMDHH (SEQ ID NO:20).

[0124] In some embodiments, each X 1 is a peptide having an amino acid sequence including any one of SEQ ID NOs: 51 to 58, 61 to 73, 81 to 85, 91 to 95, 101 to 106, and 111 to 118. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 73. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO:91.

[0125] In some embodiments, each X 2 is AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21), AEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:22), AEEEKRKAEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:23), AEEEEKKKKEEEEKKKKAGC (SEQ ID NO:24), AEAAAKEAAAKAGC (SEQ ID NO:25), PSRLEEELRRRLTEGC (SEQ ID NO:26), or A peptide linker having an amino acid sequence comprising: AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEERKKREDDEKRKKKAGC (SEQ ID NO:27).

[0126] In some embodiments, each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21).

[0127] In some embodiments, the organic linker has the structure: [ka] has.

[0128] In some embodiments, the organic linker is N-ε-maleimidocaproic acid hydrazide (EMCH): [ka] It could be.

[0129] In some embodiments, the organic linker has the structure: [ka] having wherein the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

[0130] In some embodiments, the organic linker has the structure: [ka] has. An organic linker having the above structure is known as MP2H.

[0131] In some embodiments, the random polymer of formula III has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, the random polymer of formula III has a molecular weight of about 0.7 MDa to about 1.5 MDa. In some embodiments, the random polymer of formula III has a molecular weight of about 0.8 MDa.

[0132] In some embodiments, each R 1 and R 2 is independently 1 ~C 3 Alkyl or -(C 1 ~C 3 Alkyl)-NR 3 R 4 In some embodiments, each R 1 and R 2 is ethyl or -(CH 2 ) 3 -NMe 2 In some embodiments, each R 1 is ethyl, and each R 2 Ha-(CH 2 ) 3 -NMe 2 In some embodiments, each R 1 Ha-(CH 2 ) 3 -NMe 2 And each R 2 is ethyl.

[0133] In some embodiments, each R 3 and R 4 is independently 1 ~C 3 In some embodiments, each R 3 and R 4 is methyl.

[0134] In some embodiments, subscript n is an integer between 1 and 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 1000 and is less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer between 100 and 10000. In some embodiments, subscript n is an integer between 1 and 1000 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 800 and is less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer between 100 and 10000. In some embodiments, subscript n is an integer between 10 and 450 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 300 and is less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 240 and is less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 60 and is less than about 2% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 30 and is less than about 1% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000.

[0135] In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa of Formula III: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (III) is a conjugate which is a random polymer of During the ceremony, each X is independently a peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; each XYZ 1 The part has the structure: [ka] having each Z 2 The structure: [ka] having each Z 3 Independently, the structure: [ka] having In the formula, each R 1 and R 2 is independently 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-NR 3 R 4 , or C 5 ~C 8 is cycloalkyl, Each R 3 and R 4 are independently H or C 1 ~C 6 is alkyl, each Z 3a is independently OH or Y′; each Y' is an unreacted organic linker; the subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of the subscripts n, p, and q; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; The subscript q is an integer from 100 to 10,000.

[0136] In some embodiments, each X is a peptide having an amino acid sequence comprising any one of SEQ ID NOs: 51-58, 61-73, 81-85, 91-98, 101-109, 111-131, and 141-170. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 55. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 67. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 142. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 145.

[0137] In some embodiments, the conjugate of the invention has a molecular weight of about 0.1 MDa to about 3 MDa of Formula III: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (III) is a conjugate which is a random polymer of During the ceremony, Each X is independently QVQLQES GGGLVQPGGS LRLSCAASGR TFSDHSGYTY TIGWFRQAPG KEREFVARIY WSSGNTYYAD SVKGRFAISR DIAKNTVDLT MNNLEPEDTA VYYCAARDGI PTSRSVESYN YWGQGTQVTV SSPSTPPTPS PSTPPGGCDD DDK (SEQ ID NO: 101), QVQLQES GGGLVQPGGS LRLSCAASGR TFSDHSGYTY TIGWFRQAPG KEREFVARIY WSSGNTYYAD SVKGRFAISR DIAKNTVDLT MNNLEPEDTA VYYCAARDGI PTSRSVESYN YWGQGTQVTV SSAEAAAKEA AAKEAAAKAG C (SEQ ID NO: 102), QVQLQDS GGGLVQAGGS LRLSCAASGG TFSSIIMAWF RQAPGKEREF VGAVSWSGGT TVYADSVLGR FEISRDSARK SVYLQMNSLK PEDTAVYYCA ARPYQKYNWA SASYNVWGQG TQVTVSSAEA AAKEAAAKEA AAKAGC (SEQ ID NO: 103), QVQLQES GGGLVQAGGS LRLSCAASGG TFSSIIMAWF RQAPGKEREF VGAVSWSGGT TVYADSVKGR FTISRDSARK SVYLQMNSLK PEDTAVYYCA ARPYQKYNWA SASYNVWGQG TQVTVSSAEA AAKEAAAKEA AAKAGC (SEQ ID NO: 104), CGGGVDNKFN KEVGWAFGEI GALPNLNALQ FRAFIISLWD DPSQSANLLA EAKKLNDAQA PK (SEQ ID NO: 105), or EIVMTQS PSTLSASVGD RVIITCQASQ SIDNWLSWYQ QKPGKAPKLL IYRASTLASG VPSRFSGSGS GAEFTLTISS LQPDDFATYY CQNTGGGVSI AFGQGTKLTV LGGGGGSGGG GSGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCTASGFS LSSAAMAWVR QAPGKGLEWV GIIYDSASTY YASWAKGRFT ISRDTSKNTV YLQMNSLRAE DTAVYYCARE RAIFSGDFVL WGQGTLVTVS SSPSTPPTPS PSTPPGGC (SEQ ID NO: 106) an anti-TNF-α or anti-IL-1β peptide comprising Each Y has the structure: [ka] is an organic linker having the formula each XYZ 1 The part has the structure: [ka] having each Z 2 The structure: [ka] having each Z 3 Independently, the structure: [ka] having In the formula, each R 1 and R 2 is independently 1 ~C 6 Alkyl, -(C 1 ~C 6 Alkyl)-NR 3 R 4 , or C 5 ~C 8 is cycloalkyl, Each R 3 and R 4 are independently H or C 1 ~C 6 is alkyl, each Z 3a is independently OH or Y′; Each Y' has the structure [ka] having the subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of the subscripts n, p, and q; the subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of the subscripts n, p, and q; The subscript q is an integer from 100 to 10,000.

[0138] In some embodiments, the conjugate has a molecular weight of about 0.8 MDa and has formula IIIa: (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa) is a conjugate which is a random polymer of During the ceremony, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 55; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21), Each Y has the structure: [ka] is an organic linker having the formula each X 1 -X 2 -YZ 1 The part has the structure: [ka] having each Z 2 The structure: [ka] having each Z 3 Independently, the structure: [ka] having each Z 3a is independently OH or Y′; Each Y' has the structure: [ka] having Each R 1 and R 2 is ethyl or -(CH 2 ) 3 -NMe 2 and the subscript n is an integer from 10 to 300 and is less than about 10% of the sum of the subscripts n, p, and q; the subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of the subscripts n, p, and q; The subscript q is an integer from 1000 to 3000.

[0139] In some embodiments, the conjugate has a molecular weight of about 0.8 MDa and has formula IIIa: (X 1 -X 2 -YZ 1 ) n -(Z 2 ) p -(Z 3 ) q (IIIa) is a conjugate which is a random polymer of During the ceremony, each X 1 is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO: 67; each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21), Each Y has the structure: [ka] is an organic linker having the formula each X 1 -X 2 -YZ 1 The part has the structure: [ka] having each Z 2 The structure: [ka] having each Z 3 Independently, the structure: [ka] having each Z 3a is independently OH or Y′; Each Y' has the structure: [ka] having Each R 1 and R 2 is ethyl or -(CH 2 ) 3 -NMe 2 and the subscript n is an integer from 10 to 300 and is less than about 10% of the sum of the subscripts n, p, and q; the subscript p is an integer from 1 to 15 and is less than about 0.5% of the sum of the subscripts n, p, and q; The subscript q is an integer from 1000 to 3000.

[0140] 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.

[0141] In some embodiments, the conjugates of the invention exhibit a therapeutically effective retention time in vivo of from about 12 hours to about 24 hours, from about 1 day to about 3 days, from about 3 days to about 7 days, from 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 1 month to about 3 months, or from about 3 months to about 6 months.

[0142] The biological activity of the conjugate is enhanced compared to the activity of the corresponding peptide in soluble form, e.g., compared to the activity of the peptide not conjugated to a polymer. In some embodiments, the biological activity of the conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold, or more than 1000-fold, than the biological activity of the peptide in soluble (unconjugated) form.

[0143] V. Composition In some embodiments, a pharmaceutical composition of the present invention is a pharmaceutical composition comprising a conjugate described herein and a pharma- ceutically acceptable excipient.

[0144] A. Formulation When preparing pharmaceutical compositions from the conjugates of the present invention, pharma- ceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, cachets, and dispersible granules. A solid carrier can be one or more substances that can also act as diluents, binders, preservatives, disintegrants, or encapsulating materials. Details of techniques for formulation and administration are fully described in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0145] 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 in suitable proportions with a carrier having the necessary binding properties and compressed into the desired shape and size. Powders and tablets preferably contain 5% or 10% to 70% of the conjugate of the invention.

[0146] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions.For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0147] Aqueous solutions suitable for oral use can be prepared by dissolving the conjugate of the invention in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired. Aqueous suspensions suitable for oral use may be made by dispersing the finely divided active component in water with viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, as well as dispersing or wetting agents, such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethylene oxycetanol), condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The preparations may be adjusted for osmolality.

[0148] Also included are solid form preparations that are intended to be converted immediately before use into liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavorants, stabilizers, buffers, artificial and natural sweeteners, dispersing agents, thickeners, solubilizers, etc.

[0149] Oily suspensions may be formulated by suspending the conjugate of the present invention in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or a mineral oil, such as liquid paraffin; or a mixture thereof. Oily suspensions may contain a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol. Sweetening agents, for example, glycerol, sorbitol, or sucrose, may be added to provide a palatable oral preparation. These preparations may be preserved by the addition of an antioxidant, such as ascorbic acid. For an example of an oily vehicle for injection, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical preparation of the present invention may be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil or a mineral oil, as described above, or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as gum acacia and gum tragacanth, naturally occurring phosphatides such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and the condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweetening and flavoring agents, as in the formulation of syrups and elixirs. Such formulations may also contain a demulcent, preservative, or coloring agent.

[0150] The composition of the present invention can also be delivered as microspheres for sustained release in the body. For example, microspheres can be formulated for administration by intradermal injection of drug-containing microspheres for sustained subcutaneous release (see Rao, J.Biomater Sci.Polym.Ed.7:623-645,1995); as biodegradable and injectable gel formulations (see, 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 allow constant delivery over weeks or months.

[0151] 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 intraarticular administration into a joint. The formulation for administration generally comprises a solution of the composition of the present invention dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed include water and Ringer's solution, isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil can be used, including synthetic mono-diglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used in the preparation of injectables. These solutions are sterile and generally free of undesirable substances. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, for example, 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 widely and is selected mainly based on the volume of fluid, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the patient. For intravenous, intratumoral, or intravitreal administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol.

[0152] In another embodiment, the formulation of the composition of the present invention can be delivered by using liposomes that fuse with cell membranes or are endocytosed, i.e., by using ligands attached to the liposomes or directly attached to the oligonucleotides, which bind to cell surface membrane protein receptors and trigger endocytosis. By using liposomes, the composition of the present invention can be delivered to target cells in vivo in a targeted manner, especially when a ligand specific for the target cells is carried on the liposome surface or is otherwise preferentially directed to a particular organ. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).

[0153] Lipid-based drug delivery systems include lipid solutions, lipid emulsions, lipid dispersions, self-emulsifying drug delivery systems (SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). In particular, SEDDS and SMEDDS are isotropic mixtures of lipids, surfactants and cosurfactants, which can spontaneously disperse in aqueous media to form fine emulsions (SEDDS) or microemulsions (SMEDDS). Lipids useful in the formulations of the present invention include any natural or synthetic lipids, including, but not limited to, sesame oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®.

[0154] B. Administration The conjugates and compositions of the invention can be delivered by any suitable means, including oral, parenteral, and topical methods. In some embodiments, the delivery method is intra-articular. In some embodiments, the delivery method is intravitreal. In some embodiments, the delivery method is intratumoral.

[0155] Pharmaceutical preparations are preferably in unit dosage form.In this form, the preparation is subdivided into unit doses that contain appropriate amounts of the conjugate and composition of the present invention.The unit dosage form can be a packaged preparation, the package containing individual amounts of preparations, for example, packaged tablets, capsules, and powders in vials or ampoules.

[0156] The conjugates and compositions of the present invention may 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 the other agent. Co-administration also includes administering simultaneously, about simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. Furthermore, each of the conjugates or compositions of the present invention can be administered once a day, or two, three, or more times a day to provide the preferred daily dosage.

[0157] In some embodiments, co-administration can be achieved by co-formulation, i.e., preparing a single pharmaceutical composition containing the conjugates and compositions of the invention and any other agents, or alternatively, the various components can be formulated separately.

[0158] The conjugates and compositions of the present invention, as well as any other agents, may be present in any suitable amount and may depend on a variety of factors, including but not limited to, the weight and age of the subject, the state of the disease, etc. Suitable dose 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 doses also include about 1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg. The compositions may also include other compatible therapeutic agents. The conjugates described herein may be used in combination with each other, with other active agents known to be useful in modulating the glucocorticoid receptor, or with adjuvants that may not be effective alone but may contribute to the effectiveness of the active agent.

[0159] VI. Treatment method In some embodiments, the present invention relates to methods and / or uses comprising the conjugates or compositions described herein for treating a disease or disorder in a subject in need of such treatment.

[0160] In some embodiments, the method comprises multiple administrations of the conjugate. In some embodiments, the method comprises administering the conjugate daily, every other day, every third day, or weekly. In some embodiments, the method comprises administering the conjugate weekly, every two weeks, every three weeks, or monthly. In some embodiments, the method comprises administering the conjugate monthly, every two months, or every three months. In some embodiments, the method comprises administering the conjugate two or three times a year. In some embodiments, the method comprises administering the conjugate annually.

[0161] A. Eye disorders In some embodiments, the method of the present invention is a method of treating an ocular disorder in a subject in need of such treatment, comprising administering to the subject a conjugate described herein.

[0162] In some embodiments, the methods include administering the conjugate intravitreally.

[0163] In some embodiments, the methods comprise administering the conjugate every month, every two months, or every three months.

[0164] 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.

[0165] The eye disorders that can be treated using the method of the present disclosure include, but are not limited to, uveitis, macular degeneration (also known as age-related macular degeneration (AMD)), choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, and ocular inflammation. In some embodiments, the macular degeneration is wet macular degeneration. In some embodiments, the macular degeneration is dry macular degeneration.

[0166] Ocular diseases that may be treated using the methods of the present disclosure include acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, e.g., acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, e.g., macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior segment or location; ocular tumors; retinal disorders, e.g., central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusion, retinal detachment, uveitic retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada disease; uveal diffusion diffusion); posterior ocular pathologies caused or affected by ocular laser therapy; posterior ocular pathologies caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorder; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flavimaculatus); hereditary macular degeneration; chorioretinal degeneration; Leber's congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and color vision deficiencies (including protanopia, protanopia, protanopia, protanopia, and protanopia).

[0167] In some cases, the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, diabetic retinopathy, achromatopsia, or color blindness.

[0168] In some cases, the composition comprising the conjugate is administered by an intravitreal, transscleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route of administration. In some cases, the composition comprising the conjugate is administered intravitreally. In some cases, the composition is delivered intravitreally or near the posterior segment of the eye. In some cases, the composition is administered intravitreally by injection. In some cases, the composition comprising the conjugate is administered by intraocular injection.

[0169] B. Joint diseases In some embodiments, the method of the present invention is a method of treating a joint disease or disorder in a subject in need of such treatment, comprising administering to the subject a conjugate described herein.

[0170] In some embodiments, the methods comprise administering the conjugate intra-articularly.

[0171] In some embodiments, the intra-articular 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 intra-articular half-life of the conjugate is at least 5 times longer than the half-life of the unconjugated peptide.

[0172] The present invention also provides a method for treating joint tissue diseases and disorders using the conjugate of the present invention.Examples of joint tissue diseases and disorders 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.

[0173] Many polypeptides are used as drugs to attenuate immune cell function, which has been of great use in the treatment of many joint disorders. Articular tissues are particularly vulnerable to injury and disease, because the typical cellular response to these attacks, i.e., upregulation of inflammatory mediators, is also a signal that promotes catabolism of articular cartilage and resorption of the underlying bone tissue. Degeneration of articular surfaces promotes the worsening of articular tissue injury and further upregulation of inflammatory mediators. Over time, these mechanisms create a feed-forward loop, resulting in cumulative damage to articular tissues.

[0174] Any joint in the human or animal body can be treated using the methods and conjugates of the present invention. Representative joints include, but are not limited to, fibrous joints, cartilaginous joints, synovial joints, facet joints, immobile joints, hemijoints, and mobile joints. A joint can be a simple joint with two articular surfaces, a compound joint with three or more articular surfaces, or a complex joint with two or more articular surfaces and a knee joint or meniscus. Anatomical joints that can be treated using the conjugates and methods of the present invention include, but are not limited to, the wrist (including the fingers), elbow joint, wrist joint, shoulder joint, sternum and clavicle joint, spine joint, temporomandibular joint and cranial joint, pelvic joint and hip joint, knee joint, ankle joint and ankle joint (including the toes). Joints can also be classified as planar joints, ball and socket joints, hinge joints, pivot joints, oval joints, and saddle joints. The conjugates and methods of the present invention can be used to treat tissues of joints, including, but not limited to, connective tissue, cartilage, articular surfaces, synovial cavities, menisci, and the like.

[0175] Examples of drugs designed 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, affibodies, 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.

[0176] One of the common side effects of using anti-inflammatory drugs such as those listed above is an increased risk of infection. They weaken the body's immune response, so the immune system is less able to fight bacteria, viruses, and parasites. Therefore, the benefits of using these drugs systemically must be carefully weighed against the risks associated with systemic immunosuppression. In diseases where the entire body is affected by immune hyperactivation disorders, such as rheumatoid arthritis, systemic use of immunosuppressants may be justified. However, in conditions where only one or a limited number of joints are affected, the systemic risk of infection often does not justify the use of these drugs systemically.

[0177] 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 cavity and do not provide sufficient duration of treatment after intra-articular administration. After intra-articular injection, the half-life of anti-inflammatory proteins in the synovium is short (<1.5 hours). This is evident from clinical studies in which inflammation inhibitors, including infliximab and etanercept, were administered by intra-articular injection to humans for various joint disorders. Some of these studies reported significant reductions in joint inflammation, but acknowledged that frequent (e.g., weekly) administration was necessary to obtain good results. Thus, intra-articular anti-inflammatory therapy using existing drugs is limited by the high cost and inconvenience of frequent intra-articular dosing. It is clear that methods to extend the bioactivity of anti-inflammatory drugs in the synovial fluid are needed to enable this therapeutic approach to treat joint disorders.

[0178] The main symptoms associated with joint disorders are pain, joint effusion, limited range of motion, and pathological remodeling of joint anatomy. The efficacy of a treatment for treating joint disorders may include pain relief measured by a generalized assessment, such as a visual assessment score. Efficacy may also be determined based on improved scores using systems specialized for specific joint disorders, such as the WOMAC score for osteoarthritis, the ACR20 for rheumatoid arthritis, the Psoriatic Arthritis Quality of Life for psoriatic arthritis, or the SASSS for ankylosing spondylitis. Efficacy may also be measured using functional outputs, such as increased pain-free walking distance or increased range of motion. Efficacy may also be measured based on radiographic evidence showing restoration of normal joint anatomy.

[0179] The conjugate can be administered at any suitable frequency or amount as discussed above. In some embodiments, the conjugate is injected into the joint about once a month or less. In some embodiments, the conjugate is injected into the joint about once a month to once every six months. In some embodiments, the conjugate is injected into the joint about once every two months or once every three months.

[0180] 1. Osteoarthritis In 2015, an estimated 7.75 million Americans experienced symptoms of osteoarthritis (OA) that may be related to known joint damage. Post-traumatic OA (PTOA) accounts for at least 15% of all OA cases, but it is postulated that many of the other OA diagnoses may also be related to prior joint trauma. In the absence of disease-modifying therapies, joint replacement is often the only treatment option to relieve associated discomfort and restore mobility. However, PTOA is often diagnosed in younger patients for whom joint replacement is not a viable option. Overall, the cost of treating these PTOA patients exceeds $4 billion annually in healthcare costs.

[0181] Short-term suppression of injury-related inflammation limits the long-term symptoms of PTOA. Many types of joint injury are associated with PTOA, including dislocations, ligament 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 joint-wide inflammation can promote catabolism of articular cartilage, resulting in the accumulation of further tissue damage over time and manifesting as PTOA. It is well known that TNFα and IL-1β play a role in mediating joint inflammation. These cytokines interact to promote cartilage destruction, both by downregulating the expression of cartilage matrix components and by upregulating the expression of matrix metalloproteinases (MMPs). TNFα also stimulates the recruitment of osteoclasts and induces apoptosis of osteoblasts, which form bone in an inflammatory environment, contributing to the erosion of articular cartilage tissue. TNFα and IL-1β are potent targets for moderating the inflammatory response to joint injury. Inhibition of these important acute inflammatory cytokines in the joint environment has been proposed for early intervention to slow the progression of PTOA.

[0182] 2. Inflammation caused by immune response to intra-articular microparticles Wear occurring between articular surfaces of joints can generate micron-scale particles that cause joint inflammation and osteolysis. Wear particles can be generated due to wear between endogenous surfaces such as ossified cartilage lesions, osteophytes (osteophytes), or exposed subchondral bone lesions. This type of wear particle generation is frequent in the later stages of OA, resulting in severe joint pain and lameness. This further inflammatory response accelerates the rate of degeneration of joint tissue in OA.

[0183] Wear particles can also form between the surfaces of artificial joints. In 2015, over 7 million Americans were living with artificial joints. Of these, nearly a quarter of a million would eventually require revision surgery due to eventual loosening and failure of the device caused by osteolysis of the bone surrounding the device.

[0184] Wear-related inflammation results from a foreign body response to normally inert particulates released from the articular surfaces. Macrophages within the synovial lining readily recognize wear particulates as foreign and release pro-inflammatory factors that recruit other active immune cells to the synovium, stimulating osteoclast proliferation while inhibiting bone formation. Thus, persistent inflammation triggers a feed-forward cycle in which cartilage degeneration and osteolysis lead to increased wear between the articular surfaces, as well as increased movement and physical stress, resulting in the generation of more particulates.

[0185] In some embodiments, the peptide modulates the activity of an immune cell function. In some embodiments, the peptide inhibits tumor necrosis factor-α, interleukin-1β, interleukin-6, or interferon-γ. In some embodiments, the peptide inhibits tumor necrosis factor-α.

[0186] Tumor necrosis factor (TNFα) is a potent target for controlling foreign body responses. It is well known that TNFα plays a role in mediating joint inflammation. TNFα also stimulates the recruitment of osteoclasts and induces apoptosis of bone-forming osteoblasts in an inflammatory environment, leading to osteolysis of subchondral bone. Inhibition of TNFα using a systemically administered receptor antagonist (etanercept) has been shown to reduce wear particle-induced bone resorption in mice, but the risks associated with systemic anti-TNFα are not generally considered acceptable for localized pathology. As an alternative, intra-articular anti-TNFα therapy has been proposed to prevent or inhibit the osteolytic response to intra-articular wear particles.

[0187] In some embodiments, the use of the present invention is the use of a conjugate described herein for the preparation of a medicament for a method of treating a disease or disorder in a subject.

[0188] In some embodiments, the subject is a human.

[0189] In some embodiments, the use of the present invention is for treating a disease or disorder, comprising a conjugate or pharmaceutical composition as described herein.

[0190] In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition for use in treating a disease or disorder, comprising a conjugate described herein.

[0191] In some embodiments, the conjugates of the invention are for use in the treatment of a disease or disorder described herein.

[0192] VII. Preparation method In some embodiments, the method is a method of preparing a peptide of the invention comprising: (a) translating in bacteria in a first reaction mixture a genetic sequence encoding the peptide; and (b) removing endotoxins from the first reaction mixture by forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA), thereby preparing the peptide.

[0193] In some embodiments, the method is a method of preparing a peptide of the present invention, comprising: (a) translating a genetic sequence encoding the peptide in a first reaction mixture in bacteria; (b) forming a second reaction mixture from the first reaction mixture and ethylenediaminetetraacetic acid (EDTA); and (c) filtering the second reaction mixture, thereby preparing the peptide. In some embodiments, the second reaction mixture further comprises sodium chloride. In some embodiments, the second reaction mixture further comprises sodium citrate. In some embodiments, the second reaction mixture further comprises sodium citrate pH 5.5.

[0194] In some embodiments, the bacterium is E. coli.

[0195] 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.

[0196] Filtration of the second reaction mixture can be accomplished by any method known in the art. In some embodiments, filtration of the second reaction mixture comprises a filtration membrane. In some embodiments, the filtration membrane comprises polyethersulfone (PES) or regenerated cellulose. For example, the filtration membrane can comprise a 50 kDa or 100 kDa PES membrane.

[0197] In some embodiments, the method of preparing the conjugates of the invention comprises: (a) a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa; about 0.1 to about 2 equivalents of a coupling agent per hyaluronic acid monomer; and 2 NR Y An organic linker agent of the formula Y teeth, [ka] and and subscript m is an integer from 1 to 300, whereby a first reaction mixture containing [ka] forming a first reaction mixture, from which an intermediate polymer having a plurality of monomers of (b) forming a second reaction mixture comprising the intermediate polymer and a peptide having a molecular weight of about 5 kDa to about 200 kDa, the peptide comprising one or more -SHs, thereby preparing a conjugate.

[0198] In some embodiments, the hyaluronic acid polymer has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, the hyaluronic acid polymer has a molecular weight of about 0.7 MDa to about 1.5 MDa. In some embodiments, the hyaluronic acid polymer has a molecular weight of about 0.8 MDa.

[0199] In some embodiments, the first reaction mixture includes from about 0.2 to about 1.5 equivalents of coupling agent per hyaluronic acid monomer. In some embodiments, the first reaction mixture includes from about 0.2 to about 1 equivalent of coupling agent per hyaluronic acid monomer.

[0200] In some embodiments, the coupling agent comprises a carbodiimide. In some embodiments, the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1,3-diisopropylcarbodiimide, or dicyclohexylcarbodiimide, or a salt thereof. In some embodiments, the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or a salt thereof.

[0201] In some embodiments, R Y teeth, [ka] It is.

[0202] In some embodiments, the first reaction mixture comprises about 0.2 to about 6 equivalents of organic linker agent per hyaluronic acid monomer.

[0203] In some embodiments, the first reaction mixture includes a catalyst. In some embodiments, the catalyst is ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), hydroxybenzotriazole, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), or 1-hydroxy-7-azabenzotriazole, or a salt thereof. In some embodiments, the catalyst is hydroxybenzotriazole.

[0204] In some embodiments, the second reaction mixture comprises about 0.5 to about 1.5 equivalents of peptide per organic linker.

[0205] In some embodiments, the method of preparing the conjugates of the invention comprises: (a) a hyaluronic acid polymer having a molecular weight of about 0.8 MDa; about 0.2 to about 1 equivalent of a coupling agent per hyaluronic acid monomer; and 2 NR Y An organic linker agent of the formula Y teeth, [ka] forming a first reaction mixture comprising This results in formula IV: [ka] An intermediate polymer is formed having a plurality of monomers of the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof; forming a first reaction mixture comprising about 0.2 to about 6 equivalents of an organic linker agent per hyaluronic acid monomer; and (b) forming a second reaction mixture comprising the intermediate polymer and a peptide having a molecular weight of about 5 kDa to about 200 kDa, the peptide comprising one or more -SHs, thereby preparing a conjugate.

[0206] The organic linker agent has the following structure: [ka] having It is known as 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-(3-hydrazineyl-3-oxopropoxy)ethoxy)ethyl)propanamide, also known by the abbreviation MP2H. As used herein, references to "MP2H" as an organic linker agent or organic linker are understood in the context of its use by those of skill in the art.

[0207] VIII. Examples Certain abbreviations and acronyms are used in describing the experimental details, and while most of these will be understood by those of skill in the art, the following table contains a list of many of these abbreviations and acronyms. [Table 1]

[0208] In the following examples, general methods were used.

[0209] Addition of peptide linkers for protein / conjugate stability In-frame fusion of the therapeutic protein with a C-terminal peptide linker was achieved by two methods. E. coli codon-optimized nucleotides encoding the peptide linker were added to the therapeutic ORF (containing a single C-terminal cysteine ​​residue for MVP conjugation) and ordered as a linear Geneblock (IDT or similar) with an overhang compatible for direct cloning into a protein expression plasmid. Alternatively, an oligonucleotide primer complementary to the therapeutic ORF containing the codon-optimized sequence encoding the peptide linker was extended and amplified in a PCR reaction, thereby generating a linear amplicon that was directly cloned into the protein expression plasmid. Sanger sequencing was performed on all isolated plasmids to confirm the correct positioning and integrity of the ORF containing the therapeutic fused to the peptide linker. All ORFs were expressed via an IPTG-inducible T7 promoter in a commercially available T7-compatible E. coli strain.

[0210] Cytoplasmic expression in E. coli To determine the amount of soluble protein expressed in E. coli, the protein was placed under the control of an IPTG-inducible T7 promoter (NEB Shuffle T7 Express) and grown at OD 300 in Terrific Broth. 600nm The cultures were grown to a yield of 0.6 and induced with 0.5 mM IPTG for 4 hours at 37° C. Culture sizes varied from 10 mL to 1 L depending on needs and objectives.

[0211] Expression in the E. coli periplasm For expression in the E. coli periplasm, the periplasmic targeting sequence of E. coli MalE was added to the N-terminus of the ORF, and all subsequent expression and downstream purification techniques remained unchanged.

[0212] IMAC and affinity tag removal E. coli pellets from 1 L cultures were dissolved by sonication in 25 mM HEPES, 20 mM imidazole, 400 mM sodium chloride, 0.5 mM EDTA, 5% glycerol, 0.01% Tween 20, pH 7.5, clarified at 20 k×g, and loaded onto a GE Ni-NTA HisTrap™ column. Non-specific proteins were washed off in the above buffer plus 40 mM imidazole. Proteins of interest were then eluted with a gradient to 260 mM imidazole using FPLC. Purity was confirmed 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.

[0213] Protein A purification If a polyhistidine affinity tag was not used, Protein A resin (JSR Life Sciences, Amsphere A3) was used to capture sdAbs from clarified E. coli lysates in 20 mM Tris, 25 mM sodium chloride, 0.5 mM EDTA, pH 8.5. The immobilized sdAbs were then washed in fresh lysate buffer and then eluted with 50 mM sodium citrate pH 5, 25 mM NaCl, 1 mM EDTA.

[0214] Chromatography Polishing For further purification of the protein, the pooled IMAC eluate was diluted 5-fold with nanopure water and loaded onto a GE HiTrap Q HP column pre-equilibrated with 20 mM Tris, 25 mM sodium chloride, 0.5 mM EDTA, pH 8.5. These conditions were sufficient to remove contaminating E. coli proteins from the affinity chromatography eluate pool, and the target protein remained in the column flow-through. The Q column flow-through was further diluted 2-fold with nanopure water, brought to pH 5 with acetic acid, and loaded onto a GE HiTrap SP HP column pre-equilibrated with 10 mM sodium citrate, 0.25 mM EDTA, pH 5.0. The purified protein was eluted with a gradient to 25 mM sodium citrate, 0.5 M sodium chloride, 1 mM EDTA, pH 5.5. Purity was confirmed by SDS-PAGE, and the elution peak area identified by AKTA Unicorn software was used as a comparative indicator of protein yield.

[0215] Endotoxin removal and protein polishing Pure SP elution fractions were pooled and endotoxins were removed by passing the material twice through a 100 kDa regenerated cellulose spin concentrator. The flow-through protein solution from the 100 kDa spin concentrator was then concentrated in a 3 kDa regenerated cellulose spin concentrator until the protein concentration was >175 mg / mL. Afterwards, sterile glycerol was added to the spin concentrator at 10% C. F(v / v), then flash frozen and stored at -80°C.

[0216] Alternatively, a final concentration / purification step was performed with Q anion exchange chromatography followed by cation exchange chromatography. Proteins were bound at pH 5 and eluted with a gradient from solution A (10 mM sodium citrate pH 5, 0.25 mM EDTA) to solution B (25 mM sodium citrate pH 5.5, 1 M NaCl, 1 mM EDTA), typically eluting at 10-25% B. Peak fractions were then pooled and these pooled protein solutions were then passed through a 100 kDa filter membrane (either PES or regenerated cellulose) at 3000 x g. The pure protein was then further concentrated on a 10 kDa filter membrane.

[0217] Gel analysis of free cysteine To confirm that a single free cysteine ​​was available for conjugation to the biopolymer, approximately 20 equivalents of a 1.2 kDa PEG-maleimide moiety were incubated at 42 °C for 45 min and run on a 4–20% SDS-PAGE to confirm the mobility shift by a single 1.2 kDa gel mobility shift.

[0218] Further protein / conjugate stabilizing mutations Protein sequences were obtained through BLAST queries against the PDB database. MSA operations were performed using the Jalview program, which included sequence alignment using Clustal Omega, manual curation of sequences from the alignment to include only sdAbs with the desired topology, and removal of sequence redundancy to leave only approximately 100 sequences in the MSA. Positions in the MSA with a conservation score of 9 or higher were considered as consensus and incorporated into the sdAb sequence, including a c-terminal α-helical linker peptide between the ORF and the conjugation cysteine.

[0219] Site-directed mutagenesis Complementary oligonucleotide pairs containing the desired codon-optimized amino acid substitution mutations were designed according to the guidelines published within the Agilent QuikChange site-directed mutagenesis kit protocol and purchased from IDT. SDM PCR reactions were performed on approximately 10 ng of plasmid DNA according to the manufacturer's protocol. The newly isolated plasmids were subjected to Sanger sequencing to confirm the appropriate amino acid substitution(s).

[0220] Heat precipitation of soluble lysate proteins 25 volumes of lysis buffer 25 mM HEPES, 20 mM imidazole, 400 mM sodium chloride, 0.5 mM EDTA, 5% glycerol, 0.01% Tween 20, pH 7.5 were added to E. coli pellets harvested from 25 mL TB cultures and sonicated 5 times on ice using a probe sonicator at 40% power. After lysis, the whole cell extract was clarified at 10K × g and 100 μL of the supernatant was aliquoted and subjected to incubation at 50 °C, 60 °C, 70 °C, and 80 °C for 15 min, followed by incubation on ice for 10 min. Heat-precipitated proteins were removed at 10K × g for 5 min and the soluble extract fractions were directly combined with Laemmli sample buffer, denatured, and run on 4-20% SDS-PAGE to assess yield and stability.

[0221] Example 1. Evaluation of peptide linkers Prior to conjugation to the polymer, peptides containing a biologically active peptide of interest linked to a peptide linker were expressed in Escherichia coli (E. coli) based on the expression open reading frame (ORF) in Figure 1 A. Various peptide linkers connecting the biologically active peptide to the polymer were evaluated (Table 2). [Table 2]

[0222] Figure 1B shows soluble expression of an exemplary protein, 2H10, in E. coli. Improved yields in the soluble fraction were observed with 2H10 variants containing an α-helical peptide linker. The presence of the α-helical peptide linker improved expression when a reactive cysteine ​​was present at the C-terminus of the protein.

[0223] FIG. 1C shows that increased soluble expression is evident in SDS-PAGE analysis of comparable peptides. Cultures containing autoinduction medium and carbenicillin (5 mL) were grown to saturation overnight at 37°C. Saturated cultures were harvested by centrifugation, washed with 1 mL of PBS, and harvested by repeated centrifugation. Supernatants were aspirated and cultures were frozen at -80°C. Cells were lysed on ice by probe sonication, removed by centrifugation, normalized to protein content, and run on a 4-20% SDS-PAGE. Protein expression of Hu2H10_5MUT (SEQ ID NO: 55) and Hu2H10_5MUT_CYS (SEQ ID NO: 141) was lower as evidenced by a band around 15 kDa than Hu2H10_5MUT_aH_CYS (SEQ ID NO: 142), which contains the α-helical peptide linker of SEQ ID NO: 21 at the C-terminus (band around 17 kDa).

[0224] The improvement in soluble expression was not limited to the above proteins. The anti-VEGF protein HuNb42 also showed increased soluble expression with the addition of a C-terminal α-helical peptide. Figure 1D shows the soluble expression of HuNb42_A88P (SEQ ID NO: 67) ("null") compared to HuNb42_A88P aH_Cys (SEQ ID NO: 145) ("+aH_CYS"). The total process yield per liter of culture medium containing the α-helical linker was about 4 times higher than the yield of the corresponding protein without the C-terminal α-helical peptide.

[0225] Example 2. Effect of degree of humanization on soluble expression To reduce the risk of immunogenicity, a higher degree of humanization is desirable for therapeutic peptides and proteins. However, certain residues in single domain antibodies affect the degree of humanization while simultaneously reducing stability. Therefore, we performed a systematic evaluation of specific point mutations in framework regions that are related to the degree of humanization and stability.

[0226] Sequence humanization was performed using the computational resources of the Abysis antibody analyzer and LakePharma's T20 score analyzer. For sdAbs targeting human proteins, specific amino acids in the consensus sequence were altered to achieve a T20 framework-only score of 85 or higher.

[0227] Figure 2 shows the amino acid sequences of 2H10 and point mutation variants tested for expression in E. coli. As shown in Figure 3A, variants Hu2H10 R86K A87P (SEQ ID NO: 56) and Hu2H10 R86K A87P L115Q (SEQ ID NO: 58) had higher protein expression than Hu2H10 5MUT (SEQ ID NO: 55) or Hu2H10 L115Q (SEQ ID NO: 57).

[0228] Figure 4 shows the amino acid sequences of Nb42 and point mutation variants tested for expression in E. coli. Figure 5 shows that the HuNb42 A88P variant (SEQ ID NO: 67) showed increased soluble expression. Figure 6 shows that the higher relative expression was independent of the cellular compartment, with higher relative levels of soluble expression shown in both the cytoplasmic and periplasmic compartments.

[0229] Figure 7 shows the degree of humanization of Nb42 (SEQ ID NO: 61), HuNb42 (SEQ ID NO: 62), and the HuNb42 A88P variant (SEQ ID NO: 67) compared to caplacizumab, bevacizumab, and ranibizumab. HuNb42 (SEQ ID NO: 62) and the HuNb42 A88P variant (SEQ ID NO: 67) were each comparable to literature humanized antibodies in terms of degree of humanization as measured by Z-score or T20 score.

[0230] 8 shows Coomassie Brilliant Blue (CBB) staining of E. coli cell extracts expressing aTNFaMu (SEQ ID NO: 71) or aTNFaMu_3MUT (SEQ ID NO: 72) at room temperature, 50° C., 60° C., 70° C., and 80° C. The staining shows that the three point mutations in aTNFaMu increased protein yield and thermostability up to about 70° C.

[0231] 9 shows the effect of specific point mutations in E1-1 on protein expression yield. E1-1 S49A (SEQ ID NO: 83), E1-1 F11L S49A (SEQ ID NO: 84), and E1-1 CDR (SEQ ID NO: 85) showed approximately 10-fold greater relative protein expression compared to E1-1 (SEQ ID NO: 81) or E1-1 F11L (SEQ ID NO: 82).

[0232] Figure 10 shows a gel shift assay (top graph) consistent with fewer reactive cysteines in the final protein preparation when protein synthesis was performed at higher temperatures. The reduction in reactive cysteines indicates that naturally occurring disulfide bridges were formed in the antibody, thus resulting in a more stable product. A similar gel shift (bottom graph) showed a gradient from low to high levels of disulfide bridge formation when the temperature of protein synthesis was increased.

[0233] Example 3. Endotoxin Removal Endotoxin is undesirable in protein preparations because it is carried through the drug conjugation process and is a source of contamination in animal studies (endotoxins provoke an immune response).

[0234] Endotoxin removal relies on the presence of EDTA in the buffer, which aggregates endotoxins to a consistent size that makes them filterable with minimal protein loss during filtration. Figures 11A-11B show that the endotoxin removal process removed >99.5% of the endotoxins in the samples.

[0235] Figure 11B shows the removal of endotoxin using 50 and 100 kDa filters and the recovery of anti-TNFα3 MUT VHH(mouse)-aH (SEQ ID NO: 104) content. Method: Protein solutions of approximately 15 mg / mL in 25 mM sodium citrate pH 5.5, 100 mM NaCl, 1 mM EDTA were passed through 50 kDa and 100 kDa polyethersulfone membrane (PES) filters at 15K×g for 10 minutes at room temperature. Protein concentration was measured using an A280 spectrophotometer (nanodrop) and endotoxin was measured using a Charles River Endosafe LAL cartridge.

[0236] method: 5 mL cultures were grown to saturation at 37°C in TB-autoinduction medium + antibiotics. Cells were harvested by centrifugation at 4000 RPM for 10 minutes at 4°C. The pellet was washed with 1 mL PBS and transferred to an Eppendorf tube. Cells were pelleted at 14000 RPM for 2.5 minutes at 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) at 40% power for a total of 60 seconds with 5 sec pulse on and 5 sec pulse off using a small tip sonicator. Cell lysates were normalized to total protein content using a Nanodrop A280 and run on a 4-20% SDS-PAGE under denaturing and reducing conditions. Gels were stained with InstaBlue protein stain and destained extensively with water. Densitometric signal (%) was calculated using ImageJ software and normalized across gel lanes. Overexpression was achieved when >10% of the total lane protein signal was attributable to bands around the predicted molecular weight and not seen in the uninduced sample control. An exemplary SDS-PAGE gel is shown in Figure 11C.

[0237] A summary of densitometric measurements of expression for certain peptides of the invention is shown in Table 3 below. [Table 3] § Each sequence listed is preceded by an asterisk (" * Except where indicated with a ""), each of the amino acids was covalently linked to the C-terminal alpha-helical peptide of SEQ ID NO:21.

[0238] Example 4. Consensus sequences for protein expression of single domain antibodies Based on the data presented in the examples above, the following framework sequences enable single domain antibodies to be more stably expressed and / or more human-like. Tables 4 and 5 show exemplary framework regions with tolerated amino acid substitutions. [Table 4] [Table 5-1] [Table 5-2] [Table 5-3]

[0239] Example 5. Preparation of purified thiol-reactive hyaluronic acid conjugate intermediate Sodium hyaluronate (HA, 830 kDa) was suspended at 4 mg / mL in water or 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer pH 5.7 overnight at room temperature by mixing with gentle rotation or nutation. To a solution of 3 mg (3.6 nmol, amount varies based on polymer composition and molecular weight) of HA, add hydroxybenzotriazole (HOBt) hydrate as approximately 5-100 mg / mL stock solution in DMSO, a thiol-reactive linker agent (e.g., hydrazide-X-thiol reactive group such as MP2H) in 10-100% DMSO (10-100 mg / mL stock), and a coupling agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) in 0.1 M MES buffer pH 5.7. The molar equivalents of each reactant per mole of HA and per mole of carboxylate for different and exemplary methods of carrying out the reaction are given in Tables 6 and 7 below. [Table 6] The EDC range reaction was set up using molar equivalents per mole of 830 kDa HA. * For certain intermediates, EMCH was used instead of MP2H where indicated. [Table 7] equivalents of reactant per HA monomer (approximately 2000 carboxylates per 830 kDa HA). * For certain intermediates, EMCH was used instead of MP2H where indicated.

[0240] The solution was mixed by gentle pipetting between each reagent addition, and the final reaction volume was brought up to 1 mL with buffer. The final mixture was allowed to react at room temperature for 45 min to 2 h using a rocking mixer depending on the method. After reaction, the thiol-reactive biopolymer was purified using a 5-10 mL Zeba desalting spin column with a 7 kDa MWCO equilibrated with 10% v / v glycerol (optional) pH 6.5 DPBS and 0.01% v / v polysorbate 20 (optional), loaded with 20% of the resin volume of the crude reaction. The desired intermediate was eluted into a clean conical tube using centrifugation at room temperature (elution time approximately 25-60 min). The intermediate was either used immediately for reaction with thiols or aliquoted and flash frozen on dry ice. Maleimide concentration and number of modifications per polymer were determined using UV absorbance, NMR, or a modified Ellman reaction assay.

[0241] Alternatively, the number of covalently attached thiol-reactive small molecule linkers (valency) per biopolymer was increased or decreased by changing the reaction pH or the equivalents of hydrazide linker, catalyst, and coupling agent (EDC) to higher or lower amounts.

[0242] Alternative coupling reagents such as DMTMM or Oxyma may be used in place of EDC and HOBt. The activated biopolymer intermediates can also be purified from the reactants using size exclusion chromatography, other desalting columns, tangential flow filtration, ion exchange chromatography, dialysis, or alcohol / acetone precipitation.

[0243] After purification, UV spectra (200-324 nm) were acquired for intermediates prepared using the different methods using a Take3 microspot plate on a BioTek Synergy plate reader. Maleimide concentrations can be determined by absorbance at 230 nm or by comparing the spectra to reference standard intermediates.

[0244] NMR analysis of the conjugates was performed at the Complex Carbohydrate Research Center (CCRC) of the University of Georgia using a Bruker Advance III spectrometer equipped with a 5 mm cryoprobe ( 1 H, 600.13 MHz) at 25°C. After standard preparation of the intermediate using methods 1 and 5 on a 6 mL scale, the intermediate was purified in HPLC grade water using desalting resin and shipped to CCRC on wet ice. Samples were left at 4°C for several weeks, at which time partial maleimide hydrolysis was observed in the NMR spectrum. For preparation of the NMR sample, 0.7 ml of the intermediate stock solution (2.9 mg / ml) was pipetted into a 7 ml screw-cap tube. 1.3 ml of D 2 20 mL of D2O (99.9%) was added to each sample and mixed thoroughly by vortexing. The samples were then dried using a SpeedVac vacuum concentrator at room temperature. The dried samples were then placed in 700 μL of D2O. 2 The mixture was redissolved in O (99.98%) and subjected to NMR analysis.

[0245] Chemical analyses of the reaction products of the examples are set forth in Table 8 below. [Table 8] ND=not determined; * =Measured on the day of receipt of sample by CCRC.

[0246] Tabulations of intermediates synthesized using the three different methods, along with their resulting maleimide concentrations, valences, and reaction efficiencies based on HA monomer, are shown below in Tables 9, 10, and 11. [Table 9] [Table 10] [Table 11] *Maleimide concentration and valency were quantified using UV absorbance.

[0247] 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 pH 5.7 overnight at room temperature with gentle rotation or rocking mixing. Prior to the reaction, a 4 mg / mL HA stock in 0.1 M MES was made using water and approximately 1 M MES pH 5.7 and mixed at room temperature with rocking. To a solution of 3 mg of HA (3.6 nmol, amount varies based on polymer composition and molecular weight) is added hydroxybenzotriazole (HOBt) hydrate as an approximately 5-100 mg / mL stock solution in DMSO, a thiol-reactive linker agent (e.g., hydrazide-X-thiol reactive group such as MP2H) in 1-10% DMSO (10-100 mg / mL stock), and a coupling agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) in 0.1 M MES buffer pH 5.7. The molar equivalents of each reactant per mole of HA and per mole of carboxylate for the different ways of performing the reaction, as well as the exemplary methods, are listed below in Table 12. [Table 12] equivalents of reactant per HA monomer (approximately 2000 carboxylates per 830 kDa HA).

[0248] The solution was mixed by gentle pipetting between each reagent addition, and the final reaction volume was brought up to 1 mL with buffer. The final mixture was allowed to react at room temperature for 45 min to 1.5 h on a rocking mixer depending on the method. After reaction, the thiol-reactive biopolymer was purified using a 5-10 mL Zeba desalting spin column with 7 kDa MWCO equilibrated with 10% v / v glycerol (optional) pH 6.5 DPBS, loaded with 20% of the resin volume of the crude reaction. For NMR samples, the Zeba column was equilibrated with deuterium oxide and the intermediate was eluted in deuterium oxide and not frozen. The desired intermediate was eluted into a clean conical tube using centrifugation at room temperature (elution time approximately 25-60 min). The intermediate was either used immediately for reaction with thiols or aliquoted and flash frozen on dry ice or at -80 °C. Maleimide concentration and number of modifications per polymer were determined using UV absorbance, NMR, or a modified indirect Ellman reaction assay.

[0249] The maleimide concentrations / valences for each reaction method are provided in Table 13 below. [Table 13] * The reactive maleimide concentration and approximate valency of the intermediates analyzed by NMR were determined using the Ellman reaction assay.

[0250] NMR analysis of the conjugates was performed at the Complex Carbohydrate Research Center (CCRC) of the University of Georgia using a Bruker Advance III spectrometer equipped with a 5 mm cryoprobe ( 1 H, 600.13 MHz) at 25 °C. Standard preparations of intermediates were performed using methods A-E on a 3-6 mL scale, after which the intermediates were purified to deuterium oxide using a desalting resin and shipped to the CCRC on wet ice.

[0251] 1Based on the analysis of H NMR spectra, all samples contained signals corresponding to HA, MP2H, free ethyl dimethylaminopropyl urea (EDU), a by-product of EDC hydrolysis that was not removed during purification. The abundance of MP2H, bound N-acylurea adducts, and free EDU was determined relative to HA (repeating polymer unit) by calculating the integrals of the signals from characteristic peaks. Based on the integrals, the raw abundances of HA, MP2H, free EDU, and bound N-acylurea adducts (bound EDU) were measured. The abundances of MP2H, bound N-acylurea adducts, and free EDU relative to HA are shown in Table 14. [Table 14] nd: no species detected nq: species present but not quantifiable due to low signal

[0252] Example 6. Preparation of purified peptide-polymer conjugates To obtain purified peptide-polymer conjugates, 1.1-2 equivalents of peptide per maleimide were combined with the intermediate prepared by the method of Example 1 and reacted at either 4° C. or ambient temperature for at least 2 hours to overnight with rotational or rocking mixing (most reactions were performed at room temperature to improve solubility). Optionally, the reaction pH was adjusted by adding 1M pH 7 HEPES to a final concentration of 0.1M. In some cases, prior to the conjugation reaction, any disulfide bridges between peptides were reduced by adding 10-100 equivalents per protein equivalent of a reducing agent such as DTT or TCEP HCl. This was either removed from the peptide solution prior to conjugation by desalting columns or buffer exchange, or added directly to the conjugation reaction in the form of TCEP immobilized on polymer beads. During the conjugation reaction, one or more of the following were added to improve the reaction efficiency: 0.5-10 mM EDTA to minimize oxidation of free thiols, tween 20 to stabilize the protein and / or help reduce non-specific interactions between the protein and the activated biopolymer, carbohydrates, additional buffer, or glycerol, increasing or decreasing the salt concentration to stabilize the protein and / or help reduce non-specific interactions between the protein and the activated biopolymer. Unreacted peptide was removed from the peptide-polymer conjugate by one or more of the following methods: dialysis (1:100-1:1000) against an appropriate buffer (pH should be one unit higher or lower than the pI of the peptide) with a MWCO of 50-1000 kDa at least twice for 4 h each and once for at least 4 h at 4 °C to room temperature.To purify the conjugate from unreacted peptide, tangential flow filtration into citrate buffer, DPBS pH 6-8, or 50 mM tris 150 mM NaCl pH 8-8.5 with EDTA and other additives such as tween, or trehalose depending on the peptide, FPLC polishing using a size exclusion column, FPLC polishing with an affinity chromatography column designed to bind the polymeric component of the conjugate, or selective precipitation of the conjugate can also be used. If the reaction efficiency was high enough (i.e., there was <5% unreacted protein), no purification was necessary.

[0253] Alternatively, to each solution of the intermediates of Example 1, peptide was added at the appropriate peptide:polymer molar feed ratio, and Tween-20 was added (optional) to a final concentration of up to 0.03%. The solutions were allowed to react for 2 hours to overnight with rotational (approximately 5 RPM) or rocking agitation at ambient temperature. Unreacted peptide was removed by sequential dialysis using 50-1000 kDa MWCO membranes against each of the following buffer solutions: first, phosphate buffered saline or equivalent citrate or succinate buffered saline (pH and buffer salts used are peptide dependent) with 0.01% Tween-20 (optional) for at least 4 hours, second, phosphate buffered saline with 0.01% Tween-20 overnight, and phosphate buffered saline with 0.01% Tween-20 for 4 hours at 4° C. or room temperature, plus an optional fourth dialysis step. Optionally, additives such as tween 20, EDTA, and carbohydrates were added to enhance protein stability.

[0254] After MVP purification, the conjugates were analyzed for protein concentration, protein valency, MVP radius, and binding affinity using the methods described in the stability test section. A table of MVPs synthesized using different intermediates is shown below with their resulting protein concentration, valency, dissociation constant by Biolayer Interferometry (BLI), and measured radius.

[0255] MVPs synthesized by Method 1 and Method 2 had similar or improved MVP therapeutic properties (final protein concentration, protein valency, radius, binding kinetics) compared to MVPs synthesized using Method 5 (Table 15). An example of a comparison of hydrodynamic radii of DARPin MVPs synthesized by Method 1 or Method 5 intermediates is shown in Figure 12. An example of a comparison of VEGF binding curve BLI data of anti-VEGF MVPs synthesized by Method 1 or Method 5 intermediates is shown in Figure 13A. [Table 15] * The detection limit for BLI is 0.001 nM. Samples with this value listed in the table above have a K D The BLI LOD was less than 1. ** Cy7 fluorescent dye-labeled peptide

[0256] Alternatively, to obtain purified peptide-polymer conjugates, 1.1-2 equivalents of peptide per maleimide were combined with HA conjugation substrate prepared by the method shown in Table 16. The conjugation reaction was allowed to react at ambient temperature for at least 2 hours to overnight with end-over-end or rocking mixing. The reaction pH was adjusted by adding 1 M pH 7 HEPES to a final concentration of 0.1 M. In some cases, unreacted peptide was removed from the peptide-polymer conjugate by dialysis (1:400-1:1000) at 4°C-RT for at least three times 4 hours each against an appropriate buffer (pH should be one unit higher or lower than the pI of the peptide) with a MWCO of 50-1000 kDa. [Table 16]

[0257] To confirm successful conjugation, the products of the conjugation reaction were analyzed by SDS-PAGE and DLS. SDS-PAGE was used to measure the percentage of unreacted peptide that was separated by migration into the gel consistent with its molecular weight. After the conjugation reaction, a significant proportion of the peptide was present at the top of the stacking gel as a high molecular weight conjugate that was unable to migrate through the gel due to its size (>300 kDa). DLS was used to measure the hydrodynamic radius present in the reaction products. After the conjugation reaction, the most intense peak was the R that was consistent with the conjugated substrate. h The peptides were aligned to the hyaluronic acid substrate, indicating that the peptides were conjugated to the hyaluronic acid substrate. The conjugate data are shown in Table 17. The unreacted protein (%) was determined by densitometric analysis of the SDS-PAGE bands of the unconjugated protein relative to a BSA standard of known mass and divided by the total mass loaded in each well. The hydrodynamic radius was measured using dynamic light scattering (DLS) on a Wyatt DynaPro plate reader III (25 min, 5-10 acquisitions at 5 s, n=3 samples per conjugate). Data analysis was performed with a Jupyter notebook data analysis program to extract data of adequate quality and analyze based on the most intense peaks. [Table 17]

[0258] Achieving high concentrations of pharmaceutical formulations is often required to reach therapeutic thresholds, maximize therapeutic duration, and / or minimize dosage. However, at higher concentrations, therapeutic peptides may aggregate. In the case of polymer-peptide conjugation, there is an additional concern that interactions with the polymer substrate may contribute to aggregation, or aggregation may occur at lower peptide concentrations than in the absence of conjugated polymer. The HA conjugation substrate made using method B provided polymer-peptide conjugates with no measured aggregation.

[0259] To synthesize purified conjugate #28 using Method B, 174 μL of purified conjugation intermediate from Method B was mixed with 3 μL of 2% v / v Tween 20 and 26 μL of 80 mg / mL N42 anti-VEGF VHH (SEQ ID NO: 145) (for 1.1 equivalents of peptide per maleimide) in a 2 mL V-bottom microcentrifuge tube. The reaction pH was adjusted to pH 7 by the addition of 20 μL of 1 M pH 7 HEPES to a final concentration of 0.1 M. The reaction was allowed to proceed overnight at room temperature for 16 hours with rocking mixing. Unreacted peptide was removed from the peptide-polymer conjugate by dialysis (1:1000 based on the initial reaction volume) against 25 mM citrate, pH 5.5, 100 mM NaCl, 0.03% Tween 20 at room temperature with stirring using a 200 μL microFloat-A-Lyzer dialysis cassette with 100 kDa MWCO (Repligen). A total of four dialysis steps were performed, with three buffer changes after 4 h each and one change after 16 h overnight dialysis.

[0260] To synthesize purified conjugate #30 using Method B, 186 μL of purified conjugation 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) (for 1.1 equivalents of peptide per maleimide) in a 2 mL V-bottom microcentrifuge tube. The reaction pH was adjusted to pH 7 by the addition of 20 μL of 1 M pH 7 HEPES to a final concentration of 0.1 M. The reaction was allowed to proceed overnight at room temperature for 16 hours with rocking mixing. Unreacted peptide was removed from the peptide-polymer conjugate by dialysis (1:1000 based on the initial reaction volume) against 25 mM citrate, pH 5.5, 100 mM NaCl, 0.03% Tween 20 at room temperature with stirring using a 200 μL microFloat-A-Lyzer dialysis cassette with 100 kDa MWCO (Repligen). A total of four dialysis steps were performed, with three buffer changes after 4 h each and one change after 16 h overnight dialysis.

[0261] After the fourth dialysis step was completed, the purified conjugate was removed from the dialysis cassette and stored at 4° C. The reaction products were analyzed by visual inspection, UV-Vis absorbance to measure conjugation of the purified proteins, R, as described in the Examples herein. h The conjugated proteins are characterized by DLS to measure the binding affinity, SDS-PAGE to determine the percentage of unconjugated protein, and biolayer interferometry to measure the binding affinity. [Table 18]

[0262] Example 7. Stability analysis of purified peptide-polymer conjugates The stability of MVP was assessed by setting up a long-term accelerated in vivo stability setup by maintaining MVP at 5-10x therapeutic concentrations in vitreous mimetic buffer pH 7.3 (Table 19) or PBS 0.01% Tween 20 pH 7.4 at 37° C. MVP stability was assessed using SEC MALS or SEC, DLS, and / or BLI analysis of samples removed after various time periods. [Table 19]

[0263] A long-term 37°C stability study was set up 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 mimicking buffer. This concentration is 5-fold higher than the intravitreal therapeutic concentration of our predicted clinical dose. Samples were either filtered using a sterile 0.2 μm or 5 μm spin filter or mixed with the antimicrobial agent 0.01% sodium azide prior to 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. Plates were incubated at 37°C, 5% CO in a standard tissue culture incubator. 2 At separate time points, one aliquot from each sample was removed from the plate under sterile conditions and analyzed. First, the UV-VIS spectra of the samples were acquired every 10 nm from 200-600 nm to monitor any dramatic changes in sample composition. Second, the protein concentration is measured to adjust for any volume differences that may have occurred. Binding affinity is measured using BLI. K over time on (association constant) or K D The change in radius is used to assess relative stability. To monitor the change in radius over time, samples were centrifuged at 5000 g for 5 min to remove large aggregates or dust particles, and the R was measured using DLS without any sample dilution. h Measure.

[0264] Stability test samples were analyzed using HPLC size exclusion chromatography (SEC). This method was also used to analyze MVP formation and unreacted protein (%) after purification. To assess stability by SEC, MVP was filtered to remove particulates and analyzed using Shodex 1MDa Ohpak LB-804, Shodex KW-404 or 405, or Phenomenex PolySep6000 columns with DPBS or appropriate solvent as mobile phase to obtain baseline traces at 280 nm, 230 nm, etc. Samples were removed at various time points and analyzed using the same SEC method. Increases in retention time and peak widths relative to the baseline sample indicated degradation. Additionally, decreases in MVP peak area and / or increases in peak areas of monomeric and dimeric protein species also indicate MVP degradation. Conjugation percentage (%) was quantified by comparing peak area differences over time. In the future, SEC stability analysis will be combined with MALS to quantify changes in molecular weight and valency of the conjugates due to aging at different temperatures. Representative SEC data for Method 5 or Method 1 intermediate DARPin MVP samples aged up to 71 days at 37° C. are shown in Figure 14. Due to its large size, conjugate 2 could not be analyzed on the same column used to analyze conjugate 1 in this data.

[0265] The stability test samples also measured the radius of gyration (R) of MVP at different time points after aging at 37°C. g,zThe conjugates were analyzed by combining SEC with MALS analysis for the determination of the solubility and molecular weight. For this, the conjugate stability samples were filled into glass vial inserts (250 μL volume) housed 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 (or equivalent instrument) equipped with an isocratic pump, autosampler, thermostated column compartment, and a variable wavelength detector set to monitor at 280 nm, with a guard column of Shodex KW-405-4F (4.6×300 mm, flow rate 0.35 mL / min) or LB-804 or 806 (8×300 mm, flow rate 0.4 mL / min, for the analysis of the unconjugated VHH peak), respectively. For the analysis, the column compartment was kept at 30°C and the mobile phase was prepared in HPLC grade water with 0.1 μm filtered DPBS pH 7.4, 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 using an isocratic method, with at least 2 column volumes of mobile phase eluted after sample injection or run for a total of 60 minutes. A Dawn Heleos II MALS instrument and Optilab T-rEX refractive index detector (Wyatt Technology) or equivalent instrument were placed in series with the HPLC and downstream of the UV detector. The parameters of the MALS and dRI detector for protein-polymer conjugate analysis using Astra software (Wyatt Technology) are listed in Table 20. Prior to analysis, system-specific calibration values, normalization factors, delay volumes, and band broadening terms were determined for the system. Representative SEC traces of stability samples of MVP versus EDC range are shown in Figure 14. The increase in retention time indicated size reduction / shrinkage / decomposition of MVP with aging.In Figure 14, the high EDC conjugate (conjugate 1) showed a much greater increase in retention time and a smaller radius compared to the low EDC MVP (conjugate 2). The peak broadening also suggests an increase in polydispersity of the sample with aging, indicating possible sample degradation. The decrease in radius was further verified by MALS of the sample in the accelerated aging study shown in Figure 15. [Table 20]

[0266] Stability can also be assessed using DLS to measure macromolecular size (e.g., R h) change with aging at 37°C. For stability analysis based on radius change with aging at 37°C using DLS, samples are removed from the stability test conditions at 37°C for analysis at various time points. All samples and buffers are at room temperature. Solutions are diluted in sterile 0.1 um filtered formulation buffer without polysorbate 20 to a final concentration of 100 nM in 100 μL (typically a 1:10 dilution) and mixed by gentle trituration in a 1.5 mL centrifuge tube. Large aggregates and dust particles could be removed by centrifuging the tube at 5000 g for 5 minutes in a centrifuge. For single cuvette measurements on the NanoStar, a 40 μL sample of the sample solution was filled into a Wyatt Technology disposable microcuvette with cap (Wyatt catalogue no. WNDMC), tapped to remove air bubbles and placed in the instrument for analysis. For multiple reads using a plate reader, 25-35 μL of sample was added to a clear bottom black well 384 well plate (Corning catalog number P8802-384 or similar). Air bubbles in the sample were removed by brief centrifugation in a centrifuge with a plate adapter and then removed with a pipette tip or by gentle spraying of 70% ethanol vapor from a squirt bottle. The instrument settings for analysis of this and other samples by DLS herein are presented in Table 21. Any peaks above 1000 nm should be <6% in intensity. The DLS acquisition parameters are shown below in Table 21. [Table 21]

[0267] Table 22 below shows the MVP radius change upon accelerated aging at 37° C. for MVP samples synthesized using different methods. The hydrodynamic radius or radius of gyration was determined by DLS or MALS at t=0 and at various time points after aging at 37° C. In these examples, MVP synthesized by low EDC (method 1) showed improved stability at 37° C. due to smaller radius shortening / change upon aging. This suggested that the presence of N-acylurea adducts destabilized the conjugates prepared by methods using higher amounts of EDC. [Table 22]

[0268] Changes in binding affinity over time were also measured using BioLayer Interferometry (BLI). To perform BLI experiments, samples were removed from stability testing conditions at 37°C and analyzed at various time points. All reagents were equilibrated to room temperature for at least 30 minutes before use. Two probes per sample (one for kinetic assays and one for ligand-free control) were equilibrated in 250 μL of BLI buffer (0.2 μm filtered PBS pH 7.4, 0.2% Tween and 0.2% BSA) for a minimum of 10 minutes in a Gator Bio Max plate. Ligands were diluted to fixed concentrations of 25-100 nM based on their performance in preliminary reactions in BLI buffer. Analytes were prepared in BLI buffer at the highest concentration determined in the preliminary reactions and serially diluted 1:3 two to five times further using BLI buffer. A black flat bottom non-coated 96 well plate (Greiner Bio One Cat. No. 655209 or similar) was filled, row by row, with 200 μL of ligand, analyte diluent, and BLI buffer, one row for each ligand and analyte row. One well in each analyte row should be BLI buffer used as a blank for reference subtraction. No air bubbles were present in the wells and were removed with a pipette tip or by gentle spraying of 70% ethanol vapor from a squirt bottle. The plate was placed in a Gator with a slanted platform set at 25°C. The loading and kinetics steps for the Gator K assay were set using the double reference and step times shown in Table 24. The ligand was loaded until the signal reached 0.4-0.6 nm, then returned to the buffer row for a 60 second baseline measurement. The kinetics read was initiated using the step parameters in Table 24. Once the kinetic readings were completed with the ligand-loaded probe, a ligand-free control was run using a new probe with no ligand loaded. The same kinetic assay timing and the same sample wells were used as analyzed with the ligand-loaded probe. This data was used to correct for non-specific interactions between the sample and the probe.Representative BLI data for high (Method 5) and low (Method 1) EDC MVP samples before and after accelerated aging at 37° C. are shown in FIG. 16(K). D ) and Fig. 17(K on ) as shown in

[0269] Once the kinetic assays were completed, the data was analyzed in the results and analysis section of the Gator software. Raw data was corrected to include association times from 1 second to 180 seconds. The Y-axis was aligned to the start of the association step and inter-step correction was enabled. Savitzky-Goaly filtering of the data was used. Samples were double referenced by indicating in the software which probe and well were the buffer reference. The reference subtraction formula for each assay was then edited to double reference each assay using the following formula: (Kinetics assay wells - ligand-free assay wells) - (Kinetics assay buffer reference wells - ligand-free assay buffer wells). All titrations of the same MVP were grouped by color and parameters were adjusted to a 1:1 binding model including both association and dissociation with a global, Rmax unlinked fit. The window of interest was moved to include only the 100 seconds of dissociation. Binding curves were fitted and the residuals were determined to not deviate more than 10% from the actual curve, full R 2 is > 0.98 and full X 2 The kinetics were calculated and K D , K on and the response was recorded. Different samples were D If so, the association constant K was used to distinguish the binding affinities between the different constructs. on was used (i.e., FIG. 17), where a higher association constant indicated faster binding kinetics. [Table 23] [Table 24]

[0270] Below is a table of binding kinetics upon accelerated aging at 37° C. for MVP samples synthesized using different methods. The dissociation constants of the samples were determined by BLI at various time points after aging at 37° C. In these examples, MVPs synthesized by method 1 and method 2 had similar or improved 37° C. stability based on therapeutic target binding capacity, and similar or smaller change in dissociation constant with aging. Anti-VEGF VHH peptide MVPs synthesized using intermediates from method 5 lost all binding capacity after short aging, while examples synthesized using methods 1 or 2 showed target binding capacity throughout the study, suggesting that the presence of the N-acylurea adduct destabilizes the therapeutic. [Table 25]

[0271] Example 8. In vivo half-life of purified peptide-polymer conjugates A well-established pharmacokinetic model demonstrated extended intravitreal residence time of the conjugate. New Zealand White rabbits (n=9) were divided into three groups randomized based on body weight. All animals received a 50 μL intravitreal (ITV) injection of hu_anti-TNFα_aH MVP (SEQ ID NO: 102)+HyA (850 kDa) ("anti-TNFα MVP") in the left eye and unconjugated VHH (SEQ ID NO: 102) ("anti-TNFα") in the right eye using a 31 G insulin syringe. Both eyes received equimolar doses of antibody. One hour after injection, on the 5th and 10th days, one group of 3 rabbits was sacrificed and their eyes enucleated for analysis of intravitreal VHH. Both eyes were snap frozen and vitreous, retina, and aqueous humor were isolated from the frozen eyes. Each tissue sample was then disrupted with a bead beater. After disruption, VHH concentrations were quantified either by using ELISA or by digesting peptides with trypsin and subjecting samples to LC / mass spectrometry or similar methods. Representative results of extended intravitreal half-life in rabbit eyes after bioconjugation are shown in Figure 18.

[0272] The method for fluorescent tagging of peptides for this study is as follows: Using a mouse tumor model to assess the rate of clearance of proteins from solid tumors, the intratumoral (IT) half-life of MVP was measured to maximize the parameter of tumor retention. We used antibodies tagged with amine-reactive sulfo-Cy7 NHS ester (Broadpharm catalogue no. BP-22541) or Alexa Fluor 750 near-infrared fluorescent dye by the following method, which was performed under sterile conditions. First, the dye 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 at a 3:2 volume:volume ratio. Finally, the fluorescent dye was added at a molar ratio of 1:2 protein:fluorescent dye, mixed thoroughly and incubated for 1 hour at room temperature on a rocking apparatus protected from light by covering with foil. The NHS ester was quenched by adding 10% of the reaction volume with 1.5M Tris buffer pH 8.5 and mixed for another 10 min on a rocking device. The tagged proteins were purified from unreacted fluorescent dye using a NAP-10 desalting column (illustra catalogue no. 17-0854-01) equilibrated with PBS pH 7.0 + 0.01% Tween-20 according to the manufacturer's instructions. Protein concentration and degree of Cy7 labeling were determined by absorbance at 280 nm and 750 nm. Proteins stored on ice and within 3 hours of sulfo-Cy7 labeling were used for MVP synthesis according to the protocol above. In each case, the final product was sterile filtered and stored at 4 °C protected from light until use in animal studies.

[0273] Although the above invention has been described in some detail by way of illustrations and examples for purposes of clarity of understanding, those skilled in the art will appreciate that certain changes and modifications can be made that are 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 were individually incorporated by reference. In the event of any inconsistency between this application and the references provided herein, this application shall control. [Table 26-1]

Table 26-2

Table 26-3

Table 26-4

Table 26-5

Table 26-6

Claims

1. Equation (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(I) A peptide having, In the formula, CDR1, CDR2, and CDR3 are each independently complementary determination regions; FR1 is X １０ VQLX １１ EX １２ GGGX １３ X １４ QX １５ GX １６ SLRLSCX １７ X １８ SG (SEQ ID NO: 1) (In the formula, X １０ is Q, E, or D, X １１ is V, Q, A, or E, X １２ is S or T, X １３ is L, S, or V, X １４ is V or A, X １５ is P, A, or T, X １６ is G, D, or R, X １７ is A, V, T, or E, X １８ It has an amino acid sequence containing A or V, FR2 is X ２０ X ２１ WX ２２ RQX ２３ PGKX ２４ X ２５ EX ２６ VX ２７ X ２８ I (Sequence ID 2) (In the formula, X ２０ is M, I, V, or L, X ２１ is G, S, or A, X ２２ is F, Y, or V, X ２３ is A, V, P, or T, X ２４ is E, G, A, or Q, X ２５ is R or L, X ２６ is F, G, W, or L, X ２７ is A, G, or S, X ２８ It has an amino acid sequence containing A, S, or G, FR3 is YX ３０ DSVKGRFTIS ３１ DX ３２ X ３３ KX ３４ X ３５ VX ３６ LQMX ３７ X ３８ LRX ３９ａ EDTAX ３９ｂ YYCAA (Sequence ID 3) (In the formula, X ３０ is A, G, S, or T, X ３１ is R or Q, X ３２ is N, S, or D, X ３３ is S, A, or D, X ３４ is N or K, X ３５ is either 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, X ３９ｂ It has an amino acid sequence containing V, M, L, or I, FR4, YWGX ４０ GTX ４１ VTVSS (Sequence No. 4) (In the formula, X ４０ is Q or K, X ４１ A peptide having an amino acid sequence containing L or Q.

2. The peptide according to claim 1, wherein each of the complementarity-determining regions is specific to vascular endothelial growth factor (VEGF).

3. The peptide according to claim 1, wherein the peptide comprises formula I.

4. (a) CDR1 has an amino acid sequence containing FAYSTYS (SEQ ID NO: 9), CDR2 has an amino acid sequence containing NSGTFRLW (SEQ ID NO: 10), CDR3 has an amino acid sequence containing RAWSPYSSTVDAGDFR (SEQ ID NO: 11); or (b) CDR1 has an amino acid sequence including RRFSIEA (SEQ ID NO: 12), CDR2 has an amino acid sequence containing DSGGSTD (SEQ ID NO: 13), CDR3 has an amino acid sequence containing IGGSWYGRGLD (SEQ ID NO: 14); or (c) CDR1 has an amino acid sequence including GTFSSII (SEQ ID NO: 15), CDR2 has an amino acid sequence containing SWSGGTTV (SEQ ID NO: 16), CDR3 has an amino acid sequence containing RPYQKYNWASASYNV (SEQ ID NO: 17); or (d) CDR1 has an amino acid sequence including GGSDAGT (SEQ ID NO: 18), CDR2 has an amino acid sequence containing SWAGTAWR (SEQ ID NO: 19), The peptide according to claim 1, wherein CDR3 has an amino acid sequence containing LGSYEMDHH (SEQ ID NO: 20).

5. The peptide according to claim 1, 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.

6. (a) Translating the 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), (c) filtering the second reaction mixture, A method for preparing the peptide according to claim 1, wherein the peptide is prepared by this method.

7. Formula IIa: (X) １ -X ２ (-Y) ｎ -Z (IIa) It is a conjugate of, During the ceremony, each X １ However, independently, it is the peptide described in claim 1, each X ２ However, independently, it is a peptide linker with a length of 3 to 100 amino acids. Each Y is an organic linker independently. Z is a biocompatible polymer having a molecular weight of approximately 0.1 MDa to approximately 3 MDa. The conjugate of expression IIa, where the subscript n is an integer between 1 and 1500.

8. Equation IIb: (X １ -X ２Ａ -Y) ｎ -Z (IIb) It is a conjugate of, During the ceremony, each X １ However, they are peptides that independently have molecular weights ranging from approximately 5 kDa to approximately 200 kDa. each X ２Ａ However, independently, it is a peptide linker containing an α-helix, Each Y is an organic linker independently. Z is a biocompatible polymer having a molecular weight of approximately 0.1 MDa to approximately 3 MDa. The conjugate of expression IIb, where the subscript n is an integer between 1 and 1500.

9. each X １ However, independently, the conjugate according to claim 8 is the peptide according to claim 1.

10. Each peptide linker operates independently. AEAAAAKEAAAAKAGC (Sequence ID 21), AEEEKRKAEEEKRKAEEEAGC (Sequence ID 22), AEEEKRKAEEEEKRKAEEEEKRKAEEEEAGC (Sequence ID 23), AEEEEKKKKEEEEKKKAKAGC (Sequence ID 24), AEAAAAKEAAAAKAGC (Sequence ID 25), PSRLEEELRRRRLTEGC (Sequence ID 26), or The conjugate according to claim 7, having an amino acid sequence including AEEEEKKKQQEEEEEAERLRRIQEEEMEKERKRRREEDEEERRRRKEEEEERRMKLEMEAKRKQEEEEERKKREDDEKRKKKAGC (Sequence ID 27).

11. The conjugate according to claim 7, wherein each peptide linker has an amino acid sequence containing AEAAAAAKEAAAKAGC (SEQ ID NO: 21).

12. The aforementioned organic linker has the following structure: 【Chemistry 1】 It has, The conjugate according to claim 7, wherein the subscript m is an integer from 1 to 300.

13. The aforementioned organic linker has the following structure: 【Chemistry 2】 The conjugate according to claim 7, having the following characteristics.

14. The conjugate according to claim 7, wherein the biocompatible polymer is hyaluronic acid.

15. The conjugate according to claim 7, wherein the biocompatible polymer has a molecular weight of about 0.7 MDa to about 1.5 MDa.

16. The conjugate according to claim 7, wherein the biocompatible polymer has a molecular weight of about 0.8 MDa.

17. The conjugate according to claim 7, wherein the subscript n is an integer between 10 and 100.

18. Formula IIa: (X) １ -X ２ (-Y) ｎ -Z (IIa) It is a conjugate of, During the ceremony, each X １ However, independently, it is the peptide described in claim 1, each X ２ However, it is a peptide linker having an amino acid sequence containing AEAAAAAKEAAAKAGC (SEQ ID NO: 21), Each Y has the following structure: 【Transformation 3】 It is an organic linker having, Z is a biocompatible polymer that is hyaluronic acid having a molecular weight of approximately 0.1 MDa to approximately 3 MDa. The subscript m is an integer between 1 and 300. The conjugate of expression IIa, where the subscript n is an integer between 1 and 1500.

19. A pharmaceutical composition comprising the conjugate described in claim 7 and a pharmaceutically acceptable excipient.

20. A pharmaceutical composition comprising the conjugate according to claim 7 for use in a method of treating an eye disorder in a subject requiring treatment of an eye disorder.

21. The pharmaceutical composition according to claim 20, wherein the use includes administering the pharmaceutical composition into the vitreous humor.

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

23. A pharmaceutical composition comprising the conjugate described in claim 7, for use in a method of treating a joint disease or disorder in a subject requiring treatment of a joint disease or disorder.

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