CCR2 inhibitor and method of use

Abstract

The present invention relates to a CCR2 inhibitor which is a variant CCL2 polypeptide comprising a variant N-terminal portion and a conserved C-terminal portion, as well as the peptide and its use.

Description

[Technical Field]

[0001]

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 420,780, filed on 31 October 2022.

[0003] Field of Invention

[0004] The present invention relates to polypeptides, peptides, and conjugates having CCR2 inhibitory activity. [Background technology]

[0002]

[0005] Background of the Invention

[0006] CCR2 is a receptor for the chemokine CCL2 (monocyte chemotactic protein 1, MCP-1), as well as the closely related proteins CCL8 (MCP-2), CCL7 (MCP-3), and CCL13 (MCP-4). CCL2 preferentially binds to the receptor CCR2 and mediates cellular behavior, including monocyte chemotaxis. Studies suggest CCL2-mediated monocyte infiltration in pain, cancer, and various inflammatory diseases.

[0003]

[0007] Small molecule CCR2 inhibitors have been developed and tested for use in the treatment of a variety of diseases or disorders. Small molecule CCR2 inhibitors such as AZD2423, BMS-813160, CCX-140, and Cenicriviroc have been investigated for the treatment of diseases or disorders including post-traumatic neuralgia, neuropathic pain, inflammatory diseases, chronic obstructive pulmonary disease, diabetic polyneuropathy, cancer (including colorectal cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, liver cancer, and non-small cell lung cancer), diabetic nephropathy, diabetes, HIV infection, non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis, non-alcoholic fatty liver disease, or primary sclerosing cholangitis.

[0004]

[0008] Given the potential applications of CCR2 inhibitors, there is a need for alternative CCR2 inhibitors with different pharmacological properties, such as reduced toxicity and / or improved inhibitory efficacy. [Overview of the project]

[0005]

[0009] Brief summary of the invention

[0010] The present invention provides polypeptides, peptides, and conjugates having CCR2 inhibitory activity.

[0006]

[0011] One embodiment of the present invention provides a polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 40-70 and 76-145, and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs. 71, 72, 74, or 75.

[0007]

[0012] One embodiment of the present invention provides a peptide comprising an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 40-70 and 76-145.

[0008]

[0013] One embodiment of the present invention provides a nucleic acid molecule encoding a polypeptide or peptide as described herein.

[0009]

[0014] One embodiment of the present invention provides a vector comprising a nucleic acid molecule as described herein.

[0010]

[0015] One embodiment of the present invention provides a host cell comprising a nucleic acid molecule or vector as described herein.

[0011]

[0016] One embodiment of the present invention provides a pharmaceutical composition comprising a polypeptide, peptide, nucleic acid molecule, or vector described herein, and a pharmaceutically acceptable carrier.

[0012]

[0017] In one embodiment of the present invention, there is provided a method of inhibiting CCR2 signaling in a cell, the method comprising contacting the cell with a polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition described herein.

[0013]

[0018] In one embodiment of the present invention, there is provided a method of treating or preventing a disease or disorder associated with CCR2 signaling in a subject, the method comprising administering to the subject a polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition described herein.

[0014]

[0019] In one embodiment of the present invention, there is provided the use of a polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition described herein for inhibiting CCR2 signaling in a cell.

[0015]

[0020] In one embodiment of the present invention, there is provided the use of a polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition described herein for treating or preventing a disease or disorder associated with CCR2 signaling in a subject.

[0016]

[0021] The advantages and features of the present invention will be better understood by referring to the following more detailed description and the claims in conjunction with the accompanying drawings, tables, and sequence listings.

[0017]

[0023] The embodiments are described by way of example only with reference to the accompanying drawings.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram showing the HPLC profile of purified 1P2-CCL2 (Met64Nle OB-004). [Figure 2] It is a diagram showing the MALDI-TOF / TOF spectrum of purified 1P2-CCL2 (Met64Nle OB-004). [Figure 3]This figure shows the dose-inhibition curves obtained for 1P2-CCL2 (Met64Nle OB-004) in four independent experiments. [Figure 4] This figure shows the results of an experimental example of a CCR2 inhibitory efficacy assay, testing four known small molecule CCR2 inhibitors and 1P2-CCL2 (Met64Nle OB-004). [Figure 5] This figure shows aggregated data from four experiments on CCR2 inhibitory efficacy assays that tested four known small molecule CCR2 inhibitors and 1P2-CCL2 (Met64Nle OB-004). [Figure 6] This figure shows the number of monocytes (D1) captured / released by acutely activated HUVECS after 4 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 7] This figure shows the number of monocytes (D1) captured / released by acutely activated HUVECS after 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 8] This figure shows the number of monocytes (D2) captured / released by acutely activated HUVECS after 4 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 9] This figure shows the number of monocytes (D2) captured / released by acutely activated HUVECS after 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 10] This figure shows the number of monocytes (D3) captured / released by acutely activated HUVECS after 4 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 11] This figure shows the number of monocytes (D3) captured / released by acutely activated HUVECS after 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 12] This figure shows the number of monocytes (D4) captured / released by acutely activated HUVECS after 4 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 13] This figure shows the number of monocytes (D4) captured / released by acutely activated HUVECS after 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 14] This figure shows the number of monocytes (D1-D4) captured / released by acutely activated HUVECS after 4 hours and 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 15] This figure shows the number of monocytes (D1-D4) captured / released by acutely activated HUVECS after 4 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 16] This figure shows the number of monocytes (D1-D4) captured / released by acutely activated HUVECS after 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 17] This figure shows the number of monocytes (D1-D4) captured / released by acutely activated HUVECS after 4 hours and 18 hours of stimulation with TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. [Figure 18] This figure shows that 1P2-CCL2 (Met64Nle OB-004) strongly blocks monocyte elution in HUVECS activated by TNF-alpha for 18 hours. [Figure 19] This figure shows the inhibition of CCL2 by chimeric mouse CCL2 variants 1P2 (dashed line) and 1P8 (solid line). [Figure 20] This figure shows the in vivo inhibition of monocyte / macrophage recruitment into the peritoneal cavity by the mouse chimeric CCL2 variant 1P8 polypeptide (c1P8-CCL2). [Figure 21] This figure shows body weight after control (sham) treatment, treatment with bleomycin alone, or treatment with bleomycin and a CCR2 inhibitor. Body weight was measured as a percentage of total body weight on day 2 of the study. [Figure 22A-B] Analysis of pulmonary infiltrating macrophages by flow cytometry. Total CD64+ macrophages (as percentage of cells in Figure 22A or as cell number in Figure 22B), CD64+Ly6C- / low SiglecF- stromal macrophages (as percentage of cells in Figure 22C or as cell number in Figure 22D), and CD64+Ly6C+SiglecF- monocyte-derived macrophages (as percentage of cells in Figure 22E or as cell number in Figure 22F). Comparisons between all groups and vehicle groups only; homovariance (Bartlett test); if homovariance, one-way ANOVA followed by Dunnett's post-hoc test; if heterovariance, Kruskal-Wallis test followed by Dunn's post-hoc test; data are expressed as mean + / - SEM. [Figure 22C-D] As stated above [Figure 22E-F] As stated above [Modes for carrying out the invention]

[0019]

[0046] Detailed explanation

[0047] The inventors have discovered a peptide that can be used to create a variant of CCL2 having CCR2 inhibitory activity. This peptide can be used to replace the native N-terminal portion of CCL2 or a CCL2 analog in order to create a CCL2 variant polypeptide having CCR2 inhibitory activity. As demonstrated herein by example, the CCL2 variant polypeptide in which the peptide FTNPTWAPVT (SEQ ID NO: 40) replaces the first 10 N-terminal residues of human CCL2 is IC2. 50When measured by [method], it exhibits 3.5 to 36.4 times greater potency in CCR2 inhibition compared to the small molecule CCR2 inhibitors AZD2423, BMS-813160, CCX-140, and senicliviroc (Figure 4). In another example, a chimeric CCL2 variant containing the N-terminal portion AFSIMQAPVT (SEQ ID NO: 46) and the C-terminal portion derived from mouse CCL2 (SEQ ID NO: 72) effectively inhibits the recruitment of monocytes and macrophages into the peritoneal cavity during peritonitis (Figure 20). Other peptides that contribute to CCR2 inhibitory activity are described herein.

[0020]

[0048] The arrangements, compositions, and methods for carrying out the present invention are presented in terms of the examples and embodiments described herein. However, the present invention is not limited to the examples and embodiments described herein, and those skilled in the art will understand that many other embodiments of the present invention are possible without departing from the basic concepts of the present invention, and any such methods also fall within the scope of the present invention. Other forms and configurations of the present invention are expected to be readily incorporated into the teachings of the present invention, and the configurations are shown and described for clarification and disclosure purposes, and not for the purpose of limiting the scope.

[0021]

[0049] The molecule of the present invention

[0050] This provides peptides that exhibit CCR2 inhibitory activity and can be incorporated into polypeptides such as CCL2 variants and conjugates.

[0022]

[0051] In some embodiments, polypeptides comprising an N-terminal and a C-terminal portion are provided, the polypeptide being a CCL2 variant and a CCL2 inhibitor. As used herein, “CCL2 variant,” “CCL2 variant polypeptide,” “CCL2 derivative,” or “CCL2 derivative polypeptide” according to the present invention refers to a polypeptide derived from CCL2, e.g., human CCL2 (SEQ ID NO: 1) or mouse CCL2 (SEQ ID NO: 73), wherein the N-terminal portion comprises the peptide of the present invention as described herein, and the C-terminal portion comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to SEQ ID NOs: 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises or consists of an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions to any one of SEQ ID NOs: 9-70 and 76-145. In some embodiments, the N-terminal portion contains or consists of one of the amino acid sequences from SEQ ID NOs: 146-149. As described herein, methionine residues may be conservatively substituted with non-oxidative amino acid analogs or amino acid derivatives to reduce problems caused by methionine oxidation during synthesis. In some embodiments, one or more methionine residues in the polypeptides, peptides, and conjugates of the present invention are conservatively substituted with amino acid analogs or amino acid derivatives, including but not limited to norleucine (Nle). In some embodiments, the C-terminal portion of the CCL2 variant contains a norleucine (Nle) residue at position 54 for SEQ ID NOs: 74 (position 64 for SEQ ID NOs: 1). In some embodiments, the C-terminal portion of the CCL2 variant contains a norleucine (Nle) residue at one or more of positions 9, 12, and 62 for SEQ ID NOs: 72.

[0023]

[0052] As used herein, the terms “CCR2 inhibitor” and “CCR2 antagonist” may be used interchangeably to mean polypeptides, peptides, conjugates, small molecules, or other compounds that inhibit one or more biological and / or pathological activities induced by CCR2 agonism, activation, or signaling. CCR2 inhibitors can block the binding of a ligand or pathogen by achieving partial or complete occupancy of one or more sites on CCR2 that the ligand or pathogen requires for interaction (orthosteric inhibitors). For example, a CCR2 inhibitor can block the binding of a native CCR2 ligand to CCR2, thereby preventing normal activation of CCR2, or block the binding of a synthetic CCR2 agonist to CCR2. Alternatively, a CCR2 inhibitor can block the binding of a ligand or pathogen by engaging with a site on CCR2 and inducing CCR2 to assume one or more conformations that the ligand or pathogen cannot recognize (allosteric inhibitors). CCR2 inhibitors can inhibit either the entire repertoire or a subset of the CCR2 intracellular signaling pathway and pathogenic interactions.

[0024]

[0053] 1P2-CCL2(OB-004) is an exemplary CCL2 variant with improved CCR2 inhibitory efficacy, containing the peptide FTNPTWAPVT (SEQ ID NO: 40), which was discovered by the inventors in a phage display campaign using a library of CCL2 variants in which the first six N-terminal residues were randomized (all possible amino acids except cysteine) (a common method outlined in Gaertner et al., PNAS, 2008, 105(46):17706~17711). At the end of phage library selection performed in CHO-CCR2 cells, sequencing revealed that 1P2-CCL2(OB-004) was the second most abundant clone. The N-terminal sequence (residues 1-10) of 1P2-CCL2(OB-004) was FTNPTWAPVT (SEQ ID NO: 40). A panel of CCL2 variants, including 1P2-CCL2(OB-004), was isolated by phage display and synthesized using a multiplex synthetic approach (following the general method outlined in Paolini et al., JBC, 2018, 293(49):19092~19100) with a C-terminal core fragment in which methionine residue 64 of CCL2 (residue 54 in SEQ ID NO: 74 or residue 64 in SEQ ID NO: 1) was substituted with the isosteric but non-oxidative unnatural amino acid norleucine (Nle) to reduce problems caused by methionine oxidation during synthesis. Experiments revealed that 1P2-CCL2(Met64Nle OB-004) and many other CCL2 variants inhibit CCL2 signaling activity mediated by the natural ligand CCL2.

[0025]

[0054] In one embodiment, the polypeptide of the present invention comprises an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 9-70 and 76-145. In some embodiments, the polypeptide of the present invention comprises an amino acid sequence described in any one of claims 146-149. In some embodiments, this amino acid sequence is located near the N-terminus of the polypeptide. In some embodiments, the amino acid sequence is located such that the start of the amino acid sequence is within 15 residues from the N-terminus of the polypeptide, for example, within 15, 12, 10, 8, 6, 5, 4, 3, 2, or 1 residues from the N-terminus, or is located at the N-terminus of the polypeptide.

[0026]

[0055] In some embodiments, the polypeptide according to the present invention comprises an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 9-70 and 76-145, or comprises an amino acid sequence for any one of SEQ ID NOs. 146-149, and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs. 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises the amino acid sequence FTNPTWXXXX (SEQ ID NOs. 146), where X is any amino acid, and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs. 71, 72, 74, or 75. In some embodiments, the N-terminal portion includes the amino acid sequence FTNPTW[A or D or R or S or K or Q][P or A or T or G or S or Q or R or H or E][V or F or Q or G or S or L or Y][T or V or Q or S or A] (SEQ ID NO: 147), and the C-terminal portion includes an amino acid sequence that is at least 70% identical to SEQ ID NOs: 71, 72, 74, or 75. In some embodiments, the N-terminal portion includes one of the amino acid sequences from SEQ ID NOs: 40 and 76-99, and the C-terminal portion includes an amino acid sequence that is at least 70% identical to SEQ ID NOs: 71, 72, 74, or 75. In some embodiments, the N-terminal portion includes the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148), where X1 is methionine or norleucine (Nle), X2-X5 are any amino acids, and the C-terminal portion includes an amino acid sequence that is at least 70% identical to SEQ ID NOs: 71, 72, 74, or 75. In some embodiments, the N-terminal portion comprises the amino acid sequence FPX1DGW[A or R or G or H or V or Q][P or S or G or E][V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149), where X1 is methionine or norleucine (Nle), and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs: 71, 72, 74, or 75.In some embodiments, the N-terminal portion includes one of the amino acid sequences from SEQ ID NOs. 41 and 100-145, and the C-terminal portion includes an amino acid sequence that is at least 70% identical to SEQ ID NOs. 71, 72, 74, or 75.

[0027]

[0056] In some embodiments of the polypeptide of the present invention, the N-terminal portion consists of 12 or fewer amino acids, for example, 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer amino acids. In some embodiments, the N-terminal portion consists of 11 amino acids, 10 amino acids, or 8 amino acids. In one embodiment, the N-terminus of the C-terminal portion is directly adjacent to the C-terminus of the N-terminal portion, i.e., the N-terminal portion and the C-terminal portion are directly adjacent. In some embodiments, the N-terminal portion is adjacent to the C-terminal portion via a peptide linker. In some embodiments, the N-terminal portion is located at the extreme N-terminus of the polypeptide.

[0028]

[0057] In some embodiments, peptides are provided that contain an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs: 9-70 and 76-145. In some embodiments, peptides are provided that contain an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs: 40-70 and 76-145. In some embodiments, peptides are provided that contain an amino acid sequence of any one of SEQ ID NOs: 146-149. In some embodiments, peptides are provided that contain the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), where X is any amino acid. In some embodiments, peptides are provided that contain the amino acid sequence FTNPTW[A or D or R or S or K or Q][P or A or T or G or S or Q or R or H or E][V or F or Q or G or S or H or L or Y][T or V or Q or S or A] (SEQ ID NO: 147). In some embodiments, peptides are provided that contain an amino acid sequence of any one of SEQ ID NOs: 40 and 76-99. In some embodiments, a peptide comprising the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148) is provided, where X1 is methionine or norleucine (Nle) and X2-X5 are any amino acids. In some embodiments, a peptide comprising the amino acid sequence FPX1DGW[A or R or G or H or V or Q][P or S or G or E][V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149) is provided, where X1 is methionine or norleucine (Nle). In some embodiments, a peptide comprising any one of the amino acid sequences from SEQ ID NOs: 41 and 100-145 is provided.

[0029]

[0058] In some embodiments, conjugates are provided that include a polypeptide, such as a CCL2 variant polypeptide, or a peptide described herein, partially conjugated. Where used in reference to a conjugate, the term “part” refers to the atom, molecule, or compound conjugated to the polypeptide or peptide of the present invention. In some embodiments, the conjugation of a part to a polypeptide or peptide allows for the delivery of the part to cells, tissues, and / or tumors expressing CCR2 without inducing CCR2 signaling. In some embodiments, the part is an immunoglobulin domain, such as the Fc domain of an IgG, IgM, IgE, IgD, or IgA heavy chain, or the Fc domain of a lambda or kappa light chain. In some embodiments, the part is a chemokine domain, such as the domain of mouse CCL2 or human CCL2. In some embodiments, the part is a toxin, such as a toxin, that enables the killing of CCR2-expressing cells or pathogens within CCR2-expressing cells. In some embodiments, the toxin is a cytotoxic agent, an antitumor agent, a chemotherapeutic agent, an antiviral agent, or an antibacterial agent. In some embodiments, the toxin is Pseudomonas exotoxin A, diphtheria toxin, ribosome-inactivating protein, or saporin. In some embodiments, the portion is a carrier protein such as albumin. In some embodiments, the portion is a polymer such as polyethylene glycol. In some embodiments, the portion is a lipid. In some embodiments, the portion is a detectable marker such as a fluorescent molecule (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or AlexaFluor®), a dye, or a radioisotope. The portion may be conjugated to a polypeptide or peptide by various means known in the art. "Conjugated" means that the polypeptide or peptide of the present invention associates with the portion by covalent or non-covalent bonds. In embodiments where the portion is an amino acid polymer, the polypeptide or peptide of the present invention may be conjugated to the portion by peptide bonds, optionally via a linker amino acid sequence, and / or via disulfide bonds.

[0030]

[0059] The present invention provides polypeptides, peptides, and conjugates disclosed above, and further, nucleic acid molecules encoding the polypeptides, peptides, and conjugates. In some embodiments, the nucleic acid molecules encoding the polypeptides, peptides, and / or conjugates of the present invention are RNA or DNA. Those skilled in the art can design or identify the nucleic acid molecules encoding the polypeptides, peptides, and conjugates of the present invention using methods known in the art. In some embodiments, the nucleic acid molecules encoding the polypeptides, peptides, and / or conjugates of the present invention are incorporated into vectors such as plasmids, episomes, artificial chromosomes, viruses, or viral vectors. In some embodiments, the nucleic acid molecules encoding the polypeptides, peptides, and / or conjugates of the present invention, or vectors containing the nucleic acid molecules, are contained within host cells to enable the expression of the polypeptides, peptides, and / or conjugates of the present invention. In some embodiments, the host cells are bacterial cells, yeast cells, vertebrate cells, mammalian cells, human cells, or cells of immortalized cell lines such as CHO cells, HEK cells, or HeLa cells.

[0031]

[0060] Polypeptides and peptides are polymers containing amino acids linked by peptide bonds. As used herein, the term “amino acid” is used to refer to any natural or otherwise amino acid that can be incorporated into a polypeptide or peptide. Amino acids are small molecules containing an amine (-NH2) group, a carboxyl (-COOH) group, and a variable side chain (R group) specific to each amino acid. Amino acids form polypeptides by covalent linkage via peptide bonds between the amine group of one amino acid and the carboxyl group of another. Amino acids within a polypeptide are often referred to as “residues” in the art.

[0032]

[0061] Polypeptides and peptides may include post-translational modifications such as phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, acylation, prenylation, alkylation, oxidation, or other modifications known in the art.

[0033]

[0062] Polypeptides and peptides may include amino acid analogs. As used herein, the term “amino acid analog” refers to artificial, synthetic, or unnatural amino acids beyond the 20 genetically encoded amino acids, such as the amino acid analogs described, for example, by Zou et al. (2018, Biotechnology Advances 36(7), 1917-1927). Examples of amino acid analogs that may be incorporated into the polypeptides, peptides, and conjugates of the present invention include, but are not limited to, norleucine (Nle), β-amino acids, homo-amino acids, synthetic proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids, and synthetic amino acids having an R group. Polypeptides and peptides may also include amino acid derivatives. As used herein, the term “amino acid derivative” refers to an amino acid derived from a modification of one of the 20 genetically encoded amino acids. Amino acid derivatives may be synthesized, for example, in vitro by a chemical reaction, or they may be naturally occurring in living organisms, for example, as in vivo metabolites. An example of an amino acid derivative is pyroglutamate / pyroglutamic acid, a cyclized derivative of glutamine in which the free amino group of glutamic acid is cyclized to form a lactam.

[0034]

[0063] The efficacy of the inhibitory molecule is determined by the "IC" obtained from the inhibitory efficacy assay. 50It may be measured and represented in the art from the perspective of " " value. For example, many inhibitory potency assays are known in the art, such as assays that measure competition with a labeled tracer molecule for binding to a target receptor, or assays that measure a decrease in signal transduction induced by a natural agonist. The IC 50 value of the present invention can be used for measurement. IC 50 is typically defined as the concentration of a drug at which 50% of the signal caused by a natural agonist through a receptor is inhibited by the drug. IC 50 is sometimes reported as "pIC 50 ", and pIC 50 is the negative logarithm of the IC 50 value in moles per liter (molarity or M).

[0035]

[0064] In some embodiments, the polypeptide, peptide, or conjugate of the present invention inhibits CCR2 with an IC 50 less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 30 nM, less than 10 nM, or less than 3 nM.

[0036]

[0065] In some embodiments, the polypeptide of the present invention is related to CCL2 or derived from CCL2. In some embodiments, CCL2 is human CCL2 (SEQ ID NO: 1), mouse CCL2 (SEQ ID NO:73), a part of human CCL2 (SEQ ID NOs: 2-5 and 74-75), or a part of mouse CCL2 (SEQ ID NO: 71 or 72). The polypeptide may comprise the sequences of SEQ ID NOs: 2, 3, 4, 5, 71, 72, 74, or 75, or variants, homologs (orthologs, allelic variants, derivatives, functional variants), or fragments thereof.

[0037]

[0066] According to the present invention, a sequence is said to be similar to or homologous to sequence numbers 2, 3, 4, 5, 71, 72, 74, or 75 if the sequence is more than 70% identical to sequence numbers 2, 3, 4, 5, 71, 72, 74, or 75. In some embodiments, the sequence is a C-terminal portion having more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, or 99.9% sequence identity with sequence numbers 2, 3, 4, 5, 71, 72, 74, or 75. The terms “identity” or “similarity” refer to sequence similarity between two polypeptides. Identity can be determined by comparing each position of aligned sequences. The degree of identity between amino acid sequences is, for example, a function of the number of identical or matching amino acids at positions shared by the sequences across a given region. The optimal alignment of sequences for identity comparison can be performed using a variety of algorithms, as is well known in the art, including the ClustalW program, the local homology algorithm of Smith and Waterman, 1981, Adv.Appl.Math 2:482, the homology alignment algorithm of Needleman and Wunsch, 1970, J.Mol.Biol.48:443, the similarity search method of Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 85:2444, and computer implementations of these algorithms (such as GAP, BESTFIT, FASTA, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA). Sequence identity can also be determined using the BLAST algorithm (using the publicly available default settings) described by Altschul et al., 1990, J.Mol.Biol.215:403-410. For example, you can use the "BLAST 2 Sequences" tool available through the National Center for Biotechnology Information and select the "blastp" program with the following default settings: expected threshold 10; word size 3; matrix BLOSUM62; gap cost present 11; extension 1.In another embodiment, a person skilled in the art can easily and appropriately align any given sequence and infer sequence identity, similarity, and / or homology by mere visual inspection.

[0038]

[0067] A sequence is also said to be similar to or homologous to SEQ ID NOs. 2, 3, 4, 5, 71, 72, 74, or 75 if it contains one or more conserved substitutions to SEQ ID NOs. 2, 3, 4, 5, 71, 72, 74, or 75. A conserved substitution is a substitution in the sequence of a peptide or polypeptide that does not result in a significant loss of function, or only a slight loss of function. Such a loss of function due to one or more conservative substitutions may be considered insignificant if the loss is less than 20%, less than 15%, less than 10%, less than 6%, or less than 4% of the function of the polypeptide having an unsubstituted sequence. Conservative substitutions are often those in which an amino acid side chain is replaced by an amino acid side chain that is related to the substituted residue or has similar physicochemical properties. Such conservative substitutions can be made, for example, using one of the 20 native amino acids shown in Table 2, where amino acids in the same block in the middle column, preferably in the same row in the right column, may be substituted for each other. For example, conservative substitutions can be performed using amino acid analogs or amino acid derivatives, such as the substitution of methionine with norleucine (Nle).

[0039] [Table 2]

[0068] According to the present invention, a sequence is said to be similar to or homologous to SEQ ID NOs: 2, 3, 4, 5, 71, 72, 74, or 75 if more than 30% of the residues in the sequence are identical to or conservatively substituted for SEQ ID NOs: 2, 3, 4, 5, 71, 72, 74, or 75. In some embodiments, more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the sequence are identical to or conservatively substituted for SEQ ID NOs: 2, 3, 4, 5, 71, 72, 74, or 75.

[0040]

[0069] In some embodiments, the present invention provides polypeptides comprising the fragments of SEQ ID NOs: 2, 3, 4, 5, 71, 72, 74, or 75. The fragments should contain at least "n" consecutive amino acids from SEQ ID NOs: 2, 3, 4, 5, 71, 72, 74, or 75, where, depending on the particular sequence, "n" is five or more (e.g., 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 3'0, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 55, 56, or more than 57).

[0041]

[0070] In some embodiments, the present invention provides a polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 9-70 and 76-145, and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs. 2, 3, 4, 5, 71, 72, 74, or 75. In some embodiments, the present invention provides a peptide comprising an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 9-70 and 76-145. In some embodiments of the polypeptide or peptide described above, the substitutions for any one of SEQ ID NOs. 9-70 and 76-145 are either known in the art or are conserved amino acid substitutions as defined herein.

[0042]

[0071] Preparation of polypeptides, peptides, and conjugates

[0072] The polypeptides and peptides of the present invention, either isolated or included in conjugates, can be prepared in many ways using, for example, known techniques of protein chemistry (e.g., chemical peptide synthesis) or molecular biology (i.e., genetic engineering and fermentation—in general, biotechnology).

[0043]

[0073] The polypeptides, peptides, and conjugates of the present invention can be prepared using known techniques of protein chemistry, for example, as described by Gaertner et al. (PNAS, 2008, 105(46):17706~17711).

[0044]

[0074] The present invention provides a method for preparing polypeptides, peptides, and conjugates, which includes in vitro chemical synthesis. Polypeptides, peptides, and conjugates can be synthesized in part or in whole using chemical means. For example, solid-phase peptide synthesis, such as methods based on tBoc or Fmoc chemistry, can be used. Enzymatic synthesis can also be used in part or in whole.

[0045]

[0075] In addition, the polypeptides, peptides, and conjugates of the present invention may be prepared using genetic engineering. The peptides, polypeptides, and conjugates of the present invention may be produced by culturing a host cell containing a nucleic acid molecule expressing the peptide, polypeptide, or conjugate (or the amino acid portion of the conjugate) of the present invention under conditions that induce the expression of the peptide, polypeptide, or conjugate. In some embodiments, the host cell is a bacterial cell (e.g., Escherichia coli), a yeast cell (e.g., Saccharomyces cerevisiae), or a mammalian cell (e.g., a human cell, mouse cell, CHO cell, HEK cell, HeLa cell).

[0046]

[0076] Other methods of biological synthesis besides expression in host cells may be used. For example, the polypeptides, peptides, or conjugates (or amino acid portions of conjugates) of the present invention may be produced by translation from RNA in vitro. The polypeptides, peptides, or conjugates (or amino acid portions of conjugates) of the present invention may also be prepared, for example, by digesting longer polypeptides using a protease.

[0047]

[0077] Biological methods, including genetic engineering, fermentation, and expression, are generally limited to the production of L-amino acid-based polypeptides, but the manipulation of the translation mechanism (e.g., aminoacyl-tRNA molecules) in vivo or in vitro can be used to enable the introduction of D-amino acids (or other non-natural amino acids such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.). However, when D-amino acids are involved, chemical synthesis is preferred. The polypeptides, peptides, or conjugates of the present invention may have covalent modifications at the C-terminus and / or N-terminus.

[0048]

[0078] Pharmaceutical composition

[0079] The present invention provides a pharmaceutical composition comprising a polypeptide, peptide, conjugate, or nucleic acid or vector encoding the polypeptide, peptide, or conjugate according to the present invention, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer. The pharmaceutical composition of the present invention may be provided for use as a pharmaceutical. The pharmaceutical composition of the present invention may comprise any embodiment of the present invention, namely, a polypeptide, peptide, conjugate, or nucleic acid or vector encoding the polypeptide, peptide, or conjugate according to the present invention. The preparation of the pharmaceutical composition is well known to those skilled in the art.

[0049]

[0080] The pharmaceutical compositions of the present invention can be administered to a subject in a therapeutically effective dose. As used herein, “therapeutic dose” means the amount of composition or therapeutic agent that is effective in providing a therapeutic, prophylactic, or diagnostic benefit to a subject. In some embodiments, the therapeutically effective dose of a composition is the amount that can induce a clinical response in a subject in the treatment of a particular disease or disorder. Determining the therapeutically effective dose of a composition is well within the capabilities of those skilled in the art, particularly in light of the disclosures provided herein. The therapeutically effective dose may vary depending on various factors such as the subject’s condition, weight, sex, and age.

[0050]

[0081] In one embodiment, the pharmaceutical composition of the present invention may comprise more than one polypeptide, peptide, conjugate, or nucleic acid or vector encoding the polypeptide, peptide, or conjugate. In one embodiment, the pharmaceutical composition of the present invention may comprise (a) at least one polypeptide, peptide, conjugate, or nucleic acid or vector encoding the polypeptide, peptide, or conjugate; and (b) at least one second pharmaceutical or therapeutic agent.

[0051]

[0082] In one embodiment, the second pharmaceutical or therapeutic agent may be an anti-inflammatory agent, an immunosuppressant, an antibiotic, an antiviral agent, a small molecule drug, or an antibody. In some embodiments, the second pharmaceutical or therapeutic agent may be formulated, for example, for simultaneous or sequential administration, in combination with at least one polypeptide, peptide, conjugate, or nucleic acid or vector encoding the polypeptide, peptide, or conjugate, or in a separate pharmaceutical composition.

[0052]

[0083] The pharmaceutical compositions provided herein may be prepared in a variety of pharmaceutical dosage forms, such as immediate-release, controlled-release, sustained-release, or targeted drug delivery systems. Commonly used dosage forms include, for example, liquids and suspensions, (micro)emulsions, ointments, gels, creams, pastes, foams, suppositories, vaginal suppositories (ovules), implants, patches, liposomes, tablets, sugar-coated tablets, lozenges, soft or hard-shell capsules, amorphous or crystalline powders, boiling or effervescent tablets, aerosols, and lyophilized preparations. Depending on the route of administration used, special devices may be required for the application or administration of the dosage form, such as syringes and needles, inhalers, pumps, injection pens, applicators, special containers, or other administration devices, which may be implanted in the body. The pharmaceutical dosage forms provided herein may be produced by any of the methods known in the art, for example, by conventional mixing, sieving, dissolution, melting, granulation, sugar-coated tablet production, tableting, suspension, extrusion, spray drying, wet grinding, emulsification, (nano / micro)encapsulation, encapsulation, or freeze-drying processes.

[0053]

[0084] The pharmaceutical compositions provided herein may further comprise pharmaceutically acceptable carriers, excipients, and / or stabilizers (Remington: The Science and Practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. KE. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient in dose and concentration and include, for example, buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, Alternatively, it may include proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN (trademark), PLURONICS (trademark), or polyethylene glycol (PEG).

[0054]

[0085] The pharmaceutical dosage forms provided herein may be manufactured by any method known in the art, for example, by conventional mixing, sieving, dissolution, melting, granulation, sugar-coated tablet manufacturing, tableting, suspension, extrusion, spray drying, wet grinding, emulsification, (nano / micro)encapsulation, encapsulation, or freeze-drying processes.

[0055]

[0086] Method and Use

[0087] The polypeptides, peptides, conjugates, nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention, or pharmaceutical compositions, may be used to inhibit CCR2 signaling in cells. In some embodiments, the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type 2 alveolar cells, gastric mucinous cells, NK cells, hepatocytes, Hofbauer cells, spermatocytes, fibroblasts, myeloid suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells. In some embodiments, the cells are acute leukemia, acute lymphoblastic leukemia (ALL), B cells, T cells, or FAB These are CCR2-expressing cancer cells derived from cancers such as ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and hairy cell leukemia; myelodysplastic syndrome (MDS); lymphomas such as Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma; multiple myeloma; Kaposi's sarcoma; colorectal cancers such as colon cancer; pancreatic cancers such as pancreatic cancer; renal cell carcinoma; breast cancer; prostate cancer; cervical cancer; ovarian cancer; liver cancer; kidney cancer; stomach cancer; bladder cancer; tongue cancer; esophageal cancer; nasopharyngeal cancer; malignant histiocytosis; paraneoplastic syndromes / hypercalcemia associated with malignant tumors; solid tumors; adenocarcinomas such as lung adenocarcinoma; squamous cell carcinomas such as squamous cell carcinomas and basal squamous cell carcinomas; sarcomas such as osteosarcoma; malignant melanoma; melanoma; thyroid cancer; and cystic carcinomas such as salivary gland-like carcinoma, glioma, or hemangioma. In some embodiments, cancer cells are cancerous dendritic cells, monocytes, plasma cells, macrophages (e.g., Kupffer cells or Langerhans cells), T cells, B cells, NK cells, myeloid suppressor cells, or neutrophils. "Cancerous" cells mean that the cells originate from a normal cell lineage and contain one or more genetic abnormalities that result in transformation into cancer cells.

[0056]

[0088] The polypeptides, peptides, conjugates, nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention, or pharmaceutical compositions, may be used to treat or prevent diseases or disorders in a subject, including lung diseases, cancer, inflammatory or immune-related diseases, cardiovascular diseases, neurological diseases, fibrotic conditions, wounds or tissue injuries, or infectious diseases. In some embodiments, the subject is a human subject.

[0057]

[0089] In one embodiment, lung diseases include pneumonia; lung abscess; occupational lung disease caused by dust, gas, or mist; asthma; fibrous bronchiolitis obstructing bronchitis; respiratory failure; hypersensitivity pneumonitis (exogenous allergic alveolitis), allergic bronchopulmonary aspergillosis, and hypersensitivity disorders of the lung including drug reactions; adult respiratory distress syndrome (ARDS); Goodpasture syndrome; chronic obstructive airway disorder; and chronic obstructive pulmonary disease. ; Idiopathic interstitial lung diseases such as idiopathic pulmonary fibrosis and sarcoidosis; desquamative interstitial pneumonia; acute interstitial pneumonia; interstitial lung disease with respiratory bronchiolitis; idiopathic obstructive bronchiolitis with organic pneumonia; lymphocytic interstitial pneumonia; Langerhans cell granulomatosis; idiopathic pulmonary hemosiderin deposition; acute bronchitis; alveolars; proteinosis; bronchiectasis; pleural disorders; atelectasis; cystic fibrosis; or pulmonary embolism.

[0058]

[0090] In one embodiment, cancer is acute leukemia, acute lymphoblastic leukemia (ALL), B cells, T cells, or FAB Leukemias such as ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and hairy cell leukemia; myelodysplastic syndrome (MDS); lymphomas such as Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma; multiple myeloma; Kaposi's sarcoma; colorectal cancers such as colon cancer; pancreatic cancers such as pancreatic cancer; renal cell carcinoma; breast cancer; prostate cancer; cervical cancer; ovarian cancer; liver cancer; kidney cancer; stomach cancer; bladder cancer; tongue cancer; esophageal cancer; nasopharyngeal cancer; malignant histiocytosis; paraneoplastic syndromes / hypercalcemia associated with malignant tumors; solid tumors; adenocarcinomas such as lung adenocarcinoma; squamous cell carcinomas such as squamous cell carcinoma and basal squamous cell carcinoma; sarcomas such as osteosarcoma; malignant melanoma; melanoma; thyroid cancer; salivary gland-like cystic carcinoma, glioma, or hemangioma. In one embodiment, the polypeptides, peptides, or conjugates described herein are used to inhibit CCR2 signaling in cancer cells, where the cancer cells are, for example, cancer cells such as those described above.

[0059]

[0091] In one embodiment, inflammatory diseases or immune-related diseases include: rheumatoid arthritis; psoriatic arthritis; ankylosing spondylitis; gastric ulcer; seronegative arthropathy; osteoarthritis; inflammatory bowel disease; ulcerative colitis; systemic lupus erythematosus; antiphospholipid syndrome; iridocyclitis; uveitis; optic neuritis; idiopathic pulmonary fibrosis; systemic vasculitis / Wegener's granulomatosis; sarcoidosis; orchitis; allergic atopic diseases; asthma; allergic rhinitis; allergic conjunctivitis; eczema; dermatitis; allergic conjunctivitis; hypersensitivity pneumonitis; organ transplant rejection; graft-versus-host disease; systemic inflammatory response syndrome; septic syndrome; gram-positive bacterial sepsis; gram-negative bacterial sepsis; culture-negative sepsis; fungal sepsis; neutropenic fever; urinary tract sepsis; meningococcal bacteremia; acute pancreatitis; adult respiratory distress syndrome; chronic inflammatory conditions; sarcoidosis Dosis; Crohn's disease; Diabetes; Nephrotic syndrome; Diabetic nephropathy; Diabetic retinopathy; Diabetic retinitis; Diabetic microangiopathy; Atopic diseases; Atopic dermatitis; Hypersensitivity reactions; Hay fever; Perennial rhinitis; Conjunctivitis; Endometriosis; Urticaria; Systemic anaphylaxis; Hemolytic diseases; Graves' disease; Raynaud's disease; Myasthenia gravis; Antibody-mediated cytotoxicity; Type IU hypersensitivity reactions; Polyneuropathy; Endocrine disorders; Monoclonal immunoglobulinemia; Skin change syndromes; Antiphospholipid syndrome; Pemphigus; Scleroderma; Mixed connective tissue disease; Idiopathic Addison's disease; Chronic active hepatitis; Non-alcoholic fatty liver disease; Hepatic fibrosis; Cirrhosis; Non-alcoholic fatty liver disease; Primary biliary cirrhosis; Primary sclerosing cholangitis; Vitiligo; Vasculitis; Gingivitis; Periodontitis; Periodontal disease; MI Post-cardiotomy syndrome (post-MI) Cardiotomy syndrome; type IV hypersensitivity; hypersensitivity pneumonitis; granuloma due to intracellular organisms; drug sensitivity; Wilson's disease; hemochromatosis; thyroiditis such as Hashimoto's thyroiditis; primary biliary cirrhosis; encephalomyelitis; cachexia; cystic fibrosis; neonatal chronic lung disease; chronic obstructive pulmonary disease (COPD); familial hemophagocytic lymphohistiopathy; dermatological conditions such as psoriasis and alopecia; nephrotic syndrome; nephritis such as glomerulonephritis; acute renal failure; ophthalmic (opthalmatic) disorders; pre-eclampsia; or effects of therapies including OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, and radiotherapy.

[0060]

[0092] In one embodiment, cardiovascular diseases include cardiac stun syndrome; myocardial infarction; congestive heart failure; stroke; ischemic stroke; hemorrhage; atherosclerosis; restenosis; vascular stenosis; diabetic atherosclerosis; hypertension; arterial hypertension; renovascular hypertension; syncope; shock; cardiovascular syphilis; heart failure; cor pulmonale; primary pulmonary hypertension; cardiac arrhythmia; ectopic atrial beats; atrial flutter; atrial fibrillation (persistent or paroxysmal); post-perfusion syndrome; cardiopulmonary bypass inflammatory response; disorganized atrial tachycardia or multifocal atrial tachycardia; regular narrow QRS tachycardia; ventricular fibrillation; and His bundle arrhythmia. Arrythmias); atrioventricular block; bundle branch block; myocardial ischemia; myocarditis; coronary artery disease; angina pectoris; cardiomyopathy such as dilated congestive cardiomyopathy, restrictive cardiomyopathy, and diastolic cardiomyopathy; valvular heart disease; endocarditis; pericardial disease; aortic aneurysm and peripheral artery aneurysm; aortitis; occlusion of the abdominal aorta and its branches; peripheral vascular disease; arterial occlusive disorder; peripheral atherosclerosis; thromboangiitis obliterans; functional peripheral artery disorder; acrocyanosis; erythromelopathy; venous disease; venous thrombosis; varicose veins; arteriovenous fistula; lymphedema; fatty edema; unstable angina pectoris; reperfusion injury; post-pump syndrome; ischemia-reperfusion injury; or reperfusion injury.

[0061]

[0093] In one embodiment, neurological disorders include: inflammatory pain; chronic pain; neuropathic pain such as lower back pain, hip pain, and leg pain; neuralgia such as post-traumatic neuralgia and post-herpetic neuralgia; diabetic neuropathy; neuropathic pain; acquired immunodeficiency syndrome (AIDS)-related neuropathic pain; nerve damage caused by toxins and chemotherapy; phantom limb pain; nerve root avulsion injury; painful traumatic mononeuropathy; painful polyneuropathy; thalamic pain syndrome; post-stroke pain; central nervous system injury; postoperative pain; carpal tunnel syndrome; trigeminal neuralgia; post-mastectomy syndrome; post-thoracotomy syndrome; stump pain; repetitive pain; neuropathic pain associated with hyperalgesia and allodynia; neurodegenerative diseases; migraines; demyelinating diseases such as multiple sclerosis and acute transverse myelitis; extrapyramidal disorders and cerebellar disorders such as lesions of the corticospinal system; basal ganglia disorders or cerebellar disorders; Huntington's dance. Hyperkinetic disorders such as chorea and senile chorea; drug-induced motor disorders such as those induced by drugs that block CNS dopamine receptors; hypokinetic disorders such as Parkinson's disease; progressive supranuclear palsy; structural lesions of the cerebellum; spinocerebellar degeneration such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degeneration, and multiple system degeneration (Mencel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph); motor unit disorders such as neurogenic muscular atrophy (anterior horn cell degeneration such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy); Alzheimer's disease; diffuse Lewy body disease; Lewy body senile dementia; Wernicke-Korsakoff syndrome; Creutzfeldt-Jakob disease; subacute sclerosing panencephalitis; Haller-Holden-Spats disease; or Boxer's dementia.

[0062]

[0094] In one embodiment, the fibrous condition includes hepatic fibrosis; virus-induced cirrhosis; autoimmune-induced hepatitis; pulmonary fibrosis such as idiopathic pulmonary fibrosis; renal fibrosis such as scleroderma, diabetic nephritis, glomerulonephritis, and lupus nephritis; cutaneous fibrosis such as scleroderma, hypertrophic and keloid scars, and burns; myelofibrosis; neurofibromatosis; fibroma; intestinal fibrosis; and fibrous adhesions resulting from surgical procedures.

[0063]

[0095] In one embodiment, the wound or tissue injury is a bodily injury or trauma associated with surgical procedures, including chest, abdominal, skull, or oral surgery; an acute traumatic wound; an ischemic ulcer; a pressure ulcer; a fistula; a burn; a donor site wound; or an aphthous wound.

[0064]

[0096] In one embodiment, infectious diseases include bacterial infections; parasitic infections; fungal infections; HIV infection; meningitis; hepatitis A, B, or C; septic arthritis; peritonitis; pneumonia; epiglottitis; malaria; dengue hemorrhagic fever; leishmaniasis; leprosy; toxic shock syndrome; streptococcal myositis; mycobacterial infections; Pneumocystis carinii pneumonia; pelvic inflammatory disease; orchitis; epididymitis; Legionella; Lyme disease; influenza; Epstein-Barr virus; vital-associated hemophagocytic syndrome; or encephalitis.

[0065]

[0097] The present invention provides the use of the polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates, or pharmaceutical compositions for the treatment and / or prevention (prevention) of diseases or disorders that can be treated by modulating the activity of CCR2 or by modulating the activity of cells expressing CCR2. Furthermore, the conjugates of the present invention may also be used to deliver a portion to cells expressing CCR2. In some embodiments, the portion is an immunoglobulin domain, a chemokine domain, a toxin, a carrier protein, a polymer, a lipid, or a detectable marker.

[0066]

[0098] One or more of the polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention may be administered to a subject. If more than one is administered, they may be administered together (as a mixture, or substantially simultaneously but separately) or sequentially. They may be administered in combination with one or more other pharmaceutical or therapeutic agents not contained in the polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention. The polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention may then be administered together (as a mixture, or substantially simultaneously but separately) or sequentially with the one or more other pharmaceutical or therapeutic agents.

[0067]

[0099] "To treat" or "to cure," or "to prevent" or "to prevent," as used herein, refers to a method for obtaining a beneficial or desired outcome. Beneficial or desired outcomes include, but are not limited to, the reduction or improvement of one or more symptoms or conditions, a reduction in the severity of a disease, stabilization of the disease state, prevention of the onset of a disease, prevention of the spread of a disease, delay or slowing of the progression of a disease, suppression of a disease, delay or slowing of the onset of a disease, conferring protective immunity against disease-causing factors, and improvement or mitigation of a disease state. "To treat" or "to prevent" can also mean extending the patient's survival beyond the period predicted by non-treatment, and can also mean preventing the onset of a disease by temporarily inhibiting the progression of a disease or preventing infection in the subject.

[0068]

[0100] "To treat" can be distinguished from "to prevent." "To treat" is typically done to subjects who already have a disease or disability, or who are known to have already been exposed to an infectious agent, while "to prevent" is typically done to subjects who do not have a disease or disability, or who are not known to have been exposed to an infectious agent. As can be understood, there may be overlap between treatment and prevention. For example, "treating" a disease in a subject can simultaneously "prevent" the symptoms or progression of the disease.

[0069]

[0101] Method of administration

[0102] The polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention may be delivered directly or in a pharmaceutical composition comprising a carrier, excipient, and / or stabilizer, as is known in the art. The therapeutic method of the present invention comprises administering to a subject a therapeutically effective amount of the polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention.

[0070]

[0103] For the methods and uses of the present invention, the polypeptides, peptides, conjugates, nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention, or pharmaceutical compositions, may be administered to subjects by conventional methods, such as intravenous (as a bolus or by continuous infusion over time), intramuscular, transmucosal, intraperitoneal, intracerebral, subcutaneous, intra-articular, intra-articular bursa, subarachnoid, transnasal, oral, topical, or inhalation. Other suitable routes of administration may include intra-facial or peri-facial routes.

[0071]

[0104] For intravenous injection, the polypeptides, peptides, conjugates, nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention, or pharmaceutical compositions, may be formulated into aqueous solutions, if necessary, with physiologically compatible buffers to adjust the pH of the formulation, such as phosphate, histidine, or citrate, and isotonic agents such as sodium chloride or dextrose. For transmucosal or transnasal administration, semi-solid formulations, liquid formulations, or patches may be preferred, and may contain penetration enhancers. Such penetration enhancers are generally known in the art. For oral administration, the pharmaceutical compositions provided herein may be formulated into liquid or solid dosage forms, optionally as immediate or controlled / sustained-release formulations. Suitable dosage forms for oral ingestion by subjects include tablets, capsules, pills, sugar-coated tablets, hard and soft-shell capsules, liquid formulations, gel formulations, syrup formulations, slurries, suspensions, and emulsions.

[0072]

[0105] Solid oral dosage forms can be obtained using excipients, which may include inert diluents, fillers, disintegrants, binders (dry and wet), dissolution retarders, lubricants, flow promoters, anti-adhesives, cation exchange resins, wetting agents, antioxidants, preservatives, colorants, sweeteners, and flavorings. These excipients may be derived from synthetic or natural sources. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium / sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salts, sugars (i.e., dextrose, sucrose, lactose, etc.), talc, tragacanth gum, hydrogenated vegetable oils, and waxes. Ethanol and water may function as granulation aids. In certain cases, for example, tablet coatings using taste-blocking films, gastric acid-resistant films, or release-delaying films are desired. Natural and synthetic polymers are often used in combination with colorants, sugars, and organic solvents or water to coat tablets and obtain sugar-coated tablets. When capsules are preferred over tablets, the powder, suspension, or solution of those drugs can be delivered in suitable hard or soft-shell capsules.

[0073]

[0106] Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose. Corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin. Lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to slow absorption in the gastrointestinal tract.

[0074]

[0107] Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

[0075]

[0108] In some embodiments, the polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention, or pharmaceutical compositions, may be administered topically via the skin or mucous membranes, such as through skin patches, semi-solid or liquid formulations, e.g., gels, (micro)emulsions, ointments, liquids, (nano / micro)suspensions, or foams. The penetration of the active ingredient into the target skin or mucous membranes and underlying tissues may be regulated, for example, by using penetration enhancers; by appropriate selection and combination of lipophilic, hydrophilic, and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, and emulsifiers; by pH adjustment; and by the use of complexing agents.

[0076]

[0109] In some embodiments, the polypeptides, peptides, conjugates, or nucleic acids or vectors encoding the polypeptides, peptides, or conjugates of the present invention, or pharmaceutical compositions, may be administered by inhalation or nasally from a pressurized pack or sprayer, typically with the use of a propellant, such as methane and ethane, carbon dioxide, or a halogenated carbon derived from any other suitable gas. For topical aerosols, hydrocarbons such as butane, isobutene, and pentane are useful. In the case of pressurized aerosols, appropriate dose units may be determined by providing a valve for delivering a measured amount. For use in inhalers or inhalers, for example, gelatin capsules and cartridges may be formulated. These typically contain a mixed powder of the active ingredient and a suitable powder base such as lactose or starch.

[0077]

[0110] Compositions formulated for parenteral administration by injection are typically sterile and may be present in unit dosage forms, such as ampoules, syringes, injection pens, or multi-dose containers, the latter of which typically contain preservatives. Pharmaceutical compositions suitable for parenteral administration may take the form of suspensions, liquids, or emulsions in oily or aqueous vehicles and may contain buffers, isotonic agents, viscosity enhancers, surfactants, suspensions and dispersants, antioxidants, biocompatible polymers, chelating agents, and preservatives. Depending on the injection site, the vehicle may contain water, synthetic or vegetable oil, and / or organic cosolvents. In certain cases, parenteral formulations are reconstituted or diluted prior to administration, such as by using lyophilized products or concentrates. Formulations that provide controlled or sustained release of the activator may include injectable suspensions of nano / microparticles or nano / micro or non-microparticle crystals. Polymers such as poly(lactic acid), poly(glycolic acid), or copolymers thereof, in addition to others well known in the art, can function as controlled / sustained-release matrices. Other slow-release systems may exist in the form of implantable tablets and pumps requiring incision.

[0078]

[0111] Suitable carriers for intravenous injection are well known in the art and comprise an aqueous solution containing a base, such as sodium hydroxide, for forming an ionizing agent, and sucrose or sodium chloride as an isotonic agent. The aqueous solution may contain a buffer containing phosphoric acid or histidine. A cosolvent, such as polyethylene glycol, may be added. These aqueous systems are effective in dissolving drugs and have low toxicity upon systemic administration. The ratio of components in the solution system can be changed significantly without impairing solubility and toxicity properties. Furthermore, the composition of the components can also be changed. For example, low-toxicity surfactants such as polysorbates or poloxamers may be used, such as polyethylene glycol or other cosolvents, biocompatible polymers such as polyvinylpyrrolidone may be added, and other sugars and polyols may be substituted for dextrose.

[0079]

[0112] Specific embodiments of this disclosure include, but are not limited to, the following: 1. A polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 40-70 and 76-145, and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs. 71, 72, 74, or 75. 2. The polypeptide according to Embodiment 1, wherein the amino acid substitution is a conservative substitution. 3. The polypeptide according to Embodiment 1, wherein the N-terminal portion comprises one amino acid sequence from SEQ ID NOs. 40-70 and 76-145. 4. The polypeptide according to Embodiment 1, wherein the N-terminal portion comprises the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), where X is any amino acid. 5. The polypeptide according to Embodiment 4, wherein the N-terminal portion comprises the amino acid sequence FTNPTW[A or D or R or S or K or Q][P or A or T or G or S or Q or R or H or E][V or F or Q or G or H or L or Y][T or V or Q or S or A] (Sequence ID 147). 6. The polypeptide according to Embodiment 4, wherein the N-terminal portion comprises one of the amino acid sequences from SEQ ID NO: 40 and 76-99. 7. The polypeptide according to Embodiment 1, wherein the N-terminal portion comprises the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148), where X1 is methionine or norleucine and X2-X5 are any amino acids. 8. The polypeptide according to Embodiment 7, wherein the N-terminal portion comprises the amino acid sequence FPX1DGW[A or R or G or H or V or Q][P or S or G or E][V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149), where X1 is methionine or norleucine. 9. The polypeptide according to Embodiment 7, wherein the N-terminal portion comprises one amino acid sequence from SEQ ID NOs. 41 and 100-145. 10. A polypeptide according to any one of Embodiments 1 to 4, wherein the N-terminal portion consists of 8 to 11 amino acids. 11. A polypeptide according to any one of Embodiments 1 to 5, wherein the C-terminal portion comprises the amino acid sequence of SEQ ID NOs. 71, 72, 74, or 75. 12. The polypeptide according to any one of Embodiments 1 to 11, wherein the polypeptide inhibits CCR2 with an IC50 of less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 30 nM, less than 10 nM, or less than 3 nM. 13. A peptide containing an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of the sequence numbers 40-70 and 76-145. 14. The peptide according to Embodiment 13, wherein the amino acid substitution is a conservative substitution. 15. The peptide according to Embodiment 13, comprising any one amino acid sequence from SEQ ID NOs. 40-70 and 76-145. 16. The peptide according to Embodiment 13, comprising the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), where X is any amino acid. 17. The peptide according to Embodiment 16, comprising the amino acid sequence FTNPTW[A or D or R or S or K or Q][P or A or T or G or S or Q or R or H or E][V or F or Q or G or S or L or Y][T or V or Q or S or A] (Sequence ID 147). 18. The peptide according to Embodiment 16, comprising one amino acid sequence from SEQ ID NO: 40 and 76-99. 19. The peptide according to Embodiment 13, comprising the amino acid sequence FPX1DGWX2X3X4X5 (SEQ ID NO: 148), where X1 is methionine or norleucine and X2-X5 are any amino acids. 20. The peptide according to Embodiment 13, comprising the amino acid sequence FPX1DGW[A or R or G or H or V or Q][P or S or G or E][V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149), where X1 is methionine or norleucine. 21. The peptide according to Embodiment 13, comprising one amino acid sequence from SEQ ID NO: 41 and 100-145. 22. A nucleic acid molecule encoding a polypeptide according to any one of Embodiments 1 to 12 or a peptide according to any one of Embodiments 13 to 21. 23. A vector comprising the nucleic acid molecule described in Embodiment 22. 24. A host cell containing the nucleic acid molecule described in Embodiment 22 or the vector described in Embodiment 23. 25. A pharmaceutical composition comprising a polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 13 to 21, a nucleic acid molecule according to Embodiment 22, or a vector according to Embodiment 23, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer. 26. The pharmaceutical composition according to Embodiment 25, further comprising a therapeutic agent. 27. A polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 13 to 21, a nucleic acid molecule according to Embodiment 22, a vector according to Embodiment 23, or a pharmaceutical composition according to Embodiment 25 or 26 for use in inhibiting CCR2 signaling in cells. 28. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to Embodiment 27, wherein the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type 2 alveolar cells, gastric mucus-secreting cells, NK cells, hepatocytes, Hofbauer cells, spermatocytes, fibroblasts, myeloid-derived suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells. 29. A polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to Embodiment 27 or 28, wherein cells are present in vitro. 30. A polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to Embodiment 27 or 28, wherein cells are present within the subject. 31. A polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 12 to 21, a nucleic acid molecule according to Embodiment 22, a vector according to Embodiment 23, or a pharmaceutical composition according to Embodiment 25 or 26, for use in the treatment or prevention of diseases or disorders related to CCR2 signaling in a subject. 32. A polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition of Embodiment 31, wherein the disease or disorder is a lung disease, cancer, inflammatory disease or immune-related disorder, cardiovascular disease, neurological disorder, fibrous condition, wound or tissue injury, or infectious disease. 33. A method for inhibiting CCR2 signaling in cells, comprising the step of contacting cells with a polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 13 to 21, a nucleic acid molecule according to Embodiment 22, a vector according to Embodiment 23, or a pharmaceutical composition according to Embodiment 25 or 26. 34. The method according to Embodiment 33, wherein the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type II alveolar cells, gastric mucinous cells, NK cells, hepatocytes, Hofbauer cells, spermatids, fibroblasts, myeloid-derived suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells. 35. The method according to embodiment 34, wherein the cells are in vitro. 36. The method according to embodiment 34, wherein the cells are located within the target area. 37. A method for treating or preventing a disease or disorder related to CCR2 signaling in a subject, comprising the step of administering to the subject a polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 13 to 21, a nucleic acid molecule according to Embodiment 22, a vector according to Embodiment 23, or a pharmaceutical composition according to Embodiment 25 or 26. 38. The method according to Embodiment 37, wherein the disease or disorder is a lung disease, cancer, inflammatory disease or immune-related disorder, cardiovascular disease, neurological disorder, fibrous condition, wound or tissue injury, or infectious disease. 39. Use of a polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 13 to 21, a nucleic acid molecule according to Embodiment 22, a vector according to Embodiment 23, or a pharmaceutical composition according to Embodiment 25 or 26 for inhibiting CCR2 signaling in cells. 40. The use according to Embodiment 39, wherein the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type 2 alveolar cells, gastric mucinous cells, NK cells, hepatocytes, Hofbauer cells, spermatids, fibroblasts, myeloid-derived suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells. 41. The use described in Embodiment 40, wherein the cells are in vitro. 42. The use according to Embodiment 40, wherein the cells are within the target area. 43. Use of a polypeptide according to any one of Embodiments 1 to 12, a peptide according to any one of Embodiments 13 to 21, a nucleic acid molecule according to Embodiment 22, a vector according to Embodiment 23, or a pharmaceutical composition according to Embodiment 25 or 26 for treating or preventing a disease or disorder related to CCR2 signaling in a subject. 44. The use according to Embodiment 43, wherein the disease or disorder is a lung disease, cancer, inflammatory or immune-related disease, cardiovascular disease, neurological disease, fibrotic condition, wound or tissue injury, or infectious disease.

[0080]

[0113] When understanding the scope of this disclosure, the term “comprising,” and its derivatives, as used herein, are intended to be open-ended terms that identify the presence of described features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other undescribed features, elements, components, groups, integers, and / or steps. The foregoing also applies to similar terms such as “including,” “having,” and their derivatives. As used herein, the term “consisting of,” and its derivatives, are intended to be closed terms that identify the presence of described features, elements, components, groups, integers, and / or steps, but exclude the presence of other undescribed features, elements, components, groups, integers, and / or steps. As used herein, the term “essentially from,” is intended to identify the presence of described features, elements, components, groups, integers, and / or steps, and those that do not substantially affect the basic and novel properties of the features, elements, components, groups, integers, and / or steps.

[0081]

[0114] All publications and patents cited herein are incorporated by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the event of any conflict between the definitions of terms in this disclosure and the definitions in the cited publications or patents, the definitions provided in this disclosure shall be used to describe the invention.

[0082]

[0115] The present invention will now be described by non-limiting embodiments with reference to the attached drawings. [Examples]

[0083]

[0116] Examples

[0117] Materials and methods

[0118] The CCR2 inhibitory efficacy assay was determined using the calcium flux method.

[0084]

[0119] Ca 2+ Flux measurements were performed using an FDSS microcell apparatus (HAMAMATSU). On the day of the experiment, THP-1 cells were seeded at a rate of 20,000 cells / well in the wells of a 384-well plate with a black wall and transparent bottom.

[0085]

[0120] The synthesized test sample (100 mM) was diluted in PBS supplemented with 1% BSA and 25 mM HEPES to create a dilution series for dose-response experiments: a 12-point dose-response starting at 688 nM, with a 2.5-fold dilution interval for each treatment.

[0086]

[0121] THP-1 cells were loaded with a calcium-sensitive fluorescent dye (Screen Quest® Fluo-8 No Wash Calcium Assay Kit, AAT Bioquest) according to the manufacturer's instructions for use, and then either the diluted test sample or the vehicle alone was added first. After 5 minutes, the cells were stimulated with 100 nM CCL2.

[0087]

[0122] Fluorescence signals (ex. 490 nm, em. 525 nm) were recorded throughout the entire experiment.

[0088]

[0123] Example 1: Synthesis of Fmoc-Thr(tBu)-2-chlorotrityl resin

[0124] The synthesis of polypeptides and peptides according to the present invention, such as the exemplary polypeptide 1P2-CCL2 (Met64Nle OB-004), included the synthesis of Fmoc-Thr(tBu)-2-chlorotrityl resin.

[0089]

[0125] The synthesis of 1P2-CCL2(Met64Nle OB-004) is described in detail by example. 3 g of 2-chlorotrityl resin (2CT resin load: 1.08 mmol / g) was weighed into a 50 mL peptide synthesis frit syringe and swollen with 20 mL of DMF at room temperature (RT) for 30 minutes. Fmoc-Thr(tBu)-OH (0.15 mmol, 178.9 mg) and DIPEA (392 μL, 0.45 mmol, 5 eq relative to amino acids) were dissolved in 12 mL of DMF and added to the pre-swollen resin. The reaction mixture was stirred at 6 rpm for 1.5 hours at RT. The resin load was determined by quantifying Fmoc-Thr(tBu)-OH in the flow-through using UV absorbance (UV301 nm; ε7800 M-1 cm-1), and was quantified at 0.149 mmol / g. Subsequently, 1 mL of DIPEA (2 eq relative to the resin) and 1.5 mL of MeOH were sequentially added to the reaction mixture, and the reaction was rotated at 6 rpm for 10 minutes using RT to quench the unreacted trityl chloride groups in the resin. The resin was then washed three times with 12 mL of DMF and twice with 12 mL of MeOH, and lyophilized.

[0090]

[0126] Example 2: Synthesis and purification of 1P2-CCL2 (Met64Nle OB-004) core fragment (11-76)

[0127] The C-terminal fragment of human CCL2 [11-76HumanCCL2], in which the methionine residue at position 64 of SEQ ID NO: 1 was replaced with norleucine (Nle), was synthesized using Fmoc chemistry on an automated peptide synthesizer (Prelude®, Protein Technologies, Inc.) at a 100 μmol scale on Fmoc-Thr(tBu)-2CT-resin (0.667 g; 0.15 mmol / g load). Coupling was performed twice for each amino acid [4 equivalents] using HCTU (4 equivalents) and DIPEA (10 equivalents) in 4.5 mL of N,N'-dimethylformamide (DMF) with nitrogen purging for 45 minutes. The resin was washed once with 4 mL of DMF, and unreacted amino groups were capped by incubation with 5% acetic anhydride and 0.5 M DIPEA in 8 mL of DMF with nitrogen purging for 5 minutes. The resin was washed nine times with 4 mL of DMF. The Fmoc group was removed by incubation twice with 4 mL of 20% (v / v) piperidine in DMF while purging with nitrogen for 10 minutes. The resin was washed nine times with 4 mL of DMF. At each washing step, the resin was purged with nitrogen for 30 seconds. At the end of the synthesis, the resin was manually washed three times with 8 mL of methanol and air-dried for 1 hour before peptide cleavage.

[0091]

[0128] The peptide was cleaved from the resin, and the protecting group was removed under reducing conditions by incubation with shaking at room temperature (RT) for 4 hours in 30 mL of cleavage solution [86% trifluoroacetic acid (TFA), 5% H2O, 5% phenol, and 4% triisopropylsilane (TIS)]. The resin was removed by filtration, and the filtrate was partitioned into six Falcon containers (5 mL each). The peptide was precipitated with cold diethyl ether (45 mL), incubated overnight at -20°C, and pelletized by centrifugation at 4000 g for 20 minutes.

[0092]

[0129] Core fragment peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) system (Prep 1525, Waters) using a preparative C8 reverse-phase column (10-15 μm, 250 × 22 mm; Vydac 208TP, GRACE) with a flow rate of 15 mL / min and a linear gradient of 25-40% (v / v) solvent B [solvent A: H2O containing 0.1% (v / v) TFA; solvent B: 90% (v / v) acetonitrile (ACN) / H2O containing 0.1% (v / v) TFA]. The fractions containing the desired peptides were lyophilized.

[0093]

[0130] Example 3: Synthesis of 1P2-CCL2(Met64Nle OB-004) N-terminal fragments (1-10)

[0131] The N-terminal fragment of 1P2-CCL2(Met64Nle OB-004) was synthesized on Fmoc-Cys(Trt)-SEA-2CP resin [load 0.136 mmol / g; 14.7 mg / well; scale 2 μmol; SEA = bis(2-sulfanylethyl)amino] using standard Fmoc chemistry in an Intavis MultiPep RSi parallel peptide synthesizer. Coupling was performed twice for each amino acid using HCTU and DIPEA (10 equivalents) in 59 μL of N,N'-dimethylformamide (DMF) without shaking for 30 minutes. Unreacted amino groups were capped by incubation with 5% acetic anhydride and 6% 2,6-lutidine in 50 μL of DMF for 10 minutes. The resin was washed six times with 150 μL of DMF, and the Fmoc groups were removed by incubation twice for 10 minutes with 50 μL of 20% (v / v) piperidine in DMF. The resin was washed ten times with 150 μL of DMF. At the end of the synthesis, the resin was washed ten times with 150 μL of MeOH.

[0094]

[0132] The peptide was cleaved from the resin, and the protecting group was removed under reducing conditions by incubation in 0.4 mL of cleavage solution (86% TFA, 5% H2O, 5% phenol, and 4% TIS) at 400 rpm for 3 hours with shaking at RT. The peptide was then precipitated with cold diethyl ether (1 mL), incubated overnight at -20°C, and pelletized by centrifugation at 1700 g for 30 minutes. This process was repeated three times to remove cleavage impurities, and the pellet was then air-dried at RT for 1 hour. The pellet was then dissolved in 1 mL of a 70% ACN / H2O (0.1% TFA) solution, and the resin was removed by filtration. The recovered peptide solution was freeze-dried, and the mass of the product was confirmed by MALDI-TOF / TOF using a DHB (2,5-dihydroxybenzoic acid) matrix in conjunction with linear positive ionization.

[0095]

[0133] Example 4: Fragment assembly and purification of the final product

[0134] 4.0 mg of the C-terminal core fragment [0.47 μmol] was added to 18 equivalents (8 μmol) of the N-terminal SEA-peptide fragment in 470 μL of ligation buffer (final concentration of core fragment = approximately 1 mM), and the reaction mixture was incubated overnight at 37°C with stirring [Ligation buffer: 0.2 M sodium phosphate buffer containing 6 M guanidine hydrochloride (Gn.HCl), 0.1 M TCEP.HCl, 0.1 M MPAA, and 50 mM methionine, pH 7.5]. The completion of the reaction was monitored by RP-HPLC. After the completion of the reaction, the reaction mixture was treated with 240 μL of 0.28 M TCEP.HCl solution (6 M Gn.HCl, pH 5.5) at RT for 30 minutes, and the crude product was then purified by preparative HPLC using a C8 column [Gradient method: 10-50% solvent B for 80 minutes and flow rate: 15 mL / min]. The pure fraction was recovered and freeze-dried. The ligation yield was 1.4 mg.

[0096]

[0135] For folding, the purified linear peptide was dissolved in a folding buffer at a concentration of 0.2 mg / mL, and the solution was allowed to stand at RT for 2-3 days with stirring (folding buffer: 2M Gn.HCl, 0.1M Tris, 10mM methionine, 0.5mM GSH, 0.3mM oxidized GSH, pH 8.0). The reaction was monitored by RP-HPLC. After the reaction was complete, the reaction mixture was acidified with 1 mL of 33% acetic acid solution, and the crude product was purified by HPLC using a semi-preparative C8 column [gradient method: 10-50% solvent B for 40 minutes and flow rate: 4 mL / min]. The pure fraction was collected and lyophilized.

[0097]

[0136] After final certification by analytical RP-HPLC (Figure 1) and mass spectrometry (Figure 2, MALDI-TOF / TOF m / z average calculated mass [M+H]+8772.15 and average measured mass [M+H]+8771.22), lyophilized 1P2-CCL2 (Met64Nle OB-004) was dissolved in H2O to obtain a final concentration of 100 μM.

[0098]

[0137] Example 5: CCR2 Inhibitory Efficacy Assay

[0138] Synthetic samples of 1P2-CCL2(Met64Nle OB-004) were tested in a CCR2 inhibitory efficacy assay using the calcium flux method described above. Figure 3 shows the dose-inhibition curves obtained for 1P2-CCL2(Met64Nle OB-004) in four independent experiments. Data points represent the mean fluorescence signal ± sem (n=3). The fitted IC was obtained across the four experiments. 50 The values ​​ranged from 0.9 to 3.2 nM.

[0099]

[0139] Example 6: Comparative CCR2 inhibitory efficacy assay

[0140] Figures 4 and 5 show the results of the CCR2 inhibitory efficacy assay using the calcium flux method described above, testing four known small molecule CCR2 inhibitors against the exemplary CCL2 variant polypeptide 1P2-CCL2(OB-004). Figure 4 shows the dose-inhibition curve from a representative experiment, and Figure 5 shows the mean pIC2 determined from the dose-inhibition curves obtained in four independent experiments. 50 Show the value.

[0100]

[0141] Figure 4 shows the inhibition profiles of four known small molecule CCR2 inhibitors and the exemplary CCL2 variant polypeptide 1P2-CCL2(Met64 Nle OB-004). The four known small molecule CCR2 inhibitors were AZD2423; BMS-813160; CCX-140; and senicliviroc.

[0101]

[0142] Figure 5 shows the average pIC for each of the five inhibitors. 50 The following is aggregated data from four experiments demonstrating the effects of 1P2-CCL2 (1.7 nM). The aggregated data shows that the most potent inhibitor was 1P2-CCL2 (1.7 nM). AZD2423 (6.0 nM) was 3.53 times less potent than 1P2-CCL2. BMS-813160 (9.8 nM) was 5.76 times less potent than 1P2-CCL2. CCX-140 (24.0 nM) was 14.12 times less potent than 1P2-CCL2. Cenicliviroc (62.0 nM) was 36.47 times less potent than 1P2-CCL2.

[0102]

[0143] The results of four experiments consistently and significantly demonstrate that 1P2-CCR2 (Met64Nle OB-004), an exemplary CCL2 variant polypeptide of the present invention containing the N-terminal peptide FTNPTWAPVT (SEQ ID NO: 40), is more potent than known small molecule CCR2 inhibitors tested.

[0103]

[0144] Example 7: Capture and release assay

[0145] Using a capture / release assay with human uterine vascular endothelial cells (HUVECS), we obtained data on monocyte capture, adhesion, and release using a HUVEC model.

[0104]

[0146] In the assay, HUVECS cells are introduced into a chamber slide and grown there to confluence. The HUVECS cells are then stimulated with TNF-alpha for 4 or 18 hours. The HUVEC monolayer is washed with cell culture medium and pre-incubated with the experimental compound for 20 minutes. Donor blood is collected and monocytes are purified. The purified monocyte suspension is delivered throughout the HUVEC monolayer using a calibrated pump. Monocyte capture and adhesion are recorded (for 6 minutes) using a camera and microscope operably connected to image analysis software (not shown). Some monocytes rotate throughout the HUVEC monolayer before adhering, while others do not adhere and are removed from the chamber slide. Cell culture wash medium is circulated through the chamber slide. Monocyte capture and migration are recorded at 10, 15, 20, 25, 30, 35, 40, and 60 minutes. Some monocytes adhere to the HUVEC monolayer, others partially detach from it, and still others completely detach. Monocyte capture and / or detachment are mediated by CCL2-CCR2 interactions between monocytes and HUVECs. Decreased capture and / or detachment indicates increased CCR2 inhibition.

[0105]

[0147] The test parameters for the results obtained and shown in Figures 6-18 were obtained using a blank as a negative control; 1P2-CCL2(Met64Nle OB-004) (1 μM) as the experimental compound; and GT-73 (30 μM) as a positive control. GT-73 blocks transendothelial migration of leukocytes by interfering with the function of the cell adhesion molecule PECAM-1(CD31).

[0106]

[0148] The stages of monocyte adhesion and migration are as follows: Floating monocytes adhere to the HUVEC monolayer. Some monocytes rotate before adhesion and then position themselves at cell-cell contact points. Some monocytes then begin to migrate between cell-cell contact points within the monolayer and eventually migrate completely to the opposite side of the HUVEC monolayer. Two HUVEC stimulation modes were tested: a short-term stimulation period of 4 hours with TNF-alpha; and a long-term stimulation period of 18 hours with TNF-alpha. Three stages of monocyte transport were measured: capture is shown as (1); migration % is shown as (2); and migration is shown as (3).

[0107]

[0149] Figure 6 shows the capture percentage and number of donor 1-derived monocytes captured by acutely activated HUVECS after 4 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73, but 1P2-CCL2 Met64Nle(OB-004) caused both the capture percentage and blockade of monocyte release.

[0108]

[0150] Figure 7 shows the capture percentage and number of donor 1-derived monocytes captured by acutely activated HUVECS after 18 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2 Met64Nle(OB-004) and GT-73, but 1P2-CCL2(Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0109]

[0151] Figure 8 shows the capture percentage and number of monocytes from donor 2 captured by acutely activated HUVECS after 4 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73, but 1P2-CCL2 (Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0110]

[0152] Figure 9 shows the capture percentage and number of monocytes from donor 2 captured by acutely activated HUVECS after 18 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73, but 1P2-CCL2 (Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0111]

[0153] Figure 10 shows the capture percentage and number of monocytes from donor 3 captured by acutely activated HUVECS after 4 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73, but 1P2-CCL2 (Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0112]

[0154] Figure 11 shows the capture percentage and number of monocytes from donor 3 captured in acutely activated HUVECS after 18 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73, but 1P2-CCL2 (Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0113]

[0155] Figure 13 shows the capture percentage and number of monocytes from donor 4 captured by acutely activated HUVECS after 4 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73, but 1P2-CCL2(Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0114]

[0156] Figure 13 shows the capture percentage and number of monocytes from donor 4 captured by acutely activated HUVECS after 18 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. Monocytes were captured in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73, but 1P2-CCL2 (Met64Nle OB-004) caused both the capture percentage and blockade of monocyte release.

[0115]

[0157] Figure 14 shows an overview of the AUC of the data shown in Figures 6-13 for donors 1-4. Blockade of transmission is clearly demonstrated in the presence of 1P2-CCL2 (Met64Nle OB-004).

[0116]

[0158] Figure 15 shows the number of monocytes from donors 1-4 captured and released by acutely activated HUVECS after 4 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73. The results are presented as a time-series paired analysis. Monocytes were captured in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73, but 1P2-CCL2(Met64Nle OB-004) caused blockade of monocyte release.

[0117]

[0159] Figure 16 shows the number of monocytes from donors 1-4 captured and released by acutely activated HUVECS after 18 hours of stimulation with 1000 U of TNF-alpha in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73. The results are presented as a time-series paired analysis. Monocytes were captured in the presence of 1P2-CCL2 (Met64Nle OB-004) and GT-73, but 1P2-CCL2 (Met64Nle OB-004) caused blockade of release.

[0118]

[0160] Figure 17 shows the number of monocytes from donors 1-4 captured and released by acutely activated HUVECS after 4 and 18 hours of stimulation with 1000U of TNF-alpha in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73. The results are presented as a paired analysis of AUC values. Monocytes were captured in the presence of 1P2-CCL2(Met64Nle OB-004) and GT-73, but 1P2-CCL2(Met64Nle OB-004) caused blockade of release.

[0119]

[0161] Figure 18 shows that 1P2-CCL2(1P2-CCL2(Met64Nle OB-004) strongly blocks monocyte migration in HUVECS activated with TNF-alpha for 18 hours. The results are presented as both time course and AUC. Blockade by 1P2-CCL2(1P2-CCL2(Met64Nle OB-004)) was highly effective, at approximately 99% at 1 μM.

[0120]

[0162] When provided as the N-terminal portion of a CCL2 variant polypeptide according to the present invention, numerous peptides containing 1P2 were synthesized and identified by the above method as constituting CCR2 inhibitory activity. The identified peptides were identified by measuring the IC of CCR2 inhibition for each peptide. 50 This is shown and detailed in Table 3 below.

[0163]

[0121] [Table 3] TIFF2026515249000003.tif203149 TIFF2026515249000004.tif178149

[0164] Inappropriate recruitment of CCR2-expressing immune cells underlies inflammatory conditions in many diseases and disorders. Furthermore, CCR2-expressing monocytes recruited to the tumor microenvironment (TME) can be immunosuppressive. The CCR2 inhibitors of the present invention can be used to inhibit or block the inappropriate recruitment of inflammatory or immunosuppressive CCR2-expressing cells to disease sites. For example, disease mitigation can be achieved by blocking the recruitment of inflammatory monocytes to diseased tissue using the CCR2 inhibitors of the present invention. Indeed, the migration and recruitment data presented herein demonstrate that the CCR2 inhibitors of the present invention possess extremely potent CCR2 blocking ability, thereby modifying CCR2-mediated cellular behavior.

[0122]

[0165] Example 8: Chimeric inhibitors containing mouse CCL2

[0166] In addition to the CCL2 variant polypeptides described in Examples 1-8, which include the C-terminal portion derived from human CCL2, chimeric CCL2 variants containing the C-terminal portion derived from mouse CCL2 were tested for CCL2 inhibitory activity.

[0123]

[0167] CCL2 variant polypeptides comprising an N-terminal portion derived from either 1P2 (FTNPTWAPVT, SEQ ID NO: 40) or 1P8 (AFSIMQAPVT, SEQ ID NO: 46) and a C-terminal portion derived from mouse CCL2 (SEQ ID NO: 72) were prepared according to the general methods of Examples 1-4. Chimeric CCL2 variants 1P2 and 1P8 were tested for CCR2 inhibition in vitro as described herein, except that mouse CCR2-expressing HEK cells were used to test the inhibition of mouse CCR2 activation by the agonist mouse CCL2 (mCCL2, which includes the substitution of methionine with norleucine to avoid oxidation during synthesis). As shown in Figure 19, chimeric 1P2 showed an IC50 of 2700 nM. 50 This shows that the Chimera 1P8 is an IC with a minimum impedance of 7.6 nM. 50 This was shown.

[0124]

[0168] The chimeric CCL2 variant 1P8 was further tested in an in vivo peritonitis model using male C57Bl / 6 mice. Mice were treated with either a control vehicle (physiological saline) or chimeric 1P8 containing the N-terminal portion of 1P8 AFSIMQAPVT (SEQ ID NO: 46) and the C-terminal portion derived from mouse CCL2 (SEQ ID NO: 72). Chimeric 1P8 was administered intraperitoneally in physiological saline at different doses (1 mg / kg, 10 mg / kg, 100 mg / kg) starting two days before induction of peritonitis (by intraperitoneal injection of 1 ml of 4% thioglycolate) and until euthanasia at three different time points after induction of peritonitis: 12 hours, 24 hours, and 48 hours (8 mice per dose and time point). After euthanasia, peritoneal lavage fluid was obtained by injecting 2 mL of PBS into the peritoneum, followed by aspirating the exudate and collecting immune cells. The peritoneal lavage fluid was centrifuged (450g, 5 minutes), and the cell pellet was retained for analysis by flow cytometry. The leukocyte subpopulation corresponding to the infiltrating monocytes / macrophages was then identified as Ly6G. - F4 / 80 + This was defined as follows. Dead cells and cellular debris were excluded from the analysis using a survival marker (Viobility®). In this way, the number of infiltrating monocytes / macrophages was estimated for each mouse at each dose and time point. As shown in Figure 20, administration of the chimeric CCL2 variant 1P8 effectively reduced or blocked monocyte recruitment into the peritoneal cavity in a dose-dependent manner.

[0125]

[0169] CCR2 continues to be actively investigated as a potential drug target for a wide range of diseases, from autoimmune diseases, diabetes, and chronic pain syndromes to atherosclerosis, HIV, and cancer (Fei et al., Front. Immunol., November 3, 2021). Therefore, the extremely potent CCR2 blocking ability demonstrated by the CCR2 inhibitors of the present invention identifies promising candidate molecules for the treatment of diseases or disorders related to CCR2 signaling, including post-traumatic neuralgia, neuropathic pain, inflammatory diseases, chronic obstructive pulmonary disease, diabetic polyneuropathy, cancer (including colorectal cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, liver cancer, and non-small cell lung cancer), diabetic nephropathy, diabetes, HIV infection, non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis, non-alcoholic fatty liver disease, or primary sclerosing cholangitis.

[0126]

[0170] Example 9: CCR2 Inhibitory Efficacy Assay

[0171] Further CCL2 variants were synthesized according to the methods described in Examples 1-4 and identified as constituting CCR2 inhibitory activity by the above methods. These further variants are based on the N-terminal portion 1P2 (SEQ ID NO: 40) or 2P6 (SEQ ID NO: 41), but have substitutions at positions 7-10 and are conjugated to the C-terminal fragment of human CCL2 [11-76 human CCL2], where the methionine residue at position 64 relative to SEQ ID NO: 1 is replaced with norleucine (Nle). The identified peptides were evaluated based on the IC50 of CCR2 inhibition measured for each peptide. 50 This is shown and detailed in Table 4 below.

[0172]

[0127] [Table 4] TIFF2026515249000006.tif205149 TIFF2026515249000007.tif37149

[0128]

[0173] Example 10: Evaluation of CCR2 inhibitors in bleomycin-induced pulmonary fibrosis in mice

[0174] Exemplary chimeric CCR2 inhibitors, including the N-terminal portion derived from 1P8 (AFSIMQAPVT, SEQ ID NO: 46) and the C-terminal portion derived from mouse CCL2 (SEQ ID NO: 72), as described in Example 8, were tested for their effects on the pathogenesis of bleomycin-induced pulmonary fibrosis in mouse models. Six-week-old male C567BL / 6J mice were procured from Charles River and used between 11 and 14 weeks of age at the start of the study.

[0129]

[0175] Bleomycin sulfate (MedChemExpress) was administered intratracheally at a dose of 1 U / kg (20 mg / mL per mouse, or 35 μL). Mice were administered a control vehicle or a CCR2 inhibitor (intraperitoneal injection, twice daily) on day 2 of the study, and then either the control or bleomycin was administered 5 days a week starting on day 0 of the study. Mice were euthanized for analysis on day 21 of the study.

[0130]

[0176] Group 1: Bleomycin control (fake).

[0131]

[0177] Group 2: Bleomycin + control vehicle.

[0132]

[0178] Group 3: Bleomycin + 100 mg / kg of CCR2 inhibitor.

[0133]

[0179] Group 4: Bleomycin + 33 mg / kg of CCR2 inhibitor.

[0134]

[0180] Mice were monitored for body weight throughout the study. As shown in Figure 21, mice treated with bleomycin lost a significant percentage of body weight compared to placebo mice. The weight loss due to bleomycin treatment was reduced in mice that were concurrently administered a CCR2 inhibitor.

[0135]

[0181] On day 21 of the study, lung infiltrating cells were analyzed by flow cytometry. As shown in Figure 22, combination therapy with a CCR2 inhibitor reduced the total number of infiltrating CD64+ macrophages (Figures 22A-B), the number of infiltrating CD64+Ly6C- / low SiglecF- stromal macrophages (Figures 22C-D), and the number of infiltrating monocyte-derived CD64+Ly6C+SiglecF- macrophages (Figures 22E-F).

[0136]

[0182] The foregoing description relating to specific embodiments of the present invention is provided for illustrative and explanatory purposes only. It is not intended to be exhaustive or to limit the invention and its uses to the exact forms disclosed. Many modifications and variations are possible in light of the above teachings. The described embodiments are selected and described to best illustrate the principles and practical applications of the invention, thereby enabling those skilled in the art to best utilize the invention and its various embodiments with various modifications to suit specific intended uses. Various omissions or substitutions of equivalents are contemplated, as may be suggested or advantageous in the context, but it is understood that these are intended to encompass the application or practice without departing from the spirit or scope of the claims of the invention.

Claims

1. A polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of SEQ ID NOs. 40-70 and 76-145, and the C-terminal portion comprises an amino acid sequence that is at least 70% identical to SEQ ID NOs. 71, 72, 74, or 75.

2. The polypeptide according to claim 1, wherein the amino acid substitution is a conservative substitution.

3. The polypeptide according to claim 1, wherein the N-terminal portion comprises one amino acid sequence from among SEQ ID NOs: 40-70 and 76-145.

4. The polypeptide according to claim 1, wherein the N-terminal portion comprises the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), where X is any amino acid.

5. The polypeptide according to claim 4, wherein the N-terminal portion comprises the amino acid sequence FTNPTW[A or D or R or S or K or Q][P or A or T or G or S or Q or R or H or E][V or F or Q or G or S or H or L or Y][T or V or Q or S or A] (SEQ ID NO: 147).

6. The polypeptide according to claim 4, wherein the N-terminal portion comprises one amino acid sequence from SEQ ID NO: 40 and 76-99.

7. The N-terminal portion is the amino acid sequence FPX １ DGWX ２ X ３ X ４ X ５ (Sequence number 148) is included, where X １ is methionine or norleucine, X ２ ~X ５ The polypeptide according to claim 1, wherein is any amino acid.

8. The N-terminal portion has the amino acid sequence FPX １ DGW[A or R or G or H or V or Q][P or S or G or E][V or R or L or E or T or G or Q][T or V or Q] (SEQ ID NO: 149), wherein X １ is methionine or norleucine, the polypeptide according to claim 7.

9. The polypeptide according to claim 7, wherein the N-terminal portion comprises one amino acid sequence from SEQ ID NO: 41 and 100 to 145.

10. The polypeptide according to any one of claims 1 to 4, wherein the N-terminal portion consists of 8 to 11 amino acids.

11. The polypeptide according to any one of claims 1 to 5, wherein the C-terminal portion comprises the amino acid sequence of SEQ ID NO: 71, 72, 74, or 75.

12. A polypeptide according to any one of claims 1 to 11, which inhibits CCR2 with an IC50 of less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 30 nM, less than 10 nM, or less than 3 nM.

13. A peptide comprising an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions for any one of the sequence numbers 40-70 and 76-145.

14. The peptide according to claim 13, wherein the amino acid substitution is a conservative substitution.

15. The peptide according to claim 13, comprising any one amino acid sequence from SEQ ID NOs: 40-70 and 76-145.

16. The peptide according to claim 13, comprising the amino acid sequence FTNPTWXXXX (SEQ ID NO: 146), where X is any amino acid.

17. The peptide according to claim 16, comprising the amino acid sequence FTNPTW [A or D or R or S or K or Q] [P or A or T or G or S or Q or R or H or E] [V or F or Q or G or S or H or L or Y] [T or V or Q or S or A] (SEQ ID NO: 147).

18. The peptide according to claim 16, comprising any one amino acid sequence from SEQ ID NOs. 40 and 76-99.

19. Amino acid sequence FPX １ DGWX ２ X ３ X ４ X ５ (Sequence number 148) is included, where X １ is methionine or norleucine, X ２ ~X ５ The peptide according to claim 13, wherein is any amino acid.

20. Amino acid sequence FPX １ DGW [A or R or G or H or V or Q] [P or S or G or E] [V or R or L or E or T or G or Q] [T or V or Q] (Sequence ID 149), where X １ The peptide according to claim 13, wherein is methionine or norleucine.

21. The peptide according to claim 13, comprising one amino acid sequence from SEQ ID NO: 41 and 100-145.

22. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 12 or a peptide according to any one of claims 13 to 21.

23. A vector comprising the nucleic acid molecule described in claim 22.

24. A host cell comprising the nucleic acid molecule described in claim 22 or the vector described in claim 23.

25. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 13 to 21, a nucleic acid molecule according to claim 22, or a vector according to claim 23, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

26. The pharmaceutical composition according to claim 25, further comprising a therapeutic agent.

27. A polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 13 to 21, a nucleic acid molecule according to claim 22, a vector according to claim 23, or a pharmaceutical composition according to claim 25 or 26, for use in inhibiting CCR2 signaling in cells.

28. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to claim 27, wherein the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type II alveolar cells, gastric mucus-secreting cells, NK cells, hepatocytes, Hofbauer cells, spermatocytes, fibroblasts, myeloid-derived suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells.

29. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to claim 27 or 28, wherein the cells are in vitro.

30. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to claim 27 or 28, wherein the aforementioned cells are located within the target.

31. A polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 12 to 21, a nucleic acid molecule according to claim 22, a vector according to claim 23, or a pharmaceutical composition according to claim 25 or 26, for use in the treatment or prevention of diseases or disorders related to CCR2 signaling in a subject.

32. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition according to claim 31, wherein the disease or disorder is a lung disease, cancer, inflammatory disease or immune-related disease, cardiovascular disease, neurological disease, fibrous condition, wound or tissue injury, or infectious disease.

33. A method for inhibiting CCR2 signaling in cells, comprising the step of contacting the cells with a polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 13 to 21, a nucleic acid molecule according to claim 22, a vector according to claim 23, or a pharmaceutical composition according to claim 25 or 26.

34. The method according to claim 33, wherein the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type II alveolar cells, gastric mucinous cells, NK cells, hepatocytes, Hofbauer cells, spermatids, fibroblasts, myeloid-derived suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells.

35. The method according to claim 34, wherein the cells are in vitro.

36. The method according to claim 34, wherein the cells are located within the target area.

37. A method for treating or preventing a disease or disorder related to CCR2 signaling in a subject, comprising the step of administering to the subject a polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 13 to 21, a nucleic acid molecule according to claim 22, a vector according to claim 23, or a pharmaceutical composition according to claim 25 or 26.

38. The method according to claim 37, wherein the disease or disorder is a lung disease, cancer, an inflammatory disease or immune-related disease, a cardiovascular disease, a neurological disease, a fibrous condition, a wound or tissue injury, or an infectious disease.

39. Use of a polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 13 to 21, a nucleic acid molecule according to claim 22, a vector according to claim 23, or a pharmaceutical composition according to claim 25 or 26 for inhibiting CCR2 signaling in cells.

40. The use according to claim 39, wherein the cells are dendritic cells, monocytes, plasma cells, macrophages, Kupffer cells, Langerhans cells, T cells, B cells, erythrocytes, hepatic stellate cells, cholangiocarcinomas, type II alveolar cells, gastric mucin-secreting cells, NK cells, hepatocytes, Hofbauer cells, spermatids, fibroblasts, myeloid-derived suppressor cells, neutrophils, osteoclasts, stem cells, basal keratinocytes, cardiomyocytes, endothelial cells, mammary gland cells, mammary gland myoepithelial cells, glandular cells, luminal cells, theca cells, spermatogonia, cytotrophoblasts, smooth muscle cells, adipocytes, CCR2-expressing immortalized cells, or cancer cells.

41. The use according to claim 40, wherein the cells are in vitro.

42. The use according to claim 40, wherein the aforementioned cells are within the target area.

43. Use of a polypeptide according to any one of claims 1 to 12, a peptide according to any one of claims 13 to 21, a nucleic acid molecule according to claim 22, a vector according to claim 23, or a pharmaceutical composition according to claim 25 or 26 for treating or preventing a disease or disorder related to CCR2 signaling in a subject.

44. The use according to claim 43, wherein the disease or disorder is a lung disease, cancer, an inflammatory disease or immune-related disorder, a cardiovascular disease, a neurological disorder, a fibrous condition, a wound or tissue injury, or an infectious disease.

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