Anti-PAR-2 ​​antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human PAR-2, as well as compositions comprising such antibodies or antigen-binding fragments thereof. In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to human PAR-2 ​​block the interaction between a PAR-2 ​​activating ligand and the extracellular domain of PAR-2 ​​and / or block PAR-2 ​​activation by a PAR-2 ​​activating ligand. In further embodiments, the antibodies or antigen-binding fragments can be used to treat diseases or conditions associated with increased expression of PAR-2 ​​and / or diseases or conditions that can be alleviated by antagonizing the activation of PAR-2 ​​by a PAR-2 ​​activating ligand (e.g., airway diseases, skin diseases, cancer, orofacial granulomatosis, inflammatory conditions, and pain associated with various diseases or conditions).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit as of the filing date of U.S. Provisional Patent Application No. 63 / 067,259, filed on 18 August 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] References to electronically submitted sequence listings The contents of the electronically submitted ASCII array listing text file (name: 2873_302PC01_SL_ST25.txt; size: 190,325 bytes; and creation date: August 16, 2021) filed with this application are incorporated herein by reference in their entirety.

[0003] This disclosure relates to antibodies that specifically bind to human PAR-2 ​​and their antigen-binding fragments, compositions comprising such antibodies and their antigen-binding fragments, and methods for producing and using antibodies that specifically bind to human PAR-2 ​​and their antigen-binding fragments, for example, in the treatment of respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pulmonary arterial hypertension), cancer, skin diseases, orofacial granulomatosis, inflammatory conditions, or in the relief of pain associated with various diseases or conditions. [Background technology]

[0004] G protein-coupled receptors (GPCRs) are a family of seven-transmembrane proteins that respond to extracellular stimuli via G protein activation and subsequent secondary messengers. Proteinase-activated receptor 2 (PAR-2; GPR11; F2RL1) is a class A GPCR activated by protease cleavage. In humans, the N-terminal "anchoring ligand" (e.g., amino acid sequence SLIGKV in humans) is exposed by upstream proteolytic cleavage, allowing the anchoring ligand to bind to the receptor's extracellular loop and induce signal transduction. Hollenberg MD and Compton SJ, Drug Dev. Res., vol. 59(no. 4): pp. 344-349 (2003). The N-terminal sequence of PAR-2 ​​is found to be related to trypsin (Hollenberg MD and Compton SJ, Drug Dev. Res., Vol. 59 (No. 4): pp. 344-349 (2003)), tryptase (Akers, I. et al., Am J Physiol Lung Cell Mol Physiol., Vol. 278: L193-L201 (2000)), tissue factor (Larsen, KS et al., J Biol Chem, Vol. 285: pp. 19959-19966 (2010)), neutrophil elastase (Ramachandran, R. et al., J Biol Chem., Vol. 286: pp. 24638-24648 (2011)), and matryptase-1 (Milner, JM et al., Arthritis PAR-2 ​​can be cleaved at several different sites by various serine proteases, including Rheum (Vol. 62: pp. 1955-1966 (2010)), as well as by cysteine ​​proteases such as cathepsin S (Elmariah, SB et al., PLoS One, Vol. 9 (No. 6): e99702 (2014)), papain (Liang, G. et al., J Allergy Clin Immunol, Vol. 129: pp. 1377-1386 (2012)), and Der p 1 (Asosingh, K. et al., J Clin Invest, Vol. 128 (No. 7): pp. 3116-3128 (2018)). It has also been reported that PAR-2 ​​can be transactivated by PAR-1 anchoring ligands.O'Brien, PJ, J Biol Chem, vol. 275: pp. 13502-13509 (2000).

[0005] Under normal conditions, PAR-2 ​​is expressed at very low levels on the cell surface. PAR-2 ​​is expressed in endothelial cells, smooth muscle cells, epithelial cells, keratinocytes, and fibroblasts (D'Andrea MR. et al., J Histochem Cytochem., Vol. 46 (No. 2): pp. 157-164 (1998)), as well as monocyte-derived immune cells, most notably macrophages, and neutrophils (Howells, GL et al., Journal of Cell Science, Vol. 110: pp. 881-887 (1997)). PAR-2 ​​is upregulated under inflammatory conditions, and overexpression is associated with, for example, asthma (Knight, DA et al., J Allergy Clin Immunol, Vol. 108: pp. 797-803 (2001)), chronic obstructive pulmonary disease (COPD) (Lee, KH et al., Experimental & Molecular Medicine, Vol. 50 (No. 7): pp. 1-9 (2018)), pulmonary fibrosis (Wygrecka, M. et al., Am J Respir Crit Care Med, Vol. 183: pp. 1703-1714 (2011)), rheumatoid arthritis (Tindell, AG et al., Rheumatol Int, Vol. 32: pp. 3077-3086 (2012)), and osteoarthritis (Huesa, C. et al., Ann Rheum It is associated with several diseases, including Dis, Vol. 75: pp. 1989-1997 (2016)), pancreatitis (Namkung, W. et al., Gastroenterology Vol. 126: pp. 1844-1859 (2004)), chronic pain (Mrozkova, P. et al., Physiol Res, Vol. 65: pp. 357-367 (2016)), atopic dermatitis (Lee, SE et al., Yonsei Med J, Vol. 51: pp. 808-822 (2010)), and chronic pruritus (Akiyama, T. et al., Handb Exp Pharmacol, Vol. 226: pp. 219-235 (2015)).Mice lacking PAR-2 ​​have been shown to be resistant to the induction of experimental asthma (de Boer, JD et al., Innate Immun., Vol. 20 (No. 6): 618-625 (2013)), dermatitis (Kawagoe, J. et al., Jpn J Pharmacol, Vol. 88: pp. 77-84 (2002)), fibrosis (Borensztajn, K. et al., Am J Pathol Vol. 177, pp. 2753-2764 (2010)), glomerulonephritis (Moussa, L. et al., Am J Pathol Vol. 171: pp. 800-808 (2007)), and arthritis (Busso, N. et al., Arthritis Rheum, Vol. 56: pp. 101-107 (2007)). [Prior art documents] [Patent Documents]

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[0008] Antibodies that specifically bind to the human PAR-2 ​​protein can be used for the diagnosis, prevention, and / or treatment of diseases in which PAR-2 ​​is overexpressed. Therefore, there is a need to develop potent antibodies that can broadly antagonize the activation of human PAR-2 ​​and are suitable for human administration. [Means for solving the problem]

[0009] This specification provides isolated antibodies that specifically bind to human PAR-2, antigen-binding fragments thereof, and methods for using them.

[0010] In one embodiment, an isolated antibody or an antigen-binding fragment thereof is provided that specifically binds to human PAR-2 ​​and antagonizes the activation of PAR-2 ​​by a PAR-2 ​​activating ligand. Examples of such PAR-2 ​​activating ligands include, but are not limited to, PAR-2 ​​anchoring ligands (cis or trans), PAR-1 anchoring ligands, or soluble ligands.

[0011] In some embodiments, an isolated antibody or its antigen-binding fragment is provided that specifically binds to human PAR-2 ​​but does not bind to amino acids 59-63 at the N-terminus of human PAR-2, and (a) blocks the interaction between the PAR-2 ​​activating ligand and the extracellular domain of PAR-2, and / or (b) blocks PAR-2 ​​activation by the PAR-2 ​​activating ligand. In some embodiments, the antibody or its antigen-binding fragment does not bind to the N-terminus of human PAR-2. In certain embodiments, the antibody or its antigen-binding fragment is suitable for administration to human subjects. In certain embodiments, the antibody is a humanized antibody.

[0012] In some embodiments of these models, the antibody or its antigen-binding fragment is used to activate human PAR-2 ​​by a soluble PAR-2 ​​activating ligand, and the activation of PAR-2 ​​by the ligand is measured by a PAR-2β-arrestin cell assay, resulting in an IC50 of approximately 0.1 nM to approximately 17 nM. 50 It inhibits it.

[0013] The actual form of a part is, the antibody is, the antigen-binding fragment is, the interaction between soluble PAR-2 ​​activated ligand and PAR-2, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2 nM, 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2. 5 nM, approximately 2.6 nM, approximately 2.7 nM, approximately 2.8 nM, approximately 2.9 nM, approximately 3 nM, 3.1 nM, approximately 3.2 nM, approximately 3.3 nM, approximately 3.4 nM, approximately 3.5 nM, approximately 3.6 nM, approximately 3.7 nM, approximately 3.8 nM, approximately 3.9 nM, approximately 4 nM, 4.1 nM, approximately 4.2 nM, approximately 4.3 nM, approximately 4.4 nM, approximately 4.5 nM, approximately 4.6 nM, approximately 4.7 nM, approximately 4.8 nM, approximately 4.9 nM, approximately 5 nM, 5.1 nM, approximately 5.2 nM, approximately 5.3 nM, approximately 5.4 nM, approximately 5.5 nM, approximately 5.6 nM, approximately 5.7 nM, approximately 5.8 nM, approximately 5.9 nM, approximately 6 nM, 6.1 nM, approximately 6.2 nM, approximately 6.3 nM, approximately 6.4 nM, approximately 6.5 nM, approximately 6.6 nM, approximately 6.7 nM, approximately 6.8 nM, approximately 6.9 nM, approximately 7 nM, 7.1 nM, approximately 7.2 nM, approximately 7.3 nM, approximately 7.4 nM, approximately 7.5 nM, approximately 7.6 nM, approximately 7.7 nM, approximately 7.8 nM, approximately 7.9 nM, approximately 8 nM, 8.1 nM, approximately 8.2 nM, approximately 8.3 nM, approximately 8.4 nM, approximately 8.5 nM, approximately 8.6 nM, approximately 8.7 nM, approximately 8.8 nM, approximately 8.9 nM, approximately 9 nM, 9 .1nM, approximately 9.2nM, approximately 9.3nM, approximately 9.4nM, approximately 9.5nM, approximately 9.6nM, approximately 9.7nM, approximately 9.8nM, approximately 9.9nM, approximately 10nM, 10.1nM, approximately 10.2nM, approximately 10.3nM, approximately 10.4nM, approximately 10.5nM, approximately 10.6nM, approximately 10.7nM, approximately 10.8nM, approximately 10.9nM, approximately 11nM, approximately 11.1nM, approximately 11.2nM, approximately 11.3nM, approximately 11.4nM, approximately 11.5nM, approximately 11.6nM, approximately 11.7nM, approximately 11.8nM, and approximately 11.9nM of IC. 50 で hinder す る.

[0014] In some embodiments, the antibody or its antigen-binding fragment is used to measure intracellular PAR-2 ​​activating ligand-induced and trypsin-induced calcium flux by PAR-2 ​​calcium flux cell assay, resulting in an IC50 of approximately 6 nM to approximately 11 nM. 50 This inhibits the process. In some embodiments, the cells are human lung fibroblasts or epithelial cells.

[0015] In some embodiments, the antibody or its antigen-binding fragment provides PAR-2 ​​activated ligand-inducible and trypsin-inducible calcium flux in concentrations of approximately 6 nM, 6.1 nM, 6.2 nM, 6.3 nM, 6.4 nM, 6.5 nM, 6.6 nM, 6.7 nM, 6.8 nM, 6.9 nM, 7.1 nM, 7.2 nM, 7.3 nM, 7.4 nM, 7.5 nM, 7.6 nM, 7.7 nM, 7.8 nM, 7.9 nM, 8 nM, and 8.1 nM. M, about 8.2nM, about 8.3nM, about 8.4nM, about 8.5nM, about 8.6nM, about 8.7nM, about 8.8nM, about 8.9nM, about 9nM, 9.1nM, about 9.2nM, about 9.3nM, about 9.4nM, about 9.5nM, about 9.6nM , about 9.7nM, about 9.8nM, about 9.9nM, about 10nM, 10.1nM, about 10.2nM, about 10.3nM, about 10.4nM, about 10.5nM, about 10.6nM, about 10.7nM, about 10.8nM, or about 10.9nM IC 50 It inhibits it.

[0016] In some embodiments, the antibody or its antigen-binding fragment inhibits PAR-2 ​​activating ligand-induced smooth muscle cell contraction by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% compared to a control antibody of the same isotype. In some embodiments, the smooth muscle cells are bronchial smooth muscle cells.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof inhibits the induction of pulmonary neutrophilia in cynomolgus monkeys by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% compared to a control antibody of the same isotype. 。

[0018] In some aspects, an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAR-2, wherein the antibody or antigen-binding fragment thereof has the heavy chain complementarity determining region 1 (VH CDR1), VH CDR2, VH CDR3 sequences of SEQ ID NO: 1 (GFSLX1X2YX3X4X5), SEQ ID NO: 2 (VIWGNX6NX7YYX8), and SEQ ID NO: 3 (WX9GX 10 KDX 11 PFDY), respectively, and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4 (X<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​= Isoleucine (I) or valine (V), X 14 = Tyrosine (Y), Tryptophan (W), or Phenylalanine (F), X 15 = Asparagine (N) or aspartic acid (D), X 16 = Threonine (T) or Alanine (A), X 17 = Asparagine (N), serine (S), or tyrosine (Y), X 18 = Serine (S), threonine (T), or asparagine (N), X 19 = Leucine (L) or Arginine (R), X 20 = Histidine (H) or Alanine (A), X 21 = Leucine (L) or glutamine (Q), X 22 =Asparagine (N), glycine (G), or histidine (H), and X 23 = An isolated antibody or its antigen-binding fragment, which is serine (S) or histidine (H).

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs. 10, 11, 12, 16, 17, and 18, and the VL CDR1, CDR2, and CDR3 sequences, respectively.

[0020] In some embodiments, the antibody or its antigen-binding fragment comprises VH containing the amino acid sequence of SEQ ID NO: 20 or 21, and VL containing the amino acid sequence of SEQ ID NO: 23, 24, 25, 26, or 27.

[0021] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, each containing the amino acid sequences of SEQ ID NOs. 20 and 23, SEQ ID NOs. 21 and 24, SEQ ID NOs. 21 and 25, SEQ ID NOs. 21 and 26, or SEQ ID NOs. 21 and 27, respectively.

[0022] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 or 21.

[0023] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NOs. 23, 24, 25, 26, or 27.

[0024] In certain embodiments, the antibody or antigen-binding fragment described herein has a binding affinity (K) of approximately 400 pM to approximately 1000 pM to human PAR-2. D ) has. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human and cynomolgus monkey PAR-2. In some embodiments, the antibody or its antigen-binding fragment has a binding affinity (K) of about 4 nM to about 5 nM to cynomolgus monkey PAR-2. D ) has. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human PAR-2.

[0025] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is an isotype selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region having one or more amino acid substitutions. In some embodiments, the light chain constant region is a human IgGκ light chain constant region.

[0026] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being a human IgG4 heavy chain constant region and the light chain constant region being a human IgGκ light chain constant region.

[0027] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being a human IgG4 heavy chain constant region containing one or more amino acid substitutions, and the light chain constant region being a human IgGκ light chain constant region. In some embodiments, the human IgG4 heavy chain constant region has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0028] In some embodiments, the human IgG tetraheterogeneous constant region includes an S228P substitution (according to EU numbering). In some embodiments, the human IgG tetraheterogeneous constant region includes a terminal lysine deletion (K447Δ) (according to EU numbering). In some embodiments, the human IgG tetraheterogeneous constant region includes both an S228P substitution and K447Δ (according to EU numbering).

[0029] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or its antigen-binding fragment.

[0030] In some embodiments, the antibody or its antigen-binding fragment is a full-length antibody.

[0031] In some embodiments, the antibody or its antigen-binding fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is Fab, Fab', F(ab')2, single-chain Fv(scFV), disulfide-linked Fv, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFV)2, or scFV-Fc.

[0032] In some embodiments, the antibody or antigen-binding fragment includes an Fc domain that has been manipulated to reduce its effector function.

[0033] In some embodiments, the antibody or its antigen-binding fragment includes a detectable label.

[0034] In other embodiments, isolated polynucleotides are provided that include a heavy chain variable region or a nucleic acid sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the nucleic acid molecule encodes the VH of SEQ ID NO: 20 or 21.

[0035] In some embodiments, isolated polynucleotides are provided, comprising a light chain variable region or a nucleic acid molecule encoding the light chain of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the nucleic acid molecule encodes the VL of SEQ ID NOs. 23, 24, 25, 26, or 27.

[0036] In some embodiments, an isolated polynucleotide is provided comprising a first nucleic acid molecule encoding the light chain variable region of SEQ ID NO: 23, 24, 25, 26, or 27 and a second nucleic acid molecule encoding the heavy chain variable region of SEQ ID NO: 20 or 21. In some embodiments, a mixture of isolated polynucleotides is provided comprising a first polynucleotide comprising the light chain variable region of SEQ ID NO: 23, 24, 25, 26, or 27 and a second polynucleotide comprising the heavy chain variable region of SEQ ID NO: 20 or 21.

[0037] In some embodiments, isolated polynucleotides are provided, comprising a nucleic acid molecule encoding a heavy chain variable region or heavy chain of an antibody or antigen-binding fragment disclosed herein and a light chain variable region or light chain of an antibody or antigen-binding fragment disclosed herein.

[0038] In some embodiments, isolated vectors comprising the polynucleotides disclosed herein are provided.

[0039] In some embodiments, a host cell is provided comprising (a) a polynucleotide disclosed herein, (b) a vector disclosed herein, or (c) a first vector comprising a first polynucleotide disclosed herein and a second vector comprising a second polynucleotide disclosed herein. In some embodiments, the host cell is selected from the group consisting of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, yeast, Expi293F human cells, C6 (rat glioma cell line), U2OS, Chem-1, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. In some embodiments, the host cells are CHO-K1SV cells.

[0040] In some embodiments, a method is provided for producing an antibody or antigen-binding fragment thereof that binds to human PAR-2, comprising the step of culturing host cells disclosed herein such that a nucleic acid molecule disclosed herein is expressed and an antibody or antigen-binding fragment thereof is produced, and optionally further comprising the step of isolating the antibody or antigen-binding fragment thereof from the culture.

[0041] In some embodiments, isolated antibodies or antigen-binding fragments thereof that specifically bind to human PAR-2 ​​and are encoded by polynucleotides disclosed herein or produced by methods disclosed herein are provided.

[0042] In some embodiments, a pharmaceutical composition is provided comprising an antibody or an antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable excipient.

[0043] In some embodiments, a method for inhibiting PAR-2 ​​activation by a PAR-2 ​​activating ligand in vitro or in vivo, comprising the step of blocking ligand binding to PAR-2 ​​using an antibody or antigen-binding fragment thereof as disclosed herein or a pharmaceutical composition as disclosed herein.

[0044] In certain embodiments, the PAR-2 ​​activating ligand is a soluble PAR-2 ​​activating ligand, a PAR-2 ​​anchoring ligand, or a PAR-1 anchoring ligand.

[0045] In some embodiments, antibodies or antigen-binding fragments thereof disclosed herein are provided for use in the treatment of respiratory tract diseases. In other embodiments, antibodies or antigen-binding fragments thereof disclosed herein are provided for use in the relief of pain. In other embodiments, antibodies or antigen-binding fragments thereof disclosed herein are provided for use in the treatment of cancer. In other embodiments, antibodies or antigen-binding fragments thereof disclosed herein are provided for use in the treatment of skin diseases. In other embodiments, antibodies or antigen-binding fragments thereof disclosed herein are provided for use in the treatment of inflammatory conditions.

[0046] In some embodiments, a method is provided for treating a patient's airway disease, comprising the step of administering to the patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the airway disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pulmonary arterial hypertension.

[0047] In some embodiments, a method is provided for reducing pain in a patient, comprising the step of administering to the patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the pain is selected from the group consisting of cancer pain, arthralgia, chemotherapy-induced peripheral neuropathy pain, toothache, bladder pain, pancreatitis pain, irritable bowel syndrome-related pain, visceral pain, osteoarthritis-related pain, rheumatoid arthritis-related pain, spinal cord injury pain, and migraine pain.

[0048] In some embodiments, a method is provided for treating a patient's cancer, comprising the step of administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cancer is selected from bone cancer, pancreatic cancer, gastric cancer, colon cancer, breast cancer, glioblastoma, melanoma, prostate cancer, breast cancer, colon cancer, or any combination thereof.

[0049] In some embodiments, a method is provided for treating a patient's skin disease, comprising the step of administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the skin disease is selected from the group consisting of atopic dermatitis, allergic contact dermatitis, Netherton syndrome, ichthyosis, post-injury skin barrier / permeability restoration, pruritus, skin cancer, itchiness, pigmentation associated with melanosis, and pigmentation associated with vitiligo.

[0050] In some embodiments, a method is provided for treating an oral and facial granulomatosis in a patient, comprising the step of administering to the patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as disclosed herein, or a pharmaceutical composition as disclosed herein.

[0051] In some embodiments, a method is provided for treating an inflammatory condition in a patient, comprising the step of administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof as disclosed herein, or a pharmaceutical composition as disclosed herein. In certain embodiments, the inflammatory condition is a pathology associated with rheumatoid arthritis, osteoarthritis, inflammatory-induced visceral hypersensitivity, periodontal disease, or acute coronavirus infection.

[0052] In some embodiments, a method is provided for detecting PAR-2 ​​in a sample, comprising the step of contacting the sample with an antibody or an antigen-binding fragment thereof as disclosed herein, or a pharmaceutical composition as disclosed herein. In some embodiments, the sample is obtained from a human subject, and optionally the sample is a cancer sample. In some embodiments, the sample is an in vitro sample. [Brief explanation of the drawing]

[0053] [Figure 1A] Figure 1A shows the alignment of the variable heavy chain sequences of the humanized variant of Ab309, along with the CDR enclosed in a box. [Figure 1B] Figure 1B shows the alignment of the variable light chain sequences of the humanized variant of Ab309, along with the CDR as defined herein, enclosed in a box. [Figure 2] Figure 2 shows the relative efficacy of P24E1102 in antagonizing SLIGKV-induced PAR-2 ​​calcium flux compared to the mouse anti-human PAR-2 ​​antibody MAb3949 (see Example 8). [Figure 3] Figure 3 shows the percentage change in lung resistance in cynomolgus monkeys that were loaded with Ascaris allergen after treatment with vehicle, P24E1102 (5 mg or 10 mg), or fluticasone (Floc). [Figure 4] Figure 4 shows the differences in ear caliper size in hPAR2 knock-in rats in an acute model of dermatitis. Rats were loaded with a vehicle (control); topically loaded with oxazolone; or loaded with oxazolone after administration of P24E1102, MOPC isotype control, or dexamethasone. Antibody treatment did not significantly reduce inflammation in this model. [Figure 5] Figures 5A and 5B show the response to imiquimod (IMQ) in hPAR2 knock-in rats, a model of psoriasis and dermatitis. Female and male rats were treated with imiquimod after preliminary treatment with vehicle, imiquimod, or P24E1102 or MOPC isotype controls. Figure 5A shows the PASI scores of visible skin lesions. P24E1102 or MOPC showed a significant reduction in PASI scores. Figure 5B shows the scratching episodes. Only P24E1102 significantly reduced scratching, bringing it down to below background levels. [Figure 6]Figures 6A–6C show the response of DRG neurons from wild-type rats to capsaicin after treatment with the PAR2 agonist LIGRLO or a control. LIGRLO increased the percentage of neurons that increased their response to a second capsaicin treatment (approximately 2x), despite the set sensitization threshold (120%–300%) (Figure 6A). The distribution of signals responding to the second capsaicin treatment (Figure 6B) was increased by LIGRLO (Figure 6C). [Figure 7] Figures 7A–7C show the response of DRG neurons derived from hPAR2 knock-in rats to capsaicin after treatment with the PAR2 agonist LIGRLO. Prior to LIGRLO treatment, DRGs were treated with a vehicle ("control") and 500 nM P24E1102. P24E1102 significantly reduced the number of capsaicin-sensitized neurons (p<0.05) (Figure 7A). The distribution of a second signal in response to LIGRLO treatment (Figure 7B) was reduced by P24E1102 (Figure 7C). [Figure 8] Figures 8A–8D show the effects of SLIGKV-treated or untreated cancer cells MCF (Figure 8A), MDA-MB-231 (Figure 8B), HepG2 (Figure 8C), or A549 (Figure 8D) on their viability, along with increasing concentrations of P24E1102. [Figure 9] Figures 9A–9H show that SLIGKV induces behavior consistent with changes in cell morphology and metastasis, and that this is reversed by P24E1102. Figures 9A–9D are images of untreated cells (Figure 9A), cells treated with SLIGKV (Figure 9B), cells treated with SLIGKV with 500 nM P24E1102 (Figure 9C), or cells treated with SLIGKV with 2000 nM P24E1102 (Figure 9D). Dose-dependent inhibition by P24E1102 in the presence of SLIGKV is quantified with respect to the percentage of cells treated with the process (Figure 9E), the average number of processes per cell (Figure 9F), cell migration indicated by the number of scattered cells (Figure 9G), and the entire area of ​​the cell aggregate (Figure 9H).

[0054] This specification provides antibodies (e.g., humanized antibodies) and their antigen-binding fragments that specifically bind to human PAR-2 ​​and exhibit one or more of the properties disclosed herein. Such antibodies or their antigen-binding fragments can alleviate, prevent, and / or treat diseases or conditions in which PAR-2 ​​may be increased, and / or diseases or conditions that can be alleviated by antagonizing the activation of PAR-2 ​​by PAR-2 ​​activating ligands (e.g., respiratory diseases, skin diseases, pain relief, orofacial granulomatosis, inflammatory conditions, and cancer). Such anti-human PAR-2 ​​antibodies and their antigen-binding fragments can, for example, block the interaction between PAR-2 ​​activating ligands and the extracellular domain of PAR-2, thereby blocking PAR-2 ​​activation by PAR-2 ​​activating ligands. This specification provides exemplary anti-human PAR-2 ​​antibodies exhibiting such activity.

[0055] Isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding such antibodies and their antigen-binding fragments are also provided. Vectors (e.g., expression vectors) and cells (e.g., host cells) containing such antibodies and nucleic acids (polynucleotides) encoding such antibodies and their antigen-binding fragments are further provided. Methods for producing such antibodies and their antigen-binding fragments are also provided.

[0056] In other embodiments, the Specified herein provides methods for using such antibodies to modulate PAR-2 ​​activity, for example. PAR-2 ​​activity can be modulated, for example, by antagonizing the activation of PAR-2 ​​by a PAR-2 ​​activating ligand. In some embodiments, anti-human PAR-2 ​​antibodies provided herein are used to block the binding of a PAR-2 ​​activating ligand to human PAR-2.

[0057] In a further embodiment, the anti-human PAR-2 ​​antibody provided herein is used to block the interaction between a PAR-2 ​​activating ligand and the extracellular domain of PAR-2. Such PAR-2 ​​activating ligands may include, but are not limited to, PAR-2 ​​anchoring ligands (cis or trans), PAR-1 anchoring ligands, or soluble ligands. In a further embodiment, the anti-human PAR-2 ​​antibody provided herein is used to prevent and / or treat diseases or conditions associated with increased PAR-2 ​​expression and / or increased PAR-2 ​​activation, and / or diseases or conditions that can be alleviated by antagonizing the activation of PAR-2 ​​by a PAR-2 ​​activating ligand (e.g., respiratory diseases, skin diseases, cancer, orofacial granulomatosis, inflammatory conditions, and pain associated with various diseases or conditions).

[0058] In some embodiments, such diseases or conditions include, but are not limited to, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, atopic dermatitis, allergic contact dermatitis, Netherton syndrome, ichthyosis, post-injury skin barrier / permeability restoration, pruritus, skin cancer, itchy skin, pigmentation associated with melanosis, pigmentation associated with vitiligo, cancer pain, arthralgia, chemotherapy-induced peripheral neuropathy pain, toothache, bladder pain, pancreatitis pain, irritable bowel syndrome-related pain, visceral pain, osteoarthritis-related pain, migraine pain, rheumatoid arthritis-related pain, spinal cord injury pain, bone cancer, pancreatic cancer, gastric cancer, colon cancer, breast cancer, glioblastoma, melanoma, prostate cancer, and breast cancer. Related compositions (e.g., pharmaceutical compositions), kits, and methods are also provided.

[0059] To facilitate understanding of this disclosure, several terms and phrases are defined. Additional definitions are provided throughout the detailed description.

[0060] I. Terminology Throughout this disclosure, the terms "a" or "an" entity refer to one or more such entities. For example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used synonymously herein.

[0061] Furthermore, as used herein, “and / or” should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. Accordingly, when the term “and / or” is used herein in phrases such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when the term “and / or” is used in phrases such as “A, B, and / or C,” it is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] Wherever an aspect is described in this specification as “comprising,” it should be understood that other similar aspects are also provided, which are described as “consisting of” and / or “consisting essentially of.”

[0063] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” and “having,” and their conjugations, mean “includes but not limited to.”

[0064] As used herein, the term "consisting of" means "including and limited to".

[0065] As used herein, the term “essentially derived from” means a particular substance or process of a particular method of a composition, and any additional substance or process that does not substantially affect the fundamental properties of the substance or method.

[0066] Where used herein, the terms “about” and “approximately” indicate that, when used to modify a number or range of numbers, deviations of up to 10% above and 10% below that value or range remain within the intended meaning of the stated value or range. Whenever an aspect is described herein using the terms “about” or “approximately” for a number or range, it should be understood that other similar aspects referencing a specific number or range are also provided.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure relates. For example, *The Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd edition, 1999, Academic Press; and *The Oxford Dictionary of Biochemistry and Molecular Biology*, revised edition, 2000, Oxford University Press provide many general dictionaries of the terms used herein.

[0068] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges include the numerical values ​​that define the range. Unless otherwise specified, amino acid sequences are written from left to right in the amino-carboxyl direction. The headings provided herein are not intended to limit the various aspects of this disclosure that can be grasped by referring to this specification as a whole. Thus, the terms defined immediately thereunder are more fully defined by referring to the entire specification.

[0069] The terms "protease-activated receptor 2," "PAR-2," "G protein-coupled receptor 11," "GPR11," "coagulation factor II receptor-like 1," "thrombin receptor-like 1," or "F2RL1" all refer to the same G protein-coupled receptor (GPCR) superfamily member.

[0070] As used herein, the term “PAR-2” refers to mammalian PAR-2 ​​polypeptides, including but not limited to natural PAR-2 ​​polypeptides and isoforms of PAR-2 ​​polypeptides. “PAR-2” encompasses full-length uncprocessed PAR-2 ​​polypeptides, as well as forms of PAR-2 ​​polypeptides resulting from intracellular processing. PAR-2 ​​or any of its variants and isoforms may be isolated from cells or tissues that express them naturally, or recombinantly produced using techniques well known in the art and / or techniques described herein.

[0071] As used herein, the term “human PAR-2” refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 28; naturally occurring variants of SEQ ID NO: 28, including but not limited to those having S or F at position 21, N or S at position 30, R or Q at position 270, or T or A at position 291; and processing forms of SEQ ID NO: 28, including but not limited to those lacking its signal peptide. The amino acid sequence of human PAR-2 ​​without the signal peptide is represented by the amino acid sequence of SEQ ID NO: 109. “PAR-2 ​​polynucleotide” or “PAR-2 ​​nucleic acid molecule” refers to any polynucleotide encoding PAR-2, including those described herein.

[0072] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof (e.g., glycoprotein) via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. Antibodies may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or any of their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) based on the identity of their heavy chain constant domains, which are called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have well-known subunit structures and three-dimensional configurations. The antibody may be naked, part of a fusion protein, or conjugated to other molecules such as toxins, radioisotopes, or detectable labels.

[0073] The term "antibody fragment" refers to a portion of an antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human PAR-2). It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody, for example, an anti-PAR-2 ​​antibody as described herein, include: (i) a monovalent fragment, the Fab fragment, consisting of VL, VH, CL, and CH1 domains; (ii) a bivalent fragment, the F(ab')2 fragment, containing two Fab fragments linked by a disulfide crosslink of a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody, and a disulfide-linked Fv(sdFv); (v) a dAb fragment consisting of the VH domain (Ward et al., Nature 341, 1989: pp. 544-546); and (vi) an isolated complementarity-determining region (CDR); or (vii) a combination of two or more isolated CDRs that may be optionally joined by a synthetic linker. Furthermore, even if the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombination techniques by synthetic linkers that allow the VL and VH regions to pair up and form a single protein chain that forms a monovalent molecule (known as single-chain Fv (scFV)). See, for example, Bird et al., (1988) Science 242: pp. 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: pp. 5879-5883. Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0074] The terms “PAR-2 ​​inhibitor” and “PAR-2 ​​antagonist” are used synonymously. Each is a molecule that detectably inhibits at least one function of PAR-2. Conversely, a “PAR-2 ​​agonist” is a molecule that detectably increases at least one function of PAR-2. The inhibition caused by a PAR-2 ​​inhibitor does not need to be complete, as long as it is detectable using an assay. Any assay for PAR-2 ​​function can be used, and examples are provided herein. Examples of PAR-2 ​​functions that can be inhibited by a PAR-2 ​​inhibitor or increased by a PAR-2 ​​agonist include protease activation ligand binding and downstream signaling. Examples of types of PAR-2 ​​inhibitors and PAR-2 ​​agonists include, but are not limited to, small molecules that modulate PAR-2 ​​activity, as well as PAR-2-binding polypeptides such as antigen-binding proteins (e.g., PAR-2 ​​inhibitory antigen-binding proteins), antibodies, antibody fragments, and antibody derivatives. For example, see International Publication No. 2006 / 127379, International Publication No. 2006 / 127396, U.S. Publication No. 8927503, International Publication No. 2012 / 1010453, International Publication No. 2014 / 020350, International Publication No. 2016 / 154075, International Publication No. 2010 / 017086, International Publication No. 2011 / 031695, International Publication No. 2018 / 167322, U.S. Publication No. 8236305, U.S. Publication No. 7888482, and U.S. Publication No. 8357367.

[0075] As used herein, the term "PAR-2 ​​activating ligand" refers to a ligand that binds to protease-activated receptor 2 (PAR-2) and initiates PAR-2 ​​activation and signal transduction. Such PAR-2 ​​activating ligands may include, but are not limited to, PAR-2 ​​anchoring ligands (cis or trans), PAR-1 anchoring ligands, or soluble ligands. In this context, the ligand is cis if the PAR2 molecule is activated by its own N-terminal region. The ligand is trans if PAR2 is activated by a ligand on another PAR2 molecule. Trans activation can also occur, for example, from a PAR1 ligand acting on PAR2.

[0076] As used herein, the term "PAR-2 ​​activation" refers to PAR-2 ​​activation in the presence of a PAR-2 ​​ligand (e.g., a PAR-2 ​​anchoring ligand (cis or trans), a PAR-1 anchoring ligand, or a soluble ligand (e.g., a synthetic soluble PAR-2 ​​activating ligand such as SLIGKV)).

[0077] The term "inhibit PAR-2 ​​activation" as used herein refers to the inhibition of PAR-2 ​​activation by an anti-PAR-2 ​​antibody. 50 This can be used as a measure of the inhibitory efficacy of an anti-PAR-2 ​​antibody on PAR-2 ​​activation (i.e., the nM concentration of the anti-PAR-2 ​​antibody that achieves 50% inhibition of ligand-induced PAR-2 ​​activity).

[0078] The terms “anti-PAR-2 ​​antibody,” “PAR-2 ​​antibody,” and “antibody that binds to PAR-2” refer to an antibody capable of binding to PAR-2 ​​with sufficient affinity to be useful as a diagnostic, therapeutic, and / or modulator of PAR-2 ​​activity. The degree of binding of an anti-PAR-2 ​​antibody to the PAR-2 ​​protein may be greater than that of an isotype control antibody or a non-PAR-2 ​​target-directed antibody to PAR-2, as measured, for example, by flow cytometry. The degree of binding of an anti-PAR-2 ​​antibody to an unrelated non-PAR-2 ​​protein may be equivalent to that of an isotype control antibody or a non-PAR-2 ​​target-directed antibody to PAR-2, as measured, for example, by flow cytometry. In certain embodiments, the anti-PAR-2 ​​antibody binds exclusively to PAR-2 ​​and not to PAR-1, PAR-3, and PAR-4.

[0079] A “monoclonal” antibody or its antigen-binding fragment refers to a homogeneous population of antibodies or antigen-binding fragments involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is typically in contrast to polyclonal antibodies, which contain different antibodies against different antigenic determinants. The term “monoclonal” antibody or its antigen-binding fragment encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFV) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, “monoclonal” antibodies or their antigen-binding fragments refer to such antibodies and their antigen-binding fragments produced by any number of methods, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0080] A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, pp. 1547-1553 (1992). For example, a bispecific antibody may contain one domain that binds to the PAR2 receptor binding site and another domain that binds to the N-terminal portion of PAR2 before or after protease cleavage.

[0081] As used herein, the terms “variable region” or “variable domain” are used synonymously and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, whose sequence differs significantly between antibodies and is used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while a more highly conserved region of the variable domain is called the framework region (FR). While we do not wish to be bound to any particular mechanism or theory, the CDRs of the light and heavy chains are thought to be primarily responsible for antibody-antigen interaction and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region includes rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0082] The terms "VL" and "VL domain" are used synonymously and refer to the variable region of the antibody light chain.

[0083] The terms "VH" and "VH domain" are used synonymously and refer to the variable region of the antibody's heavy chain.

[0084] The term "Kabat numbering" and similar terms are recognized in the art and refer to a method of numbering amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding fragment. In certain embodiments, the CDR can be determined according to the Kabat numbering method (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci vol. 190: pp. 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0085] The terms "Kabat amino acid numbering," "Kabat position," and their grammatical variations refer to the numbering scheme used for the heavy-chain or light-chain variable domains of a group of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991). Using this numbering scheme, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy-chain variable domain may contain a single amino acid insertion after residue 52 of CDR2 (Kabat residue 52a) and an inserted residue after heavy-chain FW residue 82 (e.g., Kabat residues 82a, 82b, and 82c). See Table 1.

[0086] [Table 1]

[0087] For all heavy chain constant region amino acid positions considered in this disclosure, the residue numbering is based on the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, Vol. 63 (I): pp. 78-85, which describes the amino acid sequence of myeloma protein EU, the first human IgG1 to be sequenced. Edelman et al.'s EU index is also shown in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda. Therefore, the phrases "the EU index shown in Kabat," "Kabat's EU index," and "position...according to the EU index shown in Kabat," and their grammatical variations, refer to the residue numbering scheme based on the human IgG1 EU antibody by Edelman et al. as shown in Kabat 1991.

[0088] As used herein, the terms “constant region” or “constant domain” are synonymous and have the common meaning in the art. The constant region is the carboxyl-terminal portion of the light and / or heavy chain of an antibody moiety, for example, which does not directly participate in the binding of the antibody to the antigen but can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a conserved amino acid sequence compared to the immunoglobulin variable domain. In certain embodiments, an antibody or antigen-binding fragment contains a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0089] As used herein, the term “heavy chain” as used in relation to antibodies can refer to any different type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain that gives rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human heavy chain.

[0090] As used herein, the term “light chain” in reference to antibodies can refer to any different type, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some embodiments, the light chain is a human light chain.

[0091] The "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc or the first component (C1q) of the classical complement system located on various cells of the immune system (e.g., effector cells). Therefore, the Fc region includes the constant region of the antibody excluding the first constant-region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody, while the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes the immunoglobulin domains Cγ2 and Cγ3, as well as the hinge between Cγ1 and Cγ2. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is typically defined as a stretch from the amino acid residue at position C226 or P230 (or the amino acid between these two) to the carboxyl terminus of the heavy chain, numbered according to the Kabat-EU index. The CH2 domain of the human IgG Fc region extends approximately from amino acid 231 to approximately amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain of the Fc region, extending approximately from amino acid 341 to approximately amino acid 447 of IgG. As used herein, the Fc region may be a natural sequence Fc containing any allotype variant, or a variant Fc (e.g., an Fc that does not exist in nature). Furthermore, Fc may refer to this region in isolation or in the context of an Fc-containing protein polypeptide (e.g., an antibody or immunoadhesion), such as an "Fc-containing binding protein" also called an "Fc fusion protein."

[0092] A "natural sequence Fc region" or "natural sequence Fc" includes an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Examples of natural sequence human Fc regions include the natural sequence human IgG1 Fc region, the natural sequence human IgG2 Fc region, the natural sequence human IgG3 Fc region, and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants. Examples of natural sequence Fc include various allotypes of Fc (see, for example, Jefferis et al., (2009) mAbs Vol. 1: p. 1; Vidarsson G. et al., Front Immunol. Vol. 5: p. 520 (published online on October 20, 2014)).

[0093] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the FcγR family of receptors, which encompass allelic variants and alternative splicing forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Human IgG1 binds to most human Fc receptors and induces the most potent Fc effector function. Human IgG1 is considered equivalent to mouse IgG2a in terms of the type of activating Fc receptor it binds to. Conversely, human IgG4 induces the least Fc effector function. (Vidarsson G. et al., Front Immunol. Vol. 5: p. 520 (published online October 20, 2014)).

[0094] The Fc region of the antibody may include modifications that modulate the serum half-life and in vivo distribution, including, but are not limited to, modifications that modulate the antibody's interaction with the neonatal Fc receptor (FcRn), a receptor that plays a crucial role in protecting IgG from catabolism and maintaining high serum antibody concentrations. Such modifications include the M252Y / S254T / T256E triple substitution described in U.S. Patent No. 7,083,784. Other substitutions may be made at positions 250 and 428 (see, for example, U.S. Patent No. 7,217,797) and positions 307, 380, and 434 (see, for example, International Publication No. 00 / 042072). Any class of antibody may have the heavy chain C-terminal lysine excluded or removed to reduce heterogeneity (ΔK). Substitution of S228P (EU numbering) in human IgG4 can stabilize antibody Fab arm exchange in vivo (Labrin et al. (2009) Nature Biotechnol. Vol. 27: No. 8; pp. 767-773).

[0095] The terms “hinge,” “hinge domain,” “hinge region,” or “antibody hinge region” are used synonymously and refer to the domain of the heavy chain constant region that junctions the CH1 domain to the CH2 domain and includes the upper, middle, and lower fragments of the hinge (Roux et al., J. Immunol. 1998, Vol. 161: p. 4083). The hinge provides varying levels of flexibility between the antibody binding region and the effector region, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, the hinge in all IgG isotypes begins at Glu216 and ends at Gly237 (Roux et al., 1998, J. Immunol. 161: p. 4083). The sequences of the wild-type IgG1, IgG2, IgG3, and IgG4 hinges are known in the art. For example, see Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. Vol. 5: p. 520 (published online on October 20, 2014). The hinge of IgG4 antibodies can be stabilized by mutating S228, such as the S228P mutation. See Silva et al. (2015) The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. J Biol Chem. Vol. 290 (No. 9): pp. 5462-5469.

[0096] The term "CH1 domain" refers to the heavy chain constant region that links the variable domain to the hinge of the heavy chain constant domain. As used herein, the CH1 domain begins at A118 and ends at V215. The term "CH1 domain" includes the wild-type CH1 domain and its naturally occurring variants (e.g., allotypes). The CH1 domain sequences (including wild-type and allotypes) of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, for example, Kabat EA et al. (1991), op. cit. and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH1 domains include those described in, for example, U.S. Patent Application Publication No. 20120100140 and the U.S. Patents and Publications and PCT Publications cited herein, which have mutations that alter the biological activity of antibodies, for example, their half-life.

[0097] The term "CH2 domain" refers to the heavy chain constant region that hinges to the CH3 domain of the heavy chain constant domain. As used herein, the CH2 domain begins at P238 and ends at K340. The term "CH2 domain" includes the wild-type CH2 domain and its naturally occurring variants (e.g., allotypes). The CH2 domain sequences (including wild-type and allotypes) of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, for example, Kabat EA et al. (1991), op. cit. and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH2 domains include those described in, for example, U.S. Patent Application Publication No. 20120100140 and the U.S. Patents and Publications and PCT Publications cited herein, which have mutations that alter the biological activity of an antibody, such as a reduction in half-life and / or Fc effector function.

[0098] The term "CH3 domain" refers to the heavy chain constant region that is C-terminal to the CH2 domain of the heavy chain constant domain. As used herein, the CH3 domain begins at G341 and ends at K447. The term "CH3 domain" includes the wild-type CH3 domain and its naturally occurring variants (e.g., allotypes). The CH3 domain sequences (including wild-type and allotypes) of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, for example, Kabat EA et al. (1991), op. cit. and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH3 domains include those with mutations that alter the biological activity of antibodies, such as half-life, as described in, for example, U.S. Patent Application Publication No. 20120100140 and the U.S. Patents and Publications and PCT Publications cited herein.

[0099] As used herein, “isotype” refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0100] An "allotype" refers to a naturally occurring variant within a particular group of isotypes, which differ in a small number of amino acids (see, e.g., Jefferis et al., (2009) mAbs 1: p. 1). The antibodies described herein may be any allotype. The allotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, for example, Kabat EA et al. (1991), op. cit., Vidarsson G. et al., Front Immunol. 5: p. 520 (published online on October 20, 2014), and Lefranc MP, mAbs 1: No. 4, pp. 1-7 (2009).

[0101] In this specification, the terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used synonymously with the term "antibody that specifically binds to an antigen."

[0102] As used herein, "isolated antibody" is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to PAR-2 ​​substantially does not contain antibodies that specifically bind to antigens other than PAR-2). However, an isolated antibody that specifically binds to the PAR-2 ​​epitope may cross-react to other PAR-2 ​​proteins from different species.

[0103] The term "chimeric" antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment whose amino acid sequence originates from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of the antibody or its antigen-binding fragment originating from one species of mammal (e.g., mouse, rat, rabbit, etc.) to have the desired specificity, affinity, and capacity, while the constant region is homologous to the sequence of the antibody or its antigen-binding fragment originating from another species (usually human) to avoid inducing an immune response in that species.

[0104] The term “humanized” antibody or its antigen-binding fragment refers to a form of non-human (e.g., mouse) antibody or its antigen-binding fragment that contains a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof containing a minimal non-human (e.g., mouse) sequence. Typically, a humanized antibody or its antigen-binding fragment is a human immunoglobulin in which residues derived from the complementarity-determining region (CDR) are replaced by residues derived from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capacity ("CDR transplanted") (Jones et al., Nature 321: pp. 522-525 (1986); Riechmann et al., Nature 332: pp. 323-327 (1988); Verhoeyen et al., Science 239: pp. 1534-1536 (1988)). Humanized antibodies or their antigen-binding fragments can be further modified by substituting additional residues in the Fv framework region and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or volume of the antibody or its antigen-binding fragment. Generally, humanized antibodies or their antigen-binding fragments will contain VH and VL, which include at least one, typically two or substantially all, of the CDR regions corresponding to non-human immunoglobulins, while all or substantially all of the FR region is from the human immunoglobulin consensus sequence. Humanized antibodies or their antigen-binding fragments may also include immunoglobulin constant regions or domains (Fc), typically at least a portion of the constant regions or domains of human immunoglobulins. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, Vol. 91(No. 3): pp. 969-973 (1994); and Roguska et al., Protein Eng. Vol. 9(No. 10): pp. 895-904 (1996). In some embodiments, “humanized antibody” is a resurfaced antibody.

[0105] The term "human" antibody (HuMAb) or its antigen-binding fragment means an antibody or its antigen-binding fragment having an amino acid sequence derived from or matching a sequence derived from a human immunoglobulin locus, and such antibodies or antigen-binding fragments are prepared using any technique known in the art. This definition of human antibody or its antigen-binding fragment includes intact or full-length antibodies and their fragments.

[0106] The term “recombinant human antibody” as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to human immunoglobulin genes or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express antibodies, e.g., transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies utilize specific human germline immunoglobulin sequences encoded by germline genes, but include variable and constant regions, including, for example, subsequent rearrangements and mutations that occur during antibody maturation. As is well known in the art (see, for example, Lonberg (2005) Nature Biotech. Vol. 23 (No. 9): pp. 1117-1125), the variable region contains antigen-binding domains encoded by various genes that are rearranged to form antibodies specific to exogenous antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (called somatic mutations or high-frequency mutations) to increase the affinity of antibodies to exogenous antigens. The constant region will change in further response to the antigen (i.e., isotype switching). Thus, nucleic acid molecules encoding light-chain and heavy-chain immunoglobulin polypeptides that have been rearranged and somatically mutated in response to the antigen may not have sequence identity with the original nucleic acid molecule, but will instead be substantially identical or similar (i.e., at least 80% identity).

[0107] "Binding affinity" generally refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). The affinity of molecule X to partner Y is generally expressed by the dissociation constant (K). D It can be expressed as (K). The affinity is the equilibrium dissociation constant (K D) and equilibrium coupling constant (K A It can be measured and / or expressed by a number of methods known in the art, including but not limited to those mentioned above. D is, k off / k on It is calculated from the quotient of K A is, k on / k off It is calculated from the quotient of k. on k refers to, for example, the binding rate constant between an antibody or its antigen-binding fragment and an antigen. off k refers to, for example, the dissociation rate constant of an antibody or its antigen-binding fragment from the antigen. on and k off This can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA®.

[0108] An antibody that is "blocking" or "blocking," or "inhibiting" or "inhibiting" is an antibody that, when bound to a target protein, reduces or inhibits (partially or completely) the binding of that target protein to one or more ligands, and / or an antibody that, when bound to a target protein, reduces or inhibits (partially or completely) the activity or function of one or more target proteins.

[0109] As used herein, “epitope” is a term of the art and refers to a localization region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope may be, for example, a sequence of amino acids in a polypeptide (linear or continuous epitope), or it may be, for example, two or more discontinuous regions of one or more polypeptides joined together (conformal, nonlinear, discontinuous, or discontinuous epitope). In some embodiments, the epitope to which an antibody or its antigen-binding fragment binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., alanine scanning or other site-specific mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr Vol. 50 (Pt 4): pp. 339-350; McPherson A (1990) Eur J Biochem Vol. 189: pp. 1-23; Chayen NE (1997) Structure Vol. 5: pp. 1269-1274; McPherson A (1976) J Biol Chem Vol. 251: pp. 6300-6303). Antibodies or their antigen-binding fragments and crystals of their antigens can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.).For example, refinement can be achieved using computer software such as Meth Enzymol (1985) Vol. 114 & 115, edited by Wyckoff HW et al.; U.S. Patent No. 2004 / 0014194), and BUSTER (see Bricogne G (1993) Acta Crystallogr D Biol Crystallogr Vol. 49 (Pt 1): pp. 37-60; Bricogne G (1997) Meth Enzymol Vol. 276A: pp. 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr Vol. 56 (Pt 10): pp. 1316-1323). Mutagenesis mapping studies can be achieved using any method known to those skilled in the art. For example, for descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see Champe M et al., (1995) J Biol Chem vol. 270: pp. 1388-1394 and Cunningham BC & Wells JA (1989) Science vol. 244: pp. 1081-1085.

[0110] The term "epitope mapping" refers to the process of identifying the molecular determinants of antibody-antigen recognition.

[0111] A PAR-2 ​​antibody that "binds to the same epitope" as a reference PAR-2 ​​antibody refers to an antibody that contacts the same PAR-2 ​​amino acid residue as the reference PAR-2 ​​antibody. The ability of a PAR-2 ​​antibody to bind to the same epitope as the reference PAR-2 ​​antibody is determined using peptide scanning mutagenesis or high-throughput alanine scanning mutagenesis (see Davidson and Doranz, Immunology 143, pp. 13-20, 2014). In the latter methodology, a comprehensive mutant library of PAR-2 ​​or a portion thereof (e.g., the extracellular domain) can be generated by mutating each individual amino acid residue to alanine (or, if the amino acid residue is alanine, to another residue such as serine) and testing each mutant for binding to an anti-PAR-2 ​​antibody or its antigen-binding fragment. The amino acid required for binding and therefore the epitope residue is identified by loss of immunoreactivity.

[0112] As used herein, the terms “specifically bind,” “specifically recognize,” “specifically bind,” “specifically recognize,” “specific binding,” “selective binding,” and “selectively bind” are synonymous terms in the context of antibodies or their antigen-binding fragments, and refer to antibodies that bind to the epitope of a given antigen. Typically, an antibody is determined, for example, by surface plasmon resonance (SPR) on a BIACORE® T200 instrument using a given antigen, e.g., recombinant human PAR-2, as the analyte and the antibody as the ligand, or by scatchard analysis of the binding of the antibody to antigen-positive cells, for about 10 -8 M, about 10 -9 M, or about 10 -10 M, or an even lower equilibrium dissociation constant (K D (ii) binds to the given antigen with an affinity at least 10 times greater than its affinity for binding to nonspecific antigens other than the given antigen or closely related antigens (e.g., BSA, casein). Therefore, an antibody that "specifically binds to human PAR-2" binds to human PAR-2 ​​(e.g., SEQ ID NO: 28 or amino acids 26-397 of SEQ ID NO: 28) at approximately 10 -8 M, about 10-9 M, or about 10 -10 M, or even lower K D This refers to an antibody that binds to PAR-2 ​​and can also bind to PAR-2 ​​derived from other species (e.g., cynomolgus monkey PAR-2). In some embodiments, the antibodies or antigen-binding fragments disclosed herein do not bind to human PAR-1, PAR-3, or PAR-4.

[0113] In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment is measured, for example, by surface plasmon resonance (SPR) technique (as described in Example 5) to have high affinity, for example, about 9.9 × 10⁻⁶ -10 M, approx. 9.8 x 10 -10 M, approx. 9.7×10 -10 M, approx. 9.6×10 -10 M, approx. 9.5 x 10 -10 M, approx. 9.4 x 10 -10 M, approx. 9.3 x 10 -10 M, approx. 9.2×10 -10 M, approx. 9.1×10 -10 M, approx. 9.0×10 -10 M, approx. 8.9 x 10 -10 M, approx. 8.8 x 10 -10 M, approx. 8.7 x 10 -10 M, approx. 8.6 x 10 -10 M, approx. 8.5 x 10 -10 M, approx. 8.4 x 10 -10 M, approx. 8.3 x 10 -10 M, approx. 8.2 x 10 -10 M, approx. 8.1 x 10 -10 M, approx. 8.0×10 -10 M, approx. 7.9×10 -10 M, approx. 7.8 x 10 -10 M, approx. 7.7 x 10 -10 M, approx. 7.6×10 -10 M, approx. 7.5 x 10 -10 M, approx. 7.4 x 10 -10 M, approx. 7.3×10 -10 M, approx. 7.2 x 10 -10 M, approx. 7.1×10 -10 M, approx. 7.0×10 -10 M, approx. 6.9 x 10 -10 M, approx. 6.8 x 10 -10 M, approx. 6.7 x 10-10 M, approximately 6.6 × 10 -10 M, approximately 6.5 × 10 -10 M, approximately 6.4 × 10 -10 M, approximately 6.3 × 10 -10 M, approximately 6.2 × 10 -10 M, approximately 6.1 × 10 -10 M, approximately 6.0 × 10 -10 M, approximately 5.9 × 10 -10 M, approximately 5.8 × 10 -10 M, approximately 5.7 × 10 -10 M, approximately 5.6 × 10 -10 M, approximately 5.5 × 10 -10 M, approximately 5.4 × 10 -10 M, approximately 5.3 × 10 -10 M, approximately 5.2 × 10 -10 M, approximately 5.1 × 10 -10 M, approximately 5.0 × 10 -10 M, approximately 4.9 × 10 -10 M, approximately 4.8 × 10 -10 M, approximately 4.7 × 10 -10 M, approximately 4.6 × 10 -10 M, approximately 4.5 × 10 -10 M, approximately 4.4 × 10 -10 M, approximately 4.3 × 10 -10 M, approximately 4.2 × 10 -10 M, approximately 4.1 × 10 -10 M, approximately 4.0 × 10 -10 M, approximately 3.9 × 10 -10 M, approximately 3.8 × 10 -10 M, approximately 3.7 × 10 -10 M, approximately 3.6 × 10 -10 M, approximately 3.5 × 10 -10 M, approximately 3.4 × 10 -10 M, approximately 3.3 × 10 -10 M, approximately 3.2 × 10 -10 M, approximately 3.1 × 10 -10 M, approximately 3.0 × 10 -10 M, approximately 2.9 × 10 -10 M, approximately 2.8 × 10 -10 M, approximately 2.7 × 10 -10 M, approximately 2.6 × 10 -10 M, approximately 2.5 × 10 -10 M, 2.4 × 10 -10 M, approximately 2.3 × 10 -10 M, approximately 2.2 × 10 -10M, approx. 2.1 x 10 -10 M, approx. 2.0×10 -10 M, approx. 1.9×10 -10 M, approx. 1.8 x 10 -10 M, approx. 1.7 x 10 -10 M, approx. 1.6 x 10 -10 M, approx. 1.5 x 10 -10 M, 1.4×10 -10 M, approx. 1.3 x 10 -10 M, approx. 1.2 x 10 -10 M, approx. 1.1×10 -10 M, approx. 1.0×10 -10 M, about 9 x 10 -11 M, about 8 x 10 -11 M, about 7 x 10 -11 M, about 6 x 10 -11 M, about 5 x 10 -11 M, approx. 4 x 10 -11 M, about 3 x 10 -11 M, approx. 2 x 10 -11 M, about 1 x 10 -11 M, about 9 x 10 -12 M, about 8 x 10 -12 M, about 7 x 10 -12 M, about 6 x 10 -12 M, about 5 x 10 -12 M, approx. 4 x 10 -12 M, about 3 x 10 -12 M, approx. 2 x 10 -12 M, about 1 x 10 -12 M, about 9 x 10 -13 M, or approximately 8 x 10 -13 M's K D It specifically binds to human PAR-2.

[0114] In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment is, for example, about 9.5 × 10 -10 M, approx. 9.4 x 10 -10 M, approx. 9.3 x 10 -10 M, approx. 9.2×10 -10 M, approx. 9.1×10 -10 M, approx. 9.0×10 -10 M, approx. 8.9 x 10 -10 M, approx. 8.8 x 10 -10 M, approx. 8.7 x 10 -10 M, approx. 8.6 x 10 -10 M, approx. 8.5 x 10-10 M, approx. 8.4 x 10 -10 M, approx. 8.3 x 10 -10 M, approx. 8.2 x 10 -10 M, approx. 8.1 x 10 -10 M, approx. 8.0×10 -10 M, approx. 7.9×10 -10 M, approx. 7.8 x 10 -10 M, approx. 7.7 x 10 -10 M, approx. 7.6×10 -10 M, approx. 7.5 x 10 -10 M, approx. 7.4 x 10 -10 M, approx. 7.3×10 -10 M, approx. 7.2 x 10 -10 M, approx. 7.1×10 -10 M, approx. 7.0×10 -10 M, approx. 6.9 x 10 -10 M, approx. 6.8 x 10 -10 M, approx. 6.7 x 10 -10 M, approx. 6.6×10 -10 M, approx. 6.5 x 10 -10 M, approx. 6.4 x 10 -10 M, approx. 6.3 x 10 -10 M, approx. 6.2 x 10 -10 M, approx. 6.1×10 -10 M, approx. 6.0×10 -10 M, approx. 5.9 x 10 -10 M, approx. 5.8 x 10 -10 M, approx. 5.7 x 10 -10 M, approx. 5.6 x 10 -10 M, approx. 5.5 x 10 -10 M, approx. 5.4 x 10 -10 M, approx. 5.3 x 10 -10 M, approx. 5.2 x 10 -10 M, approx. 5.1 x 10 -10 M, approx. 5.0 x 10 -10 M, approx. 4.9×10 -10 M, approx. 4.8 x 10 -10 M, approx. 4.7 x 10 -10 M, approx. 4.6×10 -10 M, approx. 4.5 x 10 -10 M, approx. 4.4 x 10 -10 M, or approximately 4.3 x 10 -10 M's K D It specifically binds to human PAR-2.

[0115] In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment is approximately 9.5 × 10⁻⁶ -10 M's K D It specifically binds to human PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 8.5 × 10⁻⁶. -10 M's K D It specifically binds to human PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a capacity of approximately 7.5 × 10⁻⁶. -10 M's K D It specifically binds to human PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a capacity of approximately 6.5 × 10⁻⁶. -10 M's K D It specifically binds to human PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 5.5 × 10⁻⁶. -10 M's K D It specifically binds to human PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 4.5 × 10⁻⁶. -10 M's K D It specifically binds to human PAR-2.

[0116] The terms "cross-reactive" or "cross-reactivity," as used herein, refer to the ability of an antibody described herein to bind to a PAR-2 ​​orthologue. For example, an antibody described herein that binds to human PAR-2 ​​may also bind to PAR-2 ​​of another species (e.g., cynomolgus monkey PAR-2). As used herein, cross-reactivity can be measured by detecting specific reactivity with a purified antigen in a binding assay (e.g., SPR, ELISA) or by detecting binding to or otherwise functional interaction with cells that physiologically express PAR-2. Methods for determining cross-reactivity include, for example, Biacore® surface plasmon resonance (SPR) analysis using a Biacore® T200 SPR instrument (Biacore AB, Uppsala, Sweden) or the standard binding assay described herein using flow cytometry techniques. Antibodies that "cross-react with cynomolgus monkey PAR-2" have a K of approximately 10⁻⁸ M, approximately 10⁻⁹ M, or approximately 10⁻¹⁰ M, or even lower K D This refers to antibodies that bind to cynomolgus monkey PAR-2. In some embodiments, such antibodies that do not cross-react with PAR-2 ​​from non-human species (e.g., mouse, rabbit, guinea pig, or canine PAR-2) exhibit essentially undetectable binding to these proteins in standard binding assays.

[0117] In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment is measured, for example, by surface plasmon resonance (SPR) technique (as described in Example 5), to a value of approximately 9.5 × 10⁻⁶. -9 M, approx. 9.4 x 10 -9 M, approx. 9.3 x 10 -9 M, approx. 9.2×10 -9 M, approx. 9.1×10 -9 M, approx. 9.0×10 -9 M, approx. 8.9 x 10 -9 M, approx. 8.8 x 10 -9 M, approx. 8.7 x 10 -9 M, approx. 8.6 x 10 -9 M, approx. 8.5 x 10 -9 M, approx. 8.4 x 10 -9 M, approx. 8.3 x 10-9 M, approximately 8.2 × 10 -9 M, approximately 8.1 × 10 -9 M, approximately 8.0 × 10 -9 M, approximately 7.9 × 10 -9 M, approximately 7.8 × 10 -9 M, approximately 7.7 × 10 -9 M, approximately 7.6 × 10 -9 M, approximately 7.5 × 10 -9 M, approximately 7.4 × 10 -9 M, approximately 7.3 × 10 -9 M, approximately 7.2 × 10 -9 M, approximately 7.1 × 10 -9 M, approximately 7.0 × 10 -9 M, approximately 6.9 × 10 -9 M, approximately 6.8 × 10 -9 M, approximately 6.7 × 10 -9 M, approximately 6.6 × 10 -9 M, approximately 6.5 × 10 -9 M, approximately 6.4 × 10 -9 M, approximately 6.3 × 10 -9 M, approximately 6.2 × 10 -9 M, approximately 6.1 × 10 -9 M, approximately 6.0 × 10 -9 M, approximately 5.9 × 10 -9 M, approximately 5.8 × 10 -9 M, approximately 5.7 × 10 -9 M, approximately 5.6 × 10 -9 M, approximately 5.5 × 10 -9 M, approximately 5.4 × 10 -9 M, approximately 5.3 × 10 -9 M, approximately 5.2 × 10 -9 M, approximately 5.1 × 10 -9 M, approximately 5.0 × 10 -9 M, approximately 4.9 × 10 -9 M, approximately 4.8 × 10 -9 M, approximately 4.7 × 10 -9 M, approximately 4.610 -9 M, approximately 4.5 × 10 -9 M, approximately 4.4 × 10 -9 M, approximately 4.3 × 10 -9 M, approximately 4.2 × 10 -9 M, approximately 4.1 × 10 -9 M, approximately 4.0 × 10 -9 M, approximately 3.9 × 10 -9 M, approximately 3.8 × 10 -9M, approximately 3.7 × 10 -9 M, approximately 3.6 × 10 -9 M, approximately 3.5 × 10 -9 M, approximately 3.4 × 10 -9 M, approximately 3.3 × 10 -9 M, approximately 3.2 × 10 -9 M, approximately 3.1 × 10 -9 M, approximately 3.0 × 10 -9 M, approximately 2.9 × 10 -9 M, approximately 2.8 × 10 -9 M, approximately 2.7 × 10 -9 M, approximately 2.6 × 10 -9 M, approximately 2.5 × 10 -9 M, approximately 2.4 × 10 -9 M, approximately 2.3 × 10 -9 M, approximately 2.2 × 10 -9 M, approximately 2.1 × 10 -9 M, approximately 2.0 × 10 -9 M, approximately 1.9 × 10 -9 M, approximately 1.8 × 10 -9 M, approximately 1.7 × 10 -9 M, approximately 1.6 × 10 -9 M, approximately 1.5 × 10 -9 M, approximately 1.4 × 10 -9 M, approximately 1.3 × 10 -9 M, approximately 1.2 × 10 -9 M, approximately 1.1 × 10 -9 M, approximately 1.0 × 10 -9 M, approximately 9.9 × 10 -10 M, approximately 9.8 × 10 -10 M, approximately 9.7 × 10 -10 M, approximately 9.6 × 10 -10 M, approximately 9.5 × 10 -10 M, approximately 9.4 × 10 -10 M, approximately 9.3 × 10 -10 M, approximately 9.2 × 10 -10 M, approximately 9.1 × 10 -10 M, approximately 9.0 × 10 -10 M, approximately 8.9 × 10 -10 M, approximately 8.8 × 10 -10 M, approximately 8.7 × 10 -10 M, approximately 8.6 × 10 -10 M, approximately 8.5 × 10 -10 M, approximately 8.4 × 10 -10 M, approximately 8.3 × 10 -10 M, approximately 8.2 × 10-10 M, approximately 8.1 × 10 -10 M, approximately 8.0 × 10 -10 M, approximately 7.9 × 10 -10 M, approximately 7.8 × 10 -10 M, approximately 7.7 × 10 -10 M, approximately 7.6 × 10 -10 M, approximately 7.5 × 10 -10 M, approximately 7.4 × 10 -10 M, approximately 7.3 × 10 -10 M, approximately 7.2 × 10 -10 M, approximately 7.1 × 10 -10 M, approximately 7.0 × 10 -10 M, approximately 6.9 × 10 -10 M, approximately 6.8 × 10 -10 M, approximately 6.7 × 10 -10 M, approximately 6.6 × 10 -10 M, approximately 6.5 × 10 -10 M, approximately 6.4 × 10 -10 M, approximately 6.3 × 10 -10 M, approximately 6.2 × 10 -10 M, approximately 6.1 × 10 -10 M, approximately 6.0 × 10 -10 M, approximately 5.9 × 10 -10 M, approximately 5.8 × 10 -10 M, approximately 5.7 × 10 -10 M, approximately 5.6 × 10 -10 M, approximately 5.5 × 10 -10 M, approximately 5.4 × 10 -10 M, approximately 5.3 × 10 -10 M, approximately 5.2 × 10 -10 M, approximately 5.1 × 10 -10 M, approximately 5.0 × 10 -10 M, approximately 4.9 × 10 -10 M, approximately 4.8 × 10 -10 M, approximately 4.7 × 10 -10 M, approximately 4.6 × 10 -10 M, approximately 4.5 × 10 -10 M, approximately 4.4 × 10 -10 M, approximately 4.3 × 10 -10 M, approximately 4.2 × 10 -10 M, approximately 4.1 × 10 -10 M, approximately 4.0 × 10 -10 M, approximately 3.9 × 10 -10 M, approximately 3.8 × 10 -10 M, approximately 3.7 × 10 -10M, approximately 3.6 × 10 -10 M, approximately 3.5 × 10 -10 M, approximately 3.4 × 10 -10 M, approximately 3.3 × 10 -10 M, approximately 3.2 × 10 -10 M, approximately 3.1 × 10 -10 M, approximately 3.0 × 10 -10 M, approximately 2.9 × 10 -10 M, approximately 2.8 × 10 -10 M, approximately 2.7 × 10 -10 M, approximately 2.6 × 10 -10 M, approximately 2.5 × 10 -10 M, 2.4 × 10 -10 M, approximately 2.3 × 10 -10 M, approximately 2.2 × 10 -10 M, approximately 2.1 × 10 -10 M, approximately 2.0 × 10 -10 M, approximately 1.9 × 10 -10 M, approximately 1.8 × 10 -10 M, approximately 1.7 × 10 -10 M, approximately 1.6 × 10 -10 M, approximately 1.5 × 10 -10 M, 1.4 × 10 -10 M, approximately 1.3 × 10 -10 M, approximately 1.2 × 10 -10 M, approximately 1.1 × 10 -10 M, approximately 1.0 × 10 -10 M, approximately 9×10 -11 M, approximately 8×10 -11 M, approximately 7×10 -11 M, approximately 6×10 -11 M, approximately 5×10 -11 M, approximately 4×10 -11 M, approximately 3×10 -11 M, approximately 2 × 10 -11 M, approximately 1×10 -11 M, approximately 9×10 -12 M, approximately 8×10 -12 M, approximately 7×10 -12 M, approximately 6×10 -12 M, approximately 5×10 -12 M, approximately 4×10 -12 M, approximately 3×10 -12 M, approximately 2 × 10 -12 M, approximately 1×10 -12 M, approximately 9×10 -13 M, also about 8×10 -13M binds to cynomolgus macaque PAR-2 ​​via KD.

[0118] In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment is approximately 9.5 × 10⁻⁶ -9 M's K D It specifically binds to cynomolgus monkey PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 8.5 × 10⁻⁶. -9 M's K D It specifically binds to cynomolgus monkey PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 7.5 × 10⁻⁶. -9 M's K D It specifically binds to cynomolgus monkey PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 6.5 × 10⁻⁶. -9 M's K D It specifically binds to cynomolgus monkey PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment is approximately 5.5 × 10⁻⁶. -9 M's K D It specifically binds to cynomolgus monkey PAR-2. In some embodiments, the anti-PAR-2 ​​antibody or its antigen-binding fragment has a density of approximately 4.5 × 10⁻⁶. -9 M's K D It specifically binds to cynomolgus monkey PAR-2.

[0119] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to a certain extent, thereby blocking the binding of the reference antibody to that epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0120] An antibody that "competes with another antibody for binding to the target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to the target. Whether two antibodies compete with each other for binding to the target, that is, whether one antibody inhibits the binding of the other antibody to the target, and to what extent, can be determined using known competition experiments, such as BIACORE® surface plasmon resonance (SPR) analysis. In some embodiments, an antibody competes with and inhibits the binding of another antibody to the target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody initially incubated with the target). Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA 1999. Two antibodies "cross-compete" if the antibodies block each other by at least 50% bidirectionally, i.e., regardless of whether one antibody or the other comes into contact with the antigen first in the competitive experiment.

[0121] A competitive binding assay to determine whether two antibodies compete for binding or cross-compete for binding includes, for example, flow cytometry, such as that described in the examples, to determine the competition for binding to PAR-2-expressing cells. Other methods include SPR (e.g., BIACORE®), BLI (Biolayer Interference), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology Vol. 9: p. 242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. Vol. 137: p. 3614 (1986)); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using I-125 labeling (see Morel et al., Mol. Immunol. Vol. 25 (No. 1): p. 7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., Virology See Vol. 176: p. 546 (1990); and directly labeled RIA (see Moldenhauer et al., Scand. J. Immunol. Vol. 32: p. 77 (1990)).

[0122] "k assoc " or "k a When used herein, the term "k" is intended to refer to the binding rate of a particular antibody-antigen interaction, and "k" is used to refer to the binding rate of a particular antibody-antigen interaction. dis " or "k d When used herein, the term "K" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. D The term "k" is used herein, when used in this specification. d k a The ratio to (i.e., k d / k a The term K of an antibody is intended to refer to the dissociation constant, which is obtained from the formula and expressed as molar concentration (M). DThe value can be determined using methods well established in the art. Antibody K D Methods available to determine this include surface plasmon resonance, biosensor systems such as BIACORE®, BLI (biolayer interferometry) systems, flow cytometry, and scatchard analysis.

[0123] As used herein, the term “high affinity” for IgG antibodies means 10% affinity for the target antigen. -8 M or lower, 10 -9 M or lower, or 10 -10 M or a lower K D This refers to antibodies that possess high affinity binding. However, "high affinity" binding may differ for other antibody isotypes. For example, the "high affinity" binding for IgM isotypes is 10 -10 M or lower or 10 -8 M or a lower K D This refers to antibodies that possess [a certain characteristic].

[0124] "EC" in the context of in vitro or in vivo assays 50 The term "IC" refers to the effective concentration of a drug that induces 50% of the maximum response, i.e., an intermediate response between the maximum response and the baseline. Typically, this value is used when evaluating antibody efficacy. "IC" in the context of in vitro or in vivo assays in which an antibody or its antigen-binding fragment is used. 50 The term "inhibitory potency" refers to the inhibitory concentration of an antibody or its antigen-binding fragment that inhibits the response to 50% of the maximum response, i.e., to a level midway between the maximum response and the baseline. Typically, this "inhibitory potency" value is used when evaluating antibody efficacy.

[0125] In this specification, the term “naturally occurring” as applied to an object refers to the fact that the object can be found in nature. For example, polypeptides or polynucleotide sequences that are present in living organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in the laboratory are considered naturally occurring.

[0126] "Isolated" polypeptides, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Examples of isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those purified to a degree that they are no longer found in nature. In some embodiments, isolated antibodies, polynucleotides, vectors, cells, or compositions are substantially pure. As used herein, "substantially pure" means a substance that is at least 50% pure (i.e., free of impurities), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0127] The terms “polypeptide,” “peptide,” and “protein” are used synonymously herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acid molecules. The term also includes amino acid polymers that are modified naturally or by intervention, for example, by other operations or modifications such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. For example, polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are also included in this definition. Since the polypeptides of this disclosure are antibody-based, it will be understood that in some embodiments, the polypeptides may arise as a single chain or as a chain of associated chains.

[0128] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0129] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, the predicted non-essential amino acid residues of an anti-PAR-2 ​​antibody are replaced with other amino acid residues from the same side-chain family. Methods for identifying nucleotide and amino acid conserved substitutions that do not exclude antigen binding are well known in the art (see, for example, Brummell et al., Biochem. Vol. 32: pp. 1180-1187 (1993); Kobayashi et al., Protein Eng. Vol. 12 (No. 10): pp. 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA Vol. 94: pp. 412-417 (1997)).

[0130] The percentage of identity between two arrays is a function of the number of identical positions shared by the arrays, taking into account the number of gaps that need to be introduced to optimally align the two arrays and the length of each gap (i.e., array identity % = number of identical positions / total number of positions × 100). The comparison of arrays between two arrays and the determination of the percentage of identity can be achieved using the mathematical algorithms described in the non-restrictive examples below.

[0131] The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) with the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm by E. Meyers and W. Miller (CABIOS, Vol. 4: pp. 11-17 (1989)) incorporated into the ALIGN program (version 2.0), with the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. In addition, the percentage of identity between two amino acid sequences can be determined using either the Blossum62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch (J. Mol. Biol. (Vol. 48): pp. 444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available at www.gcg.com).

[0132] The nucleic acid and protein sequences described herein can be further used as "query sequences" to identify related sequences, for example, by performing searches against public databases. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described in Altschul et al., (1990) J. Mol. Biol. Vol. 215: pp. 403-4010. A BLAST nucleotide search using the NBLAST program with a score of 100 and a word length of 12 can be performed to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search using the XBLAST program with a score of 50 and a word length of 3 can be performed to obtain amino acid sequences homologous to the protein molecules described herein. For comparative purposes, gapped alignments can be obtained using the gapped BLAST described in Altschul et al., (1997) Nucleic Acids Res. Vol. 25 (No. 17): pp. 3389-3402. When using the BLAST and gapped BLAST programs, the initial setting parameters of each program (e.g., XBLAST and NBLAST) can be used. Please refer to worldwideweb.ncbi.nlm.nih.gov.

[0133] Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are considered "isolated" or "substantially purified" if they have been purified from other cellular components or contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing and Wiley Interscience, New York (1987).

[0134] Nucleic acids, such as cDNA, can be mutated according to standard techniques for providing gene sequences. In the case of coding sequences, such mutations can, if desired, affect the amino acid sequence. In particular, DNA sequences that are substantially homologous to or derived from natural V, D, J, stationary, switch, and other such sequences described herein are intended (where “derived” indicates that the sequence is identical to or modified from another sequence).

[0135] As used herein, the terms “host cell” or “recombinant host cell” may refer to any type of cell, e.g., primary cells, cells in culture, or cells derived from a cell line. In some embodiments, the term “host cell” refers to cells transfected with nucleic acid molecules, and their offspring or potential offspring. Such offspring may not be identical to the parent cells that transfected the nucleic acid molecules, for example, due to mutations or environmental influences that may occur in subsequent generations or as a result of incorporating the nucleic acid molecules into the host cell genome.

[0136] As used herein, the term “conjugated” refers to the association of two or more molecules. The conjugation may be covalent or non-covalent. Furthermore, the conjugation may be genetic (i.e., recombinant fusion). Such conjugations can be achieved using a wide variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0137] As used herein, the term "cytokine" refers to any of a number of factors that exert various effects on cells, including, for example, inducing growth or proliferation. Non-limiting examples of cytokines that can be used alone or in combination in the practice of the present invention include interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-12 Examples of cytokines include IL-15, interleukin-18, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor-A (VEGF-A), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 beta (IL-1β), interferon-γ (IFNγ), tumor necrosis factor (TNF), MIP-11, leukemia suppressor (LIF), c-kit ligand, thrombopoietin (TPO), and flt3 ligand. Cytokines are commercially available from several companies, such as Genzyme (Framingham, Massachusetts), Genentech (South San Francisco, California), Amgen (Thousand Oaks, California), R&D Systems (Minneapolis, Minnesota), and Immunex (Seattle, Washington). Although not always explicitly stated, molecules having similar biological activity to wild-type or purified cytokines (e.g., recombinantly produced or their mutains) are intended for use within the spirit and scope of this disclosure.

[0138] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the effective biological activity of an active ingredient and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation will be administered. The preparation may be sterile.

[0139] When used herein, terms such as “administer,” “give administration,” and “give administration” refer to methods that can be used to deliver a drug, such as an anti-human PAR-2 ​​antibody or its antigen-binding fragment, to a desired site of biological action. Administration techniques that can be used with the drugs and methods described herein are found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pennsylvania. Routes of administration for antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. When used herein, the term "parenteral administration" typically refers to, but is not limited to, methods of administration other than enteral and topical administration, usually by injection, including, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraventricular, intravitreous, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via parenteral routes such as topical, epidermal, or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual, or topically. Furthermore, administration may be carried out, for example, once, multiple times, and / or over a longer period of time, one or more times.

[0140] As used herein, the terms “subject” and “patient” are used synonymously. The subject may be a non-human animal or other mammal (e.g., a cattle, pig, horse, cat, dog, rat, mouse, monkey, or other primate). In some embodiments, the subject is a human. In some embodiments, the subject is a cynomolgus macaque.

[0141] The term “effective dose” or “effective dosage” is defined as the amount of a drug, such as an anti-human PAR-2 ​​antibody or its antigen-binding fragment, sufficient to achieve the desired effect. The “therapeutic effective dose” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as manifested by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, an increase in overall survival (the period from the date of diagnosis of a disease, such as cancer, or the start of treatment, to the time when a patient diagnosed with the disease is still alive), or the prevention of functional or physical disability due to disease distress. The therapeutic effective dose or dosage of a drug includes the “preventive effective dose” or “preventive effective dosage” and is any amount of the drug that, when administered alone or in combination with another therapeutic agent, inhibits the onset or recurrence of the disease in a subject at risk of developing or experiencing a relapse of the disease. The ability of therapeutic agents to promote disease regression or inhibit the onset or recurrence of disease can be evaluated using various methods known to those skilled in the art in human subjects during clinical trials, in animal model systems where efficacy in humans is predicted, or by assaying the activity of the agent in in vitro assays.

[0142] For example, anticancer drugs are drugs that promote the regression of the target cancer. In some embodiments, a therapeutically effective dose of a drug promotes cancer regression to the extent that it eliminates the cancer. "Promoting cancer regression" means that administration of an effective dose of the drug alone or in combination with an antineoplastic agent results in a reduction of tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, an increase in overall survival, prevention of functional or physical disability due to disease distress, or otherwise improvement of the patient's disease symptoms. In addition, the terms "effective" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or biological level resulting from the administration of the drug.

[0143] As an example of tumor treatment, a therapeutically effective dose or dosage of a drug inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to an untreated subject. In some embodiments, a therapeutically effective dose or dosage of a drug completely inhibits cell proliferation or tumor growth, i.e., inhibits cell proliferation or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be evaluated using the assays described below. Alternatively, this property of a composition can be evaluated by investigating the ability of a compound to inhibit cell proliferation, and such inhibition can be measured in vitro by assays known to those skilled in the art. In some embodiments described herein, tumor regression can be observed or continued for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0144] Terms such as “to treat,” “treatment,” “to alleviate,” or “to alleviate” refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, improving, inhibiting, slowing, or preventing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with a disease, or enhancing overall survival. Treatment may be for a subject with the disease or for a subject without the disease (for example, for prevention). In certain embodiments, a subject is considered to have been successfully "treated" by the methods provided herein if the patient exhibits one or more of the following: a reduction in the number of cancer cells or their complete absence; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, including, for example, the spread of cancer to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; reduction of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumor's tumorigenicity, tumorigenicity, or tumorigenic capacity; a reduction in the number or frequency of cancer stem cells within the tumor; differentiation of tumorigenic cells into a non-tumoric state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS); a complete response (CR), a partial response (PR), stable disease (SD), a decrease in disease progression (PD), a decrease in time to progression (TTP), or any combination thereof.

[0145] In some embodiments, a patient is considered to have successfully received “treatment” for chronic obstructive pulmonary disease (COPD) by the methods provided herein if the patient exhibits one or more of the following: prevention or control of COPD symptoms, reduction in the frequency and severity of COPD exacerbations, improvement in health status, improvement in exercise tolerance, or any combination thereof. See, for example, Rabe et al., Am J Respir Crit Care Med., vol. 176: pp. 532–555 (2007).

[0146] In some embodiments, a patient is considered to have successfully received “treatment” for asthma by the methods provided herein if the patient exhibits one or more of the following: prevention or control of asthma symptoms, reduction in the frequency and severity of asthma exacerbations, improvement in health status, improvement in exercise tolerance, or any combination thereof. See, for example, Gatheral et al., Cochrane Database Syst Rev., Vol. 4, No. 4, (2017).

[0147] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by uncontrolled cell proliferation of a population of cells. Such cancers may include solid tumors, for example, solid tumors in which bone marrow cells (monocytes, macrophages, dendritic cells, granulocytes, neutrophils, microglia, or other innate immune cells) invade the tumor microenvironment. Examples of such cancers include, but are not limited to, glioblastoma, head and neck cancer, kidney cancer (e.g., clear cell carcinoma), pancreatic cancer, and breast cancer. Cancer may also be a “PAR-2 ​​positive cancer.” This term refers to cancer containing cells that express PAR-2 ​​mRNA or protein. Cancer may also have “enhanced PAR-2” mRNA or protein. This refers to cancer that has more PAR-2 ​​than healthy cells of the same tissue (e.g., on cells invaded by the cancer).

[0148] As used herein, the term "in vitro" refers to events occurring in an artificial environment, such as a test tube or reaction vessel, a cell culture, or a petri dish, rather than within a living organism (e.g., an animal, plant, or microorganism).

[0149] As used herein, the term "in vivo" refers to events occurring within a living organism (for example, an animal, plant, or microorganism, or their cells or tissues).

[0150] As used herein, the terms "ug" and "uM" are synonymous with "μg" and "μM," respectively.

[0151] Various embodiments described herein are described in further detail in the following subsections. Any composition or method provided herein may be combined with any one or more of the other compositions and methods provided herein.

[0152] II. Antibodies In some embodiments, this specification provides antibodies that specifically bind to PAR-2, such as human and cynomolgus monkey antibodies (e.g., monoclonal antibodies such as humanized antibodies), and their antigen-binding fragments. The amino acid sequences of human, cynomolgus monkey, and rat PAR-2 ​​are known in the art and are provided herein by the numbers SEQ ID NOs. 28, 30, and 32, respectively.

[0153] Human PAR-2 ​​(UniProt identification number P55085-1; SEQ ID NO: 28) is composed of 397 amino acids (including the signal peptide) and is a typical member of the class AG protein-coupled receptor superfamily, structurally possessing seven transmembrane domains, an extracellular N-terminus, and an intracellular C-terminus. A disulfide linkage exists between extracellular loop 1 and extracellular loop 2, which is thought to stabilize the structure and contribute to signal transduction. The crystal structure of PAR-2 ​​has been analyzed. See Cheng et al., Nature, vol. 545: pp. 112-115 (2017). This entire document is incorporated herein by reference.

[0154] The following is the amino acid sequence of one known human PAR-2 ​​isoform. Human PAR-2 ​​(UniProt identification number P55085-1; Sequence ID 28)

[0155] [ka]

[0156] The signal sequence of human PAR-2 ​​corresponds to amino acids 1-25 (underlined). Therefore, the mature isoform of human PAR-2 ​​isoform 1 consists of amino acids 26-397.

[0157] Human PAR-2 ​​(without signal sequence) (SEQ ID NO: 109)

[0158] [ka]

[0159] The above human PAR-2 ​​sequence (SEQ ID NO: 28) represents a natural sequence in which serine (S) is located at position 21, asparagine (N) at position 30, arginine (R) at position 270, and threonine (T) at position 291. In some embodiments, a natural variant human PAR-2 ​​sequence in which position 21 is occupied by phenylalanine (F) is intended. In some embodiments, a natural variant human PAR-2 ​​sequence in which position 30 is occupied by serine (S) is intended. In some embodiments, a natural variant human PAR-2 ​​sequence in which position 270 is occupied by glutamine (Q) is intended. In some embodiments, a natural variant human PAR-2 ​​sequence in which position 291 is occupied by alanine (A) is intended.

[0160] In some embodiments, the human PAR-2 ​​sequence is intended to lack its signal sequence. For example, the human PAR-2 ​​sequence may include amino acids 26-397 of SEQ ID NO: 28.

[0161] In some embodiments, the human PAR-2 ​​sequence is intended to lack its signal sequence (amino acids 1-25 of SEQ ID NO: 28) and propeptide sequence (amino acids 26-36 of SEQ ID NO: 28), which can be removed for receptor activation. "N-terminus of human PAR2" refers to amino acids 1-71 of SEQ ID NO: 28.

[0162] Other features of human PAR-2 ​​shown in Sequence ID No. 28 include the extracellular domain (ECD) 1 (approximately amino acids 138-149), the ECD2 (approximately amino acids 212-235), the ECD3 domain (approximately amino acids 318-323), the transmembrane domain (TM) 1 (approximately amino acids 72-101), the TM2 (approximately amino acids 109-137), the TM3 (approximately amino acids 150-177), the TM4 (approximately amino acids 184-212), the TM5 (approximately amino acids 236-269), the TM6 (approximately amino acids 278-317), and the TM7 (approximately amino acids 324-347).

[0163] Cryptomolgus macaque PAR-2 ​​(UniProt identification number E5FAJ7; SEQ ID NO: 30)

[0164] [ka]

[0165] In some embodiments, the cynomolgus monkey PAR-2 ​​sequence is intended to lack its signal sequence. For example, the cynomolgus monkey PAR-2 ​​sequence may contain amino acids 21-438 of SEQ ID NO: 31.

[0166] Cynomolgus macaque PAR-2 ​​(lacking signal sequence) (SEQ ID NO: 31)

[0167] [ka]

[0168] Rat PAR-2 ​​(UniProt identification number Q63645; SEQ ID NO: 32)

[0169] [ka]

[0170] In some embodiments, the rat PAR-2 ​​sequence is intended to lack its signal sequence. For example, the rat PAR-2 ​​sequence may contain amino acids 26-397 of SEQ ID NO: 32.

[0171] In some embodiments, the antibody or antigen-binding fragment described herein binds to human PAR-2 ​​(for example, SEQ ID NO: 28 or amino acids 26-397 of SEQ ID NO: 28, or any of the above sequences in which S or F is located at position 21, N or S is located at position 30, R or Q is located at position 270, or T or A is located at position 291).

[0172] In some embodiments, the antibody or its antigen-binding fragment binds to human PAR-2 ​​and cynomolgus monkey PAR-2 ​​(e.g., amino acids 21-438 of SEQ ID NO: 30 or SEQ ID NO: 30). In some embodiments, the antibody or its antigen-binding fragment binds to human PAR-2 ​​and rat PAR-2 ​​(e.g., amino acids 26-397 of SEQ ID NO: 32 or SEQ ID NO: 32). In some embodiments, the antibody or its antigen-binding fragment binds to human PAR-2, cynomolgus monkey PAR-2, and rat PAR-2. In some embodiments, the antibody or its antigen-binding fragment binds to human PAR-2 ​​but not to rat PAR-2 ​​(e.g., amino acids 26-397 of SEQ ID NO: 32 or SEQ ID NO: 32). In some embodiments, the antibody or its antigen-binding fragment binds to human PAR-2 ​​and optionally to rat PAR-2. In some embodiments, the antibody or its antigen-binding fragment binds to human PAR-2, cynomolgus monkey PAR-2, and optionally to rat PAR-2.

[0173] Specific antibodies that can be used in the methods disclosed herein are antibodies that specifically bind to PAR-2 ​​such as human, cynomolgus monkey, and rat PAR-2 ​​(e.g., monoclonal antibodies such as humanized antibodies) and antigen-binding fragments thereof, having the CDR and / or variable region sequences of antibodies 309-4e, P24E1102, P24E976, P24E1099, and / or P24E1103 constructed in Examples 1-4, as well as antibodies having at least 80% identity (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity) of their variable region or CDR sequence. The VH amino acid sequences of 309-4e, P24E1102, P24E976, P24E1099, and P24E1103 are shown in SEQ ID NOs. 20, 21, 21, 21, and 21, respectively. The VL amino acid sequences of 309-4e, P24E1102, P24E976, P24E1099, and P24E1103 are shown in SEQ ID NOs. 23, 24, 25, 26, and 27, respectively.

[0174] In some embodiments, the antibodies or antigen-binding fragments described herein bind specifically to human PAR-2, and the antibodies or antigen-binding fragments are, respectively, sequence numbers 1 (GFSLX1X2YX3X4X5), 2 (VIWGNX6NX7YYX8), and 3 (WX9GX). 10 KDX 11 PFDY), Sequence ID 4 (X 12 ASQNX 13 YKX 14 LD), Sequence ID 5(X 15 X 16 X 17 X 18 X 19 X 20 T), and sequence number 6(X 21 QHX 22 X 23 The sequence includes the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 sequences of the GWT, and within the sequence, X1 = Asparagine (N) or Serine (S) X2 = serine (S) or tyrosine (Y) X3 = Glycine (G) or Alanine (A) X4 = valine (V), glycine (G), or isoleucine (I) X5 = Isoleucine (I) or Serine (S) X6 = Glycine (G) or Glutamine (Q) X7 = valine (V) or threonine (T) X8 = Asparagine (N), Alanine (A), Glycine (G), or Tyrosine (Y) X9 = Arginine (R) or Lysine (K) X 10 = Tyrosine (Y), tryptophan (W), or phenylalanine (F) X 11 = Tyrosine (Y) or histidine (H) X 12 = Lysine (K) or Arginine (R) X 13 = Isoleucine (I) or valine (V) X 14 = Tyrosine (Y), tryptophan (W), or phenylalanine (F) X 15 = Asparagine (N) or aspartic acid (D), X 16 = Threonine (T) or Alanine (A) X 17 = Asparagine (N), serine (S), or tyrosine (Y) X 18 = Serine (S), threonine (T), or asparagine (N) X 19 = Leucine (L) or Arginine (R) X 20 = Histidine (H) or alanine (A) X 21 = Leucine (L) or glutamine (Q), X 22 =Asparagine (N), glycine (G), or histidine (H), and X23 = Serine (S) or Histidine (H) That is the case.

[0175] In some embodiments, X1 = serine (S), X2 = serine (S), X3 = alanine (A), X3 = isoleucine (I), X5 = serine (S), X6 = glutamine (Q), X7 = valine (V), X8 = alanine (A), X9 = lysine (K), X 10 = Tyrosine (Y), X 11 = Tyrosine (Y), X 12 =Arginine(R), X 13 = Valine (V), X 14 = Tryptophan (W), X 15 = Asparagine (N), X 16 = Alanine (A), X 17 = Asparagine (N), X 18 = Threonine (T), X 19 =Arginine(R), X 20 = Alanine (A), X 21 = Glutamine (Q), X 22 = Histidine (H), and X 23 = Serine (S).

[0176] In some embodiments, the antibody or antigen-binding fragment described herein is bound to human PAR-2 ​​and comprises six CDRs of the antibody listed in Table 2 (i.e., three VH CDRs and three VL CDRs of the same antibody listed in Table 2).

[0177] [Table 2]

[0178] The following VH CDR residues were used to define the VH CDRs in Table 2: AbM nomenclature heavy chain CDR1 (residues H26, H27, H28, H29, H30, H31, H32, H33, H34, and H35 as defined by the Kabat numbering scheme). Kabat heavy chain CDR2, excluding the last five amino acids (residues H50, H51, H52, H53, H54, H55, H56, H57, H58, H59, and H60 as defined by the Kabat numbering scheme). Kabat heavy chain CDR3, excluding the last five amino acids (residues H95, H96, H97, H98, H99, H100, H100A, H100B, H100C, H101, and H102 as defined by the Kabat numbering scheme). The following VL CDR residues were used to define the VL CDRs in Table 2: Kabat-as defined light chain CDRs (as defined by the Kabat numbering scheme, CDR1 consists of residues L24, L25, L26, L27, L28, L29, L30, L31, L32, L33, and L34; CDR2 consists of residues L50, L51, L52, L53, L54, L55, and L56; CDR3 consists of residues L89, L90, L91, L92, L93, L94, L96, and L97).

[0179] In some embodiments, the antibodies or antigen-binding fragments described herein are conjugated to human PAR-2 ​​and include the VH and VL of the antibodies listed in Table 3 (i.e., the VH and VL of the same antibodies listed in Table 3).

[0180] [Table 3-1] [Table 3-2]

[0181] Furthermore, an isolated anti-PAR-2 ​​antibody or an antigen-binding fragment thereof, comprising a heavy chain and a light chain variable region, is provided, wherein (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26; or (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 27.

[0182] This specification provides an isolated anti-PAR-2 ​​antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as SEQ ID NO: 20 or 21, and the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as SEQ ID NO: 23, 24, 25, 26, or 27.

[0183] In some aspects, this disclosure is, (a) Heavy chain and light chain variable region sequences containing sequence numbers 20 and 23, respectively (b) Heavy chain and light chain variable region sequences containing sequence numbers 21 and 24, respectively (c) Heavy chain and light chain variable region sequences containing sequence numbers 21 and 25, respectively, (d) Heavy chain and light chain variable region sequences containing sequence numbers 21 and 26, respectively, or (e) Heavy and light chain variable region arrays containing sequence numbers 21 and 27, respectively. The present invention provides an isolated anti-PAR-2 ​​antibody or its antigen-binding fragment, which contains [the specified substance].

[0184] The amino acid sequences of VH CDR1, CDR2, and CDR3 of antibodies P24E1102, P24E976, P24E1099, and P24E1103 are shown in SEQ ID NOs. 10, 11, and 12, respectively. The amino acid sequences of VH CDR1, CDR2, and CDR3 of antibody 309-4e are shown in SEQ ID NOs. 7, 8, and 9, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 of antibody P24E1102 are shown in SEQ ID NOs. 16, 17, and 18, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 of antibody P24E976 are shown in SEQ ID NOs. 16, 19, and 18, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 of antibody P24E1099 are shown in SEQ ID NOs. 16, 20, and 18, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 of antibody P24E1103 are shown in SEQ ID NOs. 16, 21, and 18, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 of antibody 309-4e are shown in SEQ ID NOs. 13, 14, and 15, respectively.

[0185] In some embodiments, an anti-PAR-2 ​​antibody or its antigen-binding fragment that specifically binds to human PAR-2 ​​is (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 10, (b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 11, (c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, (d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, (e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 17, and (f) VL CDR3 containing the amino acid sequence of SEQ ID NO: 18 Includes.

[0186] In some embodiments, an anti-PAR-2 ​​antibody or its antigen-binding fragment that specifically binds to human PAR-2 ​​is (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 10, (b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 11, (c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, (d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, (e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and (f) VL CDR3 containing the amino acid sequence of SEQ ID NO: 18 Includes.

[0187] In some embodiments, an anti-PAR-2 ​​antibody or its antigen-binding fragment that specifically binds to human PAR-2 ​​is (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 10, (b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 11, (c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, (d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, (e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 29, and (f) VL CDR3 containing the amino acid sequence of SEQ ID NO: 18 Includes.

[0188] In some embodiments, an anti-PAR-2 ​​antibody or its antigen-binding fragment that specifically binds to human PAR-2 ​​is (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 10, (b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 11, (c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, (d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, (e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and (f) VL CDR3 containing the amino acid sequence of SEQ ID NO: 18 Includes.

[0189] In some embodiments, an anti-PAR-2 ​​antibody or its antigen-binding fragment that specifically binds to human PAR-2 ​​is (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 7, (b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, (c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 9, (d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 13, (e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 14, and (f) VL CDR3 containing the amino acid sequence of SEQ ID NO: 15 Includes.

[0190] In some embodiments, antibodies comprising a heavy chain (HC) and a light chain (LC) are provided herein. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the antibody described herein that specifically binds to human PAR-2 ​​includes a heavy chain in which the amino acid sequence of the VH domain is the amino acid sequence shown in Table 3, and the constant region of the heavy chain includes the amino acid sequence of the human gamma (γ) heavy chain constant region. In some embodiments, the antibody described herein that specifically binds to human PAR-2 ​​includes a heavy chain in which the amino acid sequence of the VH domain is the amino acid sequence shown in Table 3, and the constant region of the heavy chain includes the amino acid sequence of the IgG1 heavy chain constant region. In some embodiments, the antibodies described herein that specifically bind to human PAR-2 ​​include a heavy chain in which the amino acid sequence of the VH domain is the sequence shown in Table 3, and the constant region of the heavy chain includes the amino acid sequence of the IgG2 (e.g., IgG2a or IgG2b) heavy chain constant region. In some embodiments, the antibodies described herein that specifically bind to human PAR-2 ​​include a heavy chain in which the amino acid sequence of the VH domain is the sequence shown in Table 3, and the constant region of the heavy chain includes the amino acid sequence of the IgG4 heavy chain constant region. In some embodiments, the antibodies described herein that specifically bind to human PAR-2 ​​include a heavy chain in which the amino acid sequence of the VH domain is the sequence shown in Table 3, and the constant region of the heavy chain includes the amino acids of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences are described in the art, see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al. (1991), cited above. Each of these documents is incorporated herein by reference in its entirety.

[0191] In some embodiments, the antibodies described herein that specifically bind to human PAR-2 ​​may include, but are not limited to, modifications that modulate serum half-life and in vivo distribution, including modifications that modulate the interaction of the antibody with the neonatal Fc receptor (FcRn), a receptor that plays an important role in protecting IgG from catabolism and maintaining high serum antibody concentrations. Serum half-life modulating modifications, including triple substitution of M252Y / S254T / T256E (numbered according to the EU numbering system (see Edelman, GM et al., Proc. Natl. Acad., USA, Vol. 63: pp. 78-85 (1969)) as described in U.S. Patent No. 7,083,784), may be made in the Fc region of IgG1, IgG2, or IgG4. Other substitutions may be made at positions 250 and 428 (see, for example, U.S. Patent No. 7,217,797) and positions 307, 380, and 434 (see, for example, International Publication No. 00 / 042072). Examples of constant domain amino acid substitutions that modulate subsequent functions mediated by such receptors, including Fc receptor binding, FcRn binding, and serum half-life, are described in U.S. Patent Publication No. 2009 / 0142340, U.S. Patent Publication No. 2009 / 0068175, and U.S. Patent Publication No. 2009 / 0092599.

[0192] Antibodies of any class may have their heavy chain C-terminal lysine excluded or removed to reduce heterogeneity (ΔK). The substitution of S228P (EU numbering) in human IgG4 can stabilize antibody Fab arm exchange in vivo (e.g., Labrin et al., Nature Biotechnol. Vol. 27 (No. 8), pp. 767-773 (2009)), and this substitution may be present simultaneously with M252Y / S254T / T256E and / or ΔK modifications.

[0193] The human heavy chain IgG4 constant region that can be used in this disclosure is selected from the group consisting of wild-type human IgG4 (SEQ ID NO: 33), human IgG4 (ΔK), human IgG4 S228P, human IgG4 S228P(ΔK), human IgG4 228P / 252Y / 254T / 256E, human IgG4 228P / 252Y / 254T / 256E(ΔK), human IgG4 252Y / 254T / 256E, and human IgG4 252Y / 254T / 256E(ΔK). (Human heavy chain IgG4 constant region (UniProt identification number P01861; SEQ ID NO: 33))

[0194] [ka]

[0195] In some embodiments, the anti-human PAR-2 ​​antibody includes a human IgG4 constant region, the IgG4 constant region includes an amino acid substitution corresponding to S228P (according to EU numbering). In some embodiments, the anti-human PAR-2 ​​antibody includes a human IgG4 constant region, the IgG4 constant region includes a terminal lysine deletion (K447Δ). In some embodiments, the anti-human PAR-2 ​​antibody includes a human IgG4 constant region, the IgG4 constant region includes substitutions corresponding to S228P (according to EU numbering) and a terminal lysine deletion (K447Δ) (for example, provided in SEQ ID NO: 34).

[0196] [ka]

[0197] In some embodiments, the anti-human PAR-2 ​​antibody includes a human IgG1 constant region. The human heavy chain IgG1 constant region used in this disclosure is selected from the group consisting of wild-type human IgG1 (SEQ ID NO: 35), human IgG1 (ΔK), human IgG1 252Y / 254T / 256E, human IgG1 252Y / 254T / 256E(ΔK), human IgG1 L235A / G237A, human IgG1 L235A / G237A(ΔK), human IgG1 L234A / L235A / G237A, and human IgG1 L234A / L235A / G237A(ΔK). (Human heavy chain IgG1 constant region (UniProt identification number P01857; SEQ ID NO: 35))

[0198] [ka]

[0199] In some embodiments, the anti-human PAR-2 ​​antibody includes a human IgG2 constant region. The human heavy chain IgG2 constant region usable in this disclosure is selected from the group consisting of wild-type human IgG2 (SEQ ID NO: 36), human IgG2 (ΔK), and human IgG2 A330S / P331S. (Human heavy chain IgG2 constant region (UniProt identification number P01859; SEQ ID NO: 36))

[0200] [ka]

[0201] With respect to the light chain, in some embodiments, the light chain of the antibody described herein is a kappa light chain.

[0202] In some embodiments, the antibody described herein that specifically binds to human PAR-2 ​​includes a light chain in which the amino acid sequence of the VL domain is the sequence shown in Table 3, and the constant region of the light chain includes the amino acid sequence of the human kappa light chain constant region. In some embodiments, the antibody described herein that specifically binds to human PAR-2 ​​includes a light chain in which the amino acid sequence of the VL domain is the sequence shown in Table 3, and the constant region of the light chain includes the amino acid sequence of the human kappa light chain constant region (for example, provided in SEQ ID NO: 37). Non-limiting examples of human constant region sequences are described in the Art, see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al. (1991), cited above.

[0203] [ka]

[0204] In some embodiments, the antibody described herein that specifically binds to human PAR-2 ​​comprises a VH domain and a VL domain containing the amino acid sequence of any of the anti-human PAR-2 ​​antibodies described herein, and the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the antibody described herein that specifically binds to human PAR-2 ​​comprises a VH domain and a VL domain containing the amino acid sequence of any of the anti-human PAR-2 ​​antibodies described herein, and the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b). In some embodiments, the constant region includes the amino acid sequence of the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0205] Non-restrictive examples of human constant region sequences are described in the Art of this Technology; see, for example, Kabat EA et al. (1991), op. cit.

[0206] II(a). Exemplary Fc domains The constant region can be manipulated, for example by recombinant technology, to eliminate one or more effector functions. “Effector function” refers to the interaction between the antibody Fc region and an Fc receptor or ligand, or the resulting biochemical events. Examples of “effector functions” include FcγR-mediated effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP), as well as downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be associated with a binding domain (e.g., an antibody variable domain). Therefore, the term “constant region without Fc function” includes constant regions in which one or more effector functions mediated by the Fc region are reduced or absent.

[0207] In some therapeutic uses, it may be advantageous to reduce or minimize one or more effector functions. Antibody effector functions can be reduced or avoided by various approaches. Antibody effector functions can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv(scFV), or sdAb consisting of monomeric VH or VL domains). Alternatively, so-called deglycosylated antibodies can be produced by removing sugars linked to specific residues in the Fc region, thereby reducing the antibody's effector function while maintaining other valuable attributes of the Fc region (e.g., extended half-life and heterodimerization). Deglycosylated antibodies can be produced, for example, by deleting or altering the residue to which the sugar is attached, by enzymatically removing the sugar, by producing antibodies in cells cultured in the presence of a glycosylation inhibitor, or by expressing antibodies in cells that cannot glycosylate proteins (e.g., bacterial host cells). For example, see U.S. Patent Application Publication No. 20120100140. Another approach is to use Fc regions derived from IgG subclasses that exhibit reduced effector function. For example, IgG2 and IgG4 antibodies are characterized by lower levels of Fc effector function than IgG1 and IgG3 antibodies. The residues closest to the hinge region of the CH2 domain in the Fc portion constitute a largely overlapping binding site for C1q (complement) and the IgG-Fc receptor (FcγR) on effector cells of the innate immune system, thus contributing to the antibody's effector function. Vidarsson G. et al., Front Immunol. Vol. 5: p. 520 (published online October 20, 2014).Therefore, antibodies with reduced or absent Fc effector function can be prepared, for example, by generating a chimeric Fc region containing a CH2 domain derived from an IgG4 isotype IgG antibody and a CH3 domain derived from an IgG1 isotype IgG antibody, or a chimeric Fc region containing a hinge region derived from IgG2 and a CH2 region derived from IgG4 (see, for example, Lau C. et al., J.Immunol. Vol. 191: pp. 4769-4777 (2013)), or by generating an Fc region having amino acid substitutions and / or additions and / or deletions that result in alteration of Fc effector function, such as reduction or loss of Fc function. Such Fc regions having amino acid substitutions and / or additions and / or deletions are known in the art. For example, see U.S. Patent Application Publication No. 20120100140 and the U.S. and PCT applications cited therein, An et al., mAbs Vol. 1 (No. 6): pp. 572-579 (2009), Wang et al., Protein Cell Vol. 9 (No. 1): pp. 63-73 (2018), Tam et al., Antibodies Vol. 6 (No. 3): p. 12 (2017), Vafa et al., Methods Vol. 65: pp. 114-126 (2014), and Dumet et al., mAbs Vol. 11 (No. 8): pp. 1341-1350 (2019). The disclosures in these documents are incorporated by reference in their entirety. In other uses, for example, it may be advantageous to maintain or enhance one or more effector functions when it is desirable to kill cells such as cancer cells that express PAR-2 ​​on their cell surface.

[0208] In some embodiments, the anti-PAR-2 ​​antibody, and in particular the anti-human PAR-2 ​​antibody provided herein, may include an Fc domain. In some embodiments, the Fc domain is a human IgG1, IgG2, IgG3, and / or IgG4 isotype.

[0209] In certain embodiments, the Fc domain has an IgG1 isotype. In some embodiments, the anti-PAR-2 ​​antibody contains a mouse IgG1 Fc domain. In some embodiments, the anti-human PAR-2 ​​antibody contains a human IgG1 Fc domain (hIgG1), for example, provided in SEQ ID NO: 38.

[0210] [ka]

[0211] Human IgG1 Fc domain (lacking C-terminal lysine residue (ΔK); Sequence ID No. 39)

[0212] [ka]

[0213] In some embodiments, the human IgG1 Fc domain of the anti-human PAR-2 ​​antibody binds to an activated Fc receptor. In certain embodiments, the activated Fc receptor is selected from one or more of FcγRI, FcγRIIa and IIc, and FcγRIIIa and IIIb.

[0214] In some embodiments, the human IgG1 Fc domain of an anti-human PAR-2 ​​antibody does not bind to FcγRIII (CD16) and / or C1q, or its binding is reduced. In some embodiments, the human IgG1 Fc domain of an anti-human PAR-2 ​​antibody has reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-binding activity compared to a wild-type human IgG1 Fc domain. The above effects can be achieved by certain amino acid modifications, such as the "NSLF" substitution, in which the human IgG1 Fc domain includes amino acid substitutions N325S and L328F (according to the EU numbering of the IgG1 Fc domain), as shown in, for example, SEQ ID NO: 40. In another embodiment, the human IgG1 Fc domain includes the amino acid substitution corresponding to K322A (EU numbering), as provided in, for example, SEQ ID NO: 41.

[0215] [ka]

[0216] Exemplary modifications to the human IgG1 Fc domain are listed in Table 4 below.

[0217] [Table 4]

[0218] In certain embodiments of the anti-PAR-2 ​​antibodies provided herein, the Fc domain has an IgG2 isotype. In some embodiments, the anti-human PAR-2 ​​antibody comprises a human IgG2 Fc domain (hIgG2). In some embodiments, the human IgG2 Fc domain of the anti-human PAR-2 ​​antibody binds to an activated Fc receptor. In certain embodiments, the activated Fc receptor is selected from one or more of FcγRI, FcγRIIa and IIc, and FcγRIIIa and IIIb.

[0219] In certain embodiments of the anti-PAR-2 ​​antibodies provided herein, the Fc domain has an IgG4 isotype. In some embodiments, the anti-human PAR-2 ​​antibody includes a human IgG4 Fc domain (hIgG4), as provided, for example, in SEQ ID NO: 42. In some embodiments, the human IgG4 Fc region of the anti-human PAR-2 ​​antibody binds to an activated Fc receptor. In certain embodiments, the activated Fc receptor is selected from one or more of FcγRI, FcγRIIa and IIc, and FcγRIIIa and IIIb. In certain embodiments, the human IgG4 Fc region includes an amino acid substitution corresponding to S228P (as assigned by EU numbering), as provided, for example, in SEQ ID NO: 43. In certain embodiments, the human IgG4 Fc region includes an amino acid substitution corresponding to S228P (as assigned by EU numbering) and a deletion of a terminal lysine residue (K447Δ), as provided, for example, in SEQ ID NO: 44. In certain embodiments, the human IgG4 Fc region includes S228P substitution and L235E substitution (to reduce FcγR interaction). In certain embodiments, the human IgG4 Fc includes S228P substitution, L235E substitution, and deletion of a terminal lysine residue (K447Δ). Human IgG4 Fc domain (hIgG4)

[0220] [ka]

[0221] Human IgG4 Fc domain containing S228P

[0222] [ka]

[0223] Human IgG4 Fc domain having S228P and K447Δ

[0224] [ka]

[0225] In some embodiments, any of the constant-chain region mutations or modifications described herein can be introduced into one or both heavy-chain constant-chain regions of an antibody or antigen-binding fragment thereof described herein having two heavy-chain constant-chain regions.

[0226] In some embodiments, the antibody described herein or its antigen-binding fragment that specifically binds to human PAR-2 ​​comprises a heavy chain and a light chain, (i) the heavy chain comprises a VH domain comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences of the antibody listed in Table 2 (e.g., SEQ ID NOs. 10, 11, and 12, respectively), (ii) the light chain comprises a VL domain comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences of the same antibody listed in Table 2 (e.g., SEQ ID NOs. 16, 17, and 18, respectively), (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of the human IgG4 heavy chain, or the amino acid sequence of the constant domain of the human IgG4 heavy chain comprising amino acid substitutions corresponding to S228P (as assigned by EU) and terminal lysine deletion (K447Δ), and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of the human kappa light chain.

[0227] In some embodiments, the antibody described herein or its antigen-binding fragment that specifically binds to human PAR-2 ​​comprises a heavy chain and a light chain, (i) the heavy chain comprises a VH domain comprising the amino acid sequence of the antibody listed in Table 3 (e.g., SEQ ID NO: 21), (ii) the light chain comprises a VL domain comprising the amino acid sequence of the same antibody listed in Table 3 (e.g., SEQ ID NO: 24), (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of the human IgG4 heavy chain, or the amino acid sequence of the constant domain of the human IgG4 heavy chain comprising amino acid substitutions corresponding to S228P (according to EU numbering) and terminal lysine deletion (K447Δ), and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of the human kappa light chain.

[0228] II(b). Anti-PAR-2 ​​antibody activity Antibodies or antigen-binding fragments thereof described herein that specifically bind to human PAR-2 ​​can block the ligand-binding site of PAR-2. Blocking the binding of human PAR-2 ​​to its ligand reduces PAR-2 ​​signaling. PAR-2 ​​transmits signals via G proteins and β-arrestin. PAR-2 ​​transmits signals via various Gα proteins, most notably Gα q Gα i , and Gα 12 / 13 These G proteins transmit signals via multiple pathways, including ERK, NF-κB, cAMP, and p85 / p110. Rothmeier, AS and Ruf, W., Seminars in Immunopathology, vol. 34: pp. 133-149 (2012). β-arrestin recruitment terminates G protein-mediated signaling and initiates new signaling pathways. Nichols, HL et al., Proc Natl Acad Sci USA, vol. 109: pp. 16660-16665 (2012). β-arrestin can activate signaling via Raf and ERK, and can also induce actin rearrangement, thus affecting cell motility. Pal, K. et al., J Biol Chem, vol. 288: pp. 3265-3274 (2013).

[0229] In some embodiments, the antibodies or antigen-binding fragments described herein that specifically bind to human PAR-2 ​​(a) block the interaction between the PAR-2 ​​activating ligand and the extracellular domain of PAR-2, and / or (b) block PAR-2 ​​activation by the PAR-2 ​​activating ligand, and do not bind to amino acids 59-63 at the N-terminus of human PAR-2. Examples of such PAR-2 ​​activating ligands include, but are not limited to, PAR-2 ​​anchoring ligands (cis or trans), PAR-1 anchoring ligands, or soluble ligands (e.g., SLIGKV (SEQ ID NO: 45), SLIGRL (SEQ ID NO: 46), or synthetic soluble PAR-2 ​​activating ligands such as 2-Froyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2 trifluoroacetate (2-Froyl-LIGRLO)). Various proteases (e.g., serine proteases including trypsin, tryptase, tissue factor, neutrophil elastase, and matryptase, as well as cysteine ​​proteases including cathepsin S, papain, and Der p) can expose various PAR-2 ​​anchoring ligands that can bind to and activate PAR-2. Soluble PAR-2 ​​and PAR-1 ligands can also activate PAR-2. Furthermore, PAR-1 can transactivate PAR-2.

[0230] Anti-human PAR-2 ​​antibodies that antagonize PAR-2 ​​activation by PAR-2 ​​activating ligands can be identified, for example, by testing PAR-2 ​​activation in the presence of a PAR-2 ​​ligand (e.g., a synthetic soluble PAR-2 ​​activating ligand such as SLIGKV) and the inhibition of PAR-2 ​​activation by an anti-PAR-2 ​​antibody. 50 This is used as a measure of the inhibitory efficacy of anti-PAR-2 ​​antibodies on PAR-2 ​​activation in this system (i.e., the nM concentration of anti-PAR-2 ​​antibody that achieves 50% inhibition of ligand-induced PAR-2).

[0231] In some embodiments, the interaction between the antibody or its antigen-binding fragment and PAR-2 ​​in an intracellularly soluble PAR-2 ​​activating ligand (e.g., SLIGKV) is measured by the PAR-2β-arrestin cell assay described in Example 2.1, resulting in an IC50 of approximately 0.1 nM to approximately 17 nM. 50 It inhibits it.

[0232] In some embodiments, the antibody or antigen-binding fragment described herein that specifically binds to human PAR-2 ​​enables interaction between soluble PAR-2 ​​activating ligand (e.g., SLIGKV) and PAR-2 ​​at concentrations of approximately 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, 2 nM, and 2.1 nM. M, about 2.2nM, about 2.3nM, about 2.4nM, about 2.5nM, about 2.6nM, about 2.7nM, about 2.8nM, about 2.9nM, about 3nM, 3.1nM, about 3.2nM, about 3.3nM, about 3.4nM, about 3.5nM, about 3.6nM, about 3.7nM, about 3. 8nM, approximately 3.9nM, approximately 4nM, 4.1nM, approximately 4.2nM, approximately 4.3nM, approximately 4.4nM, approximately 4.5nM, approximately 4.6nM, approximately 4.7nM, approximately 4.8nM, approximately 4.9nM, approximately 5nM, 5.1nM, approximately 5.2nM, approximately 5.3nM, approximately 5.4nM, approximately 5.5n M, about 5.6nM, about 5.7nM, about 5.8nM, about 5.9nM, about 6nM, 6.1nM, about 6.2nM, about 6.3nM, about 6.4nM, about 6.5nM, about 6.6nM, about 6.7nM, about 6.8nM, about 6.9nM, about 7nM, 7.1nM, about 7.2nM , about 7.3nM, about 7.4nM, about 7.5nM, about 7.6nM, about 7.7nM, about 7.8nM, about 7.9nM, about 8nM, 8.1nM, about 8.2nM, about 8.3nM, about 8.4nM, about 8.5nM, about 8.6nM, about 8.7nM, about 8.8nM, about 8.9 nM, approximately 9nM, 9.1nM, approximately 9.2nM, approximately 9.3nM, approximately 9.4nM, approximately 9.5nM, approximately 9.6nM, approximately 9.7nM, approximately 9.8nM, approximately 9.9nM, approximately 10nM, 10.1nM, approximately 10.2nM, approximately 10.3nM, approximately 10.4nM, approximately 10.5nM , about 10.6nM, about 10.7nM, about 10.8nM, about 10.9nM, about 11nM, about 11.1nM, about 11.2nM, about 11.3nM, about 11.4nM, about 11.5nM, about 11.6nM, about 11.7nM, about 11.8nM, or about 11.9nM IC 50 This inhibits the interaction between soluble PAR-2 ​​ligands and PAR-2. The inhibition of this interaction may be dose-dependent on the anti-human PAR-2 ​​antibody.

[0233] In some embodiments, the antibodies or antigen-binding fragments described herein that specifically bind to human PAR-2 ​​measure intracellular PAR-2 ​​activation ligand-inducible and trypsin-inducible calcium flux by the PAR-2 ​​calcium flux cell assay described in Example 2.2, resulting in an IC50 of approximately 6 nM to approximately 11 nM. 50 It inhibits within a certain range.

[0234] In some embodiments, the antibodies or antigen-binding fragments described herein that specifically bind to human PAR-2 ​​stimulate intracellular PAR-2-activated ligand-induced and trypsin-induced calcium fluxes at concentrations of approximately 6 nM, 6.1 nM, 6.2 nM, 6.3 nM, 6.4 nM, 6.5 nM, 6.6 nM, 6.7 nM, 6.8 nM, 6.9 nM, 7.1 nM, 7.2 nM, 7.3 nM, 7.4 nM, 7.5 nM, 7.6 nM, 7.7 nM, 7.8 nM, and 7.9 nM. nM, approximately 8nM, 8.1nM, approximately 8.2nM, approximately 8.3nM, approximately 8.4nM, approximately 8.5nM, approximately 8.6nM, approximately 8.7nM, approximately 8.8nM, approximately 8.9nM, approximately 9nM, 9.1nM, approximately 9.2nM, approximately 9.3nM, approximately 9.4nM, approximately 9.5nM, IC of about 9.6nM, about 9.7nM, about 9.8nM, about 9.9nM, about 10nM, 10.1nM, about 10.2nM, about 10.3nM, about 10.4nM, about 10.5nM, about 10.6nM, about 10.7nM, about 10.8nM, or about 10.9nM 50 It inhibits within a certain range.

[0235] In some embodiments, the antibodies or antigen-binding fragments described herein that specifically bind to human PAR-2 ​​inhibit PAR-2-activated ligand-induced mucin production in cells (e.g., human lung epithelial cells or cells derived from cynomolgus monkey bronchoalveolar lavage fluid) by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% compared to PAR-2-activated ligand-induced cytokine production in the absence of the antibody or fragment or in the presence of a control antibody or fragment of the same isotype. Inhibition of PAR-2-activated ligand-induced mucin production in cells can be measured, for example, using the assay described in Example 7. Inhibition of PAR-2-activated ligand-induced mucin production in cells may be dose-dependent of the anti-human PAR-2 ​​antibody.

[0236] In some embodiments, antibodies or antigen-binding fragments thereof described herein that specifically bind to human PAR-2 ​​inhibit PAR-2-activated ligand-induced contraction of smooth muscle cells (e.g., bronchial smooth muscle cells) by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100%, compared to PAR-2-activated ligand-induced smooth muscle cell contraction in the absence of the antibody or fragment thereof or in the presence of a control antibody or fragment thereof of the same isotype. PAR-2-activated ligand-induced muscle cell contraction can be measured, for example, using the assay described in Example 7. Inhibition of PAR-2-activated ligand-induced muscle cell contraction may be dose-dependent of the anti-human PAR-2 ​​antibody.

[0237] In some embodiments, the antibodies or antigen-binding fragments described herein that specifically bind to human PAR-2 ​​increase pulmonary neutrophils in cynomolgus monkeys. SymptomsInhibits induction by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% compared to induction of pulmonary neutrophilia in the absence of the antibody or fragment thereof or in the presence of a control antibody or fragment of the same isotype.

[0238] II(c). Antigen-binding fragment In some embodiments, antigen-binding fragments of anti-PAR-2 ​​antibodies described herein, such as anti-human PAR-2 ​​antibodies, are provided. Exemplary antigen-binding fragments include Fab, Fab', F(ab')2, and scFv, where Fab, Fab', F(ab')2, or scFv are, but are not limited to, the heavy chain variable region sequences and light chain variable region sequences of anti-human PAR-2 ​​antibodies described herein. Fab, Fab', F(ab')2, or scFv can be produced by any technique known to those skilled in the art, including, but not limited to, those discussed in Section III below. In some embodiments, antigen-binding fragments such as Fab, Fab', F(ab')2, or scFv further include a portion that extends the half-life of the antibody in vivo. This portion is also referred to as the "half-life extension portion." Any portion known to those skilled in the art can be used to extend the half-life of antigen-binding fragments such as Fab, Fab', F(ab')2, or scFv in vivo. For example, the half-life extension portion may include an Fc region, a polymer, albumin, or an albumin-binding protein or compound. Examples of polymers include naturally or synthetically substituted linear or branched polyalkylenes, polyalkenes, polyoxylalkylenes, polysaccharides, polyethylene glycols, polypropylene glycols, polyvinyl alcohols, methoxypolyethylene glycols, lactose, amylose, dextran, glycogen, or derivatives thereof. Substituents may include one or more hydroxyl groups, methyl groups, or methoxy groups. In some embodiments, antigen-binding fragments such as Fab, Fab', F(ab')2, or scFV may be modified by adding one or more C-terminal amino acids for attachment of the half-life extension portion. In some embodiments, the half-life extension portion is polyethylene glycol or human serum albumin. In some embodiments, antigen-binding fragments such as Fab, Fab', F(ab')2, or scFV are fused to the Fc region.

[0239] Anti-PAR-2 ​​antibodies (such as anti-human PAR-2 ​​antibodies) or their antigen-binding fragments may be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels such as glucose oxidase; iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Examples include radioisotopes such as Tc; luminescence labels such as luminol; fluorescent labels such as fluorescein and rhodamine; and biotin. Such labeled antibodies or antigen-binding fragments thereof can be used to detect PAR-2 ​​(e.g., human PAR-2) proteins. See, for example, sections IV and V below.

[0240] III. Anti-PAR-2 ​​antibody production Antibodies that specifically bind to human PAR-2 ​​and their antigen-binding fragments can be produced by any method known in the art for synthesizing antibodies and antigen-binding fragments, for example, by chemical synthesis or recombinant expression techniques. Unless otherwise indicated, the methods described herein utilize conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. Such techniques are described and adequately explained in the references cited herein, for example. For example, see Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Eckstein (ed.), (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.), (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0241] In some embodiments, the Specified Method provides for producing an antibody or antigen-binding fragment that specifically binds to human PAR-2, comprising the step of culturing cells or host cells described herein (e.g., cells or host cells containing a polynucleotide encoding the antibody or antigen-binding fragment described herein). In certain embodiments, the Specified Method provides for producing an antibody or antigen-binding fragment that specifically binds to human PAR-2, comprising the step of expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment using cells or host cells described herein (e.g., cells or host cells containing a polynucleotide encoding the antibody or antigen-binding fragment described herein). In some embodiments, the cells are isolated cells. In some embodiments, the encoding polynucleotide has been introduced into the cells. In some embodiments, the Method further comprises the step of purifying the antibody or antigen-binding fragment expressed by the cells or host cells.

[0242] Monoclonal antibodies or their antigen-binding fragments can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, yeast-based presentation techniques, or combinations thereof. For example, monoclonal antibodies or their antigen-binding fragments can be produced using hybridoma techniques, including techniques known in the art, as taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981); or as described in Kohler G & Milstein C (1975) Nature 256:495. Examples of yeast-based presentation methods that can be used to select and generate the antibodies described herein include, for example, those disclosed in International Publication No. 2009 / 036379A2, International Publication No. 2010 / 105256, and International Publication No. 2012 / 009568. Each of these publications is incorporated herein by reference in its entirety.

[0243] In some embodiments, the monoclonal antibody or antigen-binding fragment is an antibody or antigen-binding fragment produced by clonal cells (e.g., a hybridoma or host cell producing a recombinant antibody or antigen-binding fragment) that specifically binds to human PAR-2, as determined, for example, by ELISA or other antigen-binding assays known in the art or provided herein. In some embodiments, the monoclonal antibody or its antigen-binding fragment may be a chimeric or humanized antibody or its antigen-binding fragment. In some embodiments, the monoclonal antibody or its antigen-binding fragment may be a Fab fragment or an F(ab')2 fragment. The monoclonal antibodies or their antigen-binding fragments described herein may be prepared, for example, by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256: 495, or isolated, for example, from a phage library using the techniques described herein. Other methods for preparing clonal cell lines, monoclonal antibodies expressed therein, and their antigen-binding fragments are well known in the art (see, for example, Short Protocols in Molecular Biology, 5th edition (2002), Ausubel FM et al., Chapter 11 above).

[0244] The antigen-binding fragments of the antibodies described herein can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of an immunoglobulin molecule using an enzyme such as papain (for producing the Fab fragment) or pepsin (for producing the F(ab')2 fragment). The Fab fragment corresponds to one of the two identical arms of the tetrameric antibody molecule and includes a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment includes the two antigen-binding arms of the tetrameric antibody molecule linked by a disulfide bond in the hinge region.

[0245] Furthermore, the antibodies or their antigen-binding fragments described herein can also be generated using various phage display methods and / or yeast-based presentation methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles having a polynucleotide sequence encoding the protein. In particular, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). The DNA encoding the VH and VL domains is recombinant with an scFV linker by PCR and cloned into a phagemide vector. The vector is electroporated into Escherichia coli (E. coli) to infect the E. coli with helper phages. The phages used in these methods are typically filamentous phages containing fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. Phages expressing antibodies or their antigen-binding fragments that bind to a specific antigen can be selected or identified by antigen, for example, using a labeled antigen or an antigen bound to or captured on a solid surface or beads.Examples of phage display methods that can be used to produce the antibodies or fragments described herein include: Brinkman U et al., (1995) J Immunol Methods Vol. 182: pp. 41-50; Ames RS et al., (1995) J Immunol Methods Vol. 184: pp. 177-186; Kettleborough CA et al. (1994) Eur J Immunol Vol. 24: pp. 952-958; Persic L et al. (1997) Gene Vol. 187: pp. 9-18; Burton DR & Barbas CF (1994) Advan Immunol Vol. 57: Pages 191-280; PCT application PCT / GB91 / 001134; International Publications 90 / 02809, 91 / 10737, 92 / 01047, 92 / 18619, 93 / 11236, 95 / 15982, 95 / 20401, and 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403, Examples include those disclosed in U.S. Patent Nos. 484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0246] The humanized antibody or its antigen-binding fragment can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.

[0247] Methods for producing multispecific (e.g., bispecific) antibodies are described. See, for example, U.S. Patent Nos. 7,951,917, 7,183,076, 8,227,577, 5,837,242, 5,989,830, 5,869,620, 6,132,992, and 8,586,713.

[0248] III(a). Polynucleotides In certain embodiments, the Specified herein provides polynucleotides comprising nucleotide sequences encoding antibodies described herein that specifically bind to human PAR-2, or antigen-binding fragments thereof, or domains thereof (e.g., variable light chain regions and / or variable heavy chain regions), and vectors, such as vectors comprising such polynucleotides for recombinant expression in host cells (e.g., Escherichia coli and mammalian cells).

[0249] In some embodiments, the Specified Information provides antibodies or antigen-binding fragments thereof that specifically bind to human PAR-2 ​​and contain the amino acid sequences described herein, as well as polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments that compete with such antibodies or antigen-binding fragments for binding to human PAR-2 ​​(e.g., in a dose-dependent manner) or that bind to the same epitope as such antibodies or antigen-binding fragments.

[0250] Furthermore, this specification provides isolated polynucleotides comprising a nucleic acid sequence encoding a heavy chain variable region or heavy chain of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the nucleic acid molecule encodes the VH of SEQ ID NO: 20 or 21.

[0251] In some embodiments, isolated polynucleotides are provided, comprising a light chain variable region or a nucleic acid molecule encoding the light chain of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the nucleic acid molecule encodes the VL of SEQ ID NOs. 23, 24, 25, 26, or 27.

[0252] In some embodiments, an isolated polynucleotide is provided comprising a first nucleic acid molecule encoding the light chain variable region of SEQ ID NO: 23, 24, 25, 26, or 27 and a second nucleic acid molecule encoding the heavy chain variable region of SEQ ID NO: 20 or 21. In some embodiments, a mixture of isolated polynucleotides is provided comprising a first polynucleotide comprising the light chain variable region of SEQ ID NO: 23, 24, 25, 26, or 27 and a second polynucleotide comprising the heavy chain variable region of SEQ ID NO: 20 or 21.

[0253] In some embodiments, the isolated polynucleotide comprises nucleic acid molecules encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding fragment disclosed herein and the light chain variable region or light chain of the antibody or antigen-binding fragment disclosed herein.

[0254] Furthermore, this specification provides a kit, vector, or host cell comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 20, and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 23. Furthermore, this specification provides a kit, vector, or host cell comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 21, and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 24. Furthermore, this specification provides a kit, vector, or host cell comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 21, and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 25. Furthermore, this specification provides a kit, vector, or host cell comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 21, and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 26. Furthermore, this specification provides a kit, vector, or host cell comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 21, and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 27. In a kit containing such first and second polynucleotides, the first and second polynucleotides may be present in the same vector or in different vectors. In a host cell containing such first and second polynucleotides, the first and second polynucleotides may be present in the same vector or in different vectors.

[0255] In some embodiments, the Specified provides a polynucleotide comprising nucleotide sequences encoding three VH domain CDRs, for example, a polypeptide comprising VH CDR1, VH CDR2, and VH CDR3 of any one of the antibodies described herein (see, for example, Table 2), wherein, for example, the three VH domain CDRs are present in a VH background. In some embodiments, the Specified provides a polynucleotide comprising nucleotide sequences encoding three VL domain CDRs, for example, a polypeptide comprising VL CDR1, VL CDR2, and VL CDR3 of any one of the antibodies described herein (see, for example, Table 3), wherein, for example, the three VL domain CDRs are present in a VL background. In some embodiments, the Specified provides a polynucleotide (or combination of polynucleotides) comprising a polypeptide comprising (i) three VH domain CDRs, for example, VH CDR1, VH CDR2, and VH CDR3 of any one of the antibodies described herein (see, for example, Table 2), wherein the three VH domain CDRs are present in a VH background, and (ii) a polypeptide comprising three VL domain CDRs, for example, VL CDR1, VL CDR2, and VL CDR3 of any one of the antibodies described herein (see, for example, Table 2), wherein the three VL domain CDRs are present in a VL background, and a polynucleotide (or combination of polynucleotides) comprising a nucleotide sequence encoding an anti-human PAR-2 ​​antibody or an antigen-binding fragment thereof.

[0256] In some embodiments, the polynucleotide comprises a heavy chain variable region (e.g., a VH comprising the amino acid sequence of SEQ ID NO: 20 or 21) and a heavy chain constant region, e.g., a human gamma (γ) heavy chain constant region, which comprises a nucleic acid sequence.

[0257] In some embodiments, the polynucleotide includes a light chain variable region (e.g., a VL comprising the amino acid sequence of SEQ ID NOs. 23, 24, 25, 26, or 27) and a light chain constant region, such as the human kappa light chain constant region, which comprises a nucleic acid sequence.

[0258] Furthermore, this specification provides polynucleotides encoding anti-human PAR-2 ​​antibodies or their antigen-binding fragments or domains as described herein, optimized, for example, by codon / RNA optimization, substitution with heterogeneous signal sequences, and elimination of mRNA unstable elements. Methods for generating optimized nucleic acids encoding anti-human PAR-2 ​​antibodies or their antigen-binding fragments or domains (e.g., heavy chain, light chain, VH domain, or VL domain) to be recombinantly expressed by introducing codon changes (e.g., codon changes encoding the same amino acid for degeneracy of the gene code) and / or eliminating inhibitory regions of mRNA can be carried out by appropriately applying the optimization methods described in, for example, U.S. Patents 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, each of which is incorporated herein by reference in whole.

[0259] The antibodies, antigen-binding fragments thereof, or polynucleotides encoding their domains described herein can be generated from nucleic acids of a suitable source (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be carried out using genomic DNA obtained from hybridoma cells producing the antibody of interest. Using such PCR amplification methods, nucleic acids containing sequences encoding the light and / or heavy chains of the antibody or its antigen-binding fragment can be obtained. Using such PCR amplification methods, nucleic acids containing sequences encoding the variable light and / or variable heavy chain regions of the antibody or its antigen-binding fragment can be obtained. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning to generate, for example, chimeric and humanized antibodies or their antigen-binding fragments.

[0260] The polynucleotides provided herein may be, for example, in the form of RNA or DNA. Examples of DNA include cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. In the case of single-stranded DNA, the DNA may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is cDNA or DNA lacking one or more endogenous introns. In some embodiments, the polynucleotide is a polynucleotide that does not exist in nature. In some embodiments, the polynucleotide is recombinantly produced. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from natural components.

[0261] III(b). Cells and Vectors In some embodiments, the Specified Public Service provides vectors (e.g., expression vectors) containing polynucleotides that encode anti-human PAR-2 ​​antibodies or their antigen-binding fragments or their domains, for recombinant expression in host cells (e.g., mammalian cells). The Specified Public Service also provides cells, e.g., host cells, containing such vectors for recombinant expression of the anti-human PAR-2 ​​antibodies or their antigen-binding fragments (e.g., human or humanized antibodies or their antigen-binding fragments) described herein. In some embodiments, the Specified Public Service provides a method for producing the antibodies or their antigen-binding fragments described herein, comprising the step of expressing such antibodies or their antigen-binding fragments in host cells.

[0262] In some embodiments, recombinant expression of the antibody described herein or its antigen-binding fragment or its domain (e.g., the heavy chain or light chain described herein) that specifically binds to human PAR-2 ​​involves constructing an expression vector containing a polynucleotide encoding the antibody or its antigen-binding fragment or its domain. Once a polynucleotide encoding the antibody or its antigen-binding fragment or its domain (e.g., the heavy chain or light chain variable domain) described herein is obtained, a vector for producing the antibody or its antigen-binding fragment can be produced by recombinant DNA techniques using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing an antibody or its antigen-binding fragment or its domain (e.g., the light chain or heavy chain) encoding a nucleotide sequence is described herein. Using methods well known to those skilled in the art, an expression vector containing the antibody or its antigen-binding fragment or its domain (e.g., the light chain or heavy chain) coding sequence and appropriate transcription and translational regulatory signals can be constructed. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Furthermore, a replicable vector is provided comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment, heavy chain or light chain, heavy chain or light chain variable domain, or heavy chain or light chain CDR described herein, operably linked to a promoter. Such a vector may, for example, include a nucleotide sequence encoding the constant region of the antibody or its antigen-binding fragment (see, for example, International Publication Nos. 86 / 05807 and International Publication Nos. 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody or its antigen-binding fragment can be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain.

[0263] An expression vector can be introduced into cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce the antibodies or their antigen-binding fragments described herein (e.g., antibodies or their antigen-binding fragments containing six CDRs of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain) or their domains (e.g., VH, VL, VH and VL, heavy chain, or light chain of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e). Accordingly, this specification provides host cells comprising polynucleotides that encode an antibody or its antigen-binding fragment (e.g., an antibody or its antigen-binding fragment comprising six CDRs of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain) or its domain (e.g., VH, VL, VH and VL, heavy chain, or light chain of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e) and are operably linked to a promoter for expressing such sequences in host cells. In some embodiments, when expressing a double-chain antibody or its antigen-binding fragment, vectors encoding both the heavy chain and light chain individually can be co-expressed in the host cell to express the entire immunoglobulin, as detailed below. In some embodiments, the host cell comprises a vector containing polynucleotides encoding both the heavy and light chains of the antibodies described herein (e.g., the heavy and light chains of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e) or their domains (e.g., the VH and VL of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e).In some embodiments, the host cell comprises two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or its antigen-binding fragment described herein, and the second vector comprising a polynucleotide encoding the light chain or light chain variable region or domain of an antibody described herein (e.g., an antibody comprising six CDRs of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e). In some embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or its antigen-binding fragment described herein, and a second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of the antibody or its antigen-binding fragment described herein (e.g., an antibody or its antigen-binding fragment comprising six CDRs of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e). In some embodiments, the heavy chain / heavy chain variable region expressed by the first cell, in conjunction with the light chain / light chain variable region of the second cell, forms the human PAR-2 ​​antibody or its antigen-binding fragment described herein (e.g., an antibody or its antigen-binding fragment comprising six CDRs of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e). In some embodiments, the Specified Information provides a population of host cells including such first host cells and such second host cells.

[0264] In some embodiments, the Specified

[0265] Various host-expression vector systems can be used to express the antibodies and their antigen-binding fragments described herein (e.g., antibodies or their antigen-binding fragments containing CDRs of P24E1102, P24E976, P24E1099, P24E1103, or 309-4e) (see, for example, U.S. Patent No. 5,807,715). Such host-expression systems are vehicles that can produce and subsequently purify the coding sequence of the desired type, but are also cells that, upon transformation or transfecting with the appropriate nucleotide coding sequence, can express the antibodies or their antigen-binding fragments described herein in situ. Such host-expression systems include, but are not limited to, the following: microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell lines infected with recombinant virus expression vectors containing antibody coding sequences (e.g., baculovirus); and plant cell lines (e.g., Chlamydomonas) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid). Mammalian cell lines containing recombinant expression constructs including green algae such as reinhardtii); or promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter) (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells).In some embodiments, the cells used to express the antibodies described herein and their antigen-binding fragments (e.g., antibodies containing the CDR of hPA-002, hPA-005, hPA-004, or hPA-001, or their antigen-binding fragments) are CHO cells, for example, CHO GS System® or CHO K1SV® (Lonza) cells. In some embodiments, the cells used to express the antibodies described herein are human cells, for example, human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC® or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) are used to express the recombinant antibody molecules, particularly to express the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, used in conjunction with vectors such as major pre-initial gene promoter elements derived from human cytomegalovirus, are effective antibody expression systems (Foecking MK & Hofstetter H (1986) Gene 45: pp. 101-105; and Cockett MI et al., (1990) Biotechnology 8: pp. 662-667). In some embodiments, the antibodies or their antigen-binding fragments described herein are produced by CHO cells or NS0 cells.

[0266] In addition, host cell lines can be selected that modulate the expression of the inserted sequence or modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may contribute to the protein's function. Therefore, eukaryotic host cells with cellular mechanisms for proper processing, glycosylation, and phosphorylation of the primary transcript and gene product can be used. Examples of such mammalian host cells include, but are not limited to, Expi293F human cells, C6 (rat glioma cell line), U2OS, Chem-1, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In some embodiments, the anti-human PAR-2 ​​antibodies described herein or their antigen-binding fragments (e.g., antibodies containing CDRs of hPA-002, hPA-005, hPA-004, or hPA-001, or their antigen-binding fragments) are produced in mammalian cells such as CHO cells.

[0267] Once the antibodies or antigen-binding fragments described herein are produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly chromatography based on affinity to a specific antigen after protein A, and sizing column chromatography), centrifugation, solubility difference, or any other standard technique for purifying proteins. Furthermore, the antibodies or antigen-binding fragments described herein may be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0268] In some embodiments, the antibodies or antigen-binding fragments described herein are isolated or purified. Generally, isolated antibodies or antigen-binding fragments substantially do not contain other antibodies or their antigen-binding fragments having different antigen specificity than the isolated antibody or antigen-binding fragment. For example, in some embodiments, preparations of antibodies or antigen-binding fragments described herein substantially do not contain cellular material and / or chemical precursors.

[0269] IV. Bispecific molecules The anti-PAR-2 ​​antibodies described herein can be used to form bispecific molecules. The anti-PAR-2 ​​antibody or its antigen-binding fragment may be derivatized or ligated to another functional molecule, such as another peptide or protein (e.g., a ligand for another antibody or receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. For example, the anti-PAR-2 ​​antibody may be ligated to an antibody or scFV that specifically binds to any protein that can be used as a potential target for combination therapy. The antibodies described herein may, in practice, be derived from or ligated to more than one other functional molecule so as to produce a polyspecific molecule that binds to more than two different binding sites and / or target molecules. Such polyspecific molecules are also intended to be included in the term “bispecific molecule” as used herein. To produce the bispecific molecules described herein, the antibodies described herein can be functionally ligated to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic (e.g., by chemical coupling, gene fusion, non-covalent attachment, or otherwise), so as to yield the bispecific molecule.

[0270] Accordingly, this specification provides a bispecific molecule comprising at least one first binding specificity to PAR-2 ​​and a second binding specificity to a second target epitope. In some embodiments described herein where the bispecific molecule is polyspecific, the molecule may further comprise a third binding specificity.

[0271] In some embodiments, the bispecific molecules described herein include at least one antibody or its antigen-binding fragment, which, as binding specificity, includes, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv(scFV). The antibody may also be a light-chain or heavy-chain dimer, or any smallest fragment thereof, such as Fv or a single-chain construct as described in Ladner et al., U.S. Patent No. 4,946,778.

[0272] The bispecific molecules described herein can be prepared by conjugating binding specificity components using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated together. When the binding specificity is a protein or peptide, various coupling agents or crosslinking agents can be used in the covalent conjugation. See, for example, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648. Other methods include those described by Paulus (1985) Behring Ins. Mitt. Vol. 78, pp. 118-132; Brennan et al. (1985) Science Vol. 229: pp. 81-83; and Glennie et al. (1987) J. Immunol. Vol. 139: pp. 2367-2375. Some conjugates are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co., Ltd. (Rockford, Illinois).

[0273] V. Pharmaceutical Compositions This specification provides compositions comprising an anti-PAR-2 ​​antibody (such as an anti-human PAR-2 ​​antibody) or an antigen-binding fragment thereof as described herein. In some embodiments, the antibody or antigen-binding fragment thereof having a desired degree of purity is present, for example, in a formulation containing a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, Pennsylvania). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosage and concentration used. Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostatic agents, and solutes which make the formulation isotonic with the blood of the recipient to whom the formulation is intended, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0274] In some embodiments, the pharmaceutical composition comprises an anti-human PAR-2 ​​antibody or its antigen-binding fragment as described herein, and a pharmaceutically acceptable carrier (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd edition, Pharmaceutical Press (2000)). In some embodiments, the pharmaceutical compositions described herein are intended for use as pharmaceuticals. Compositions used for in vivo administration may be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0275] The pharmaceutical compositions described herein can be used to exert biological effects in vivo or in vitro. For example, the pharmaceutical compositions described herein can be used to block the interaction between a PAR-2 ​​activating ligand and the extracellular domain of PAR-2, and / or to block PAR-2 ​​activation by a PAR-2 ​​activating ligand. Examples of such PAR-2 ​​activating ligands include, but are not limited to, PAR-2 ​​anchoring ligands (cis or trans), PAR-1 anchoring ligands, or soluble ligands (e.g., synthetic soluble PAR-2 ​​activating ligands such as SLIGKV, SLIGRL, or 2-froyl-LIGRLO).

[0276] The pharmaceutical compositions described herein can be used to treat diseases or conditions that can be alleviated by antagonizing the activation of PAR-2 ​​by PAR-2 ​​activating ligands.

[0277] In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as respiratory tract diseases. Examples of respiratory tract diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pulmonary arterial hypertension.

[0278] In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as skin diseases. Examples of skin diseases include, but are not limited to, inflammatory skin conditions, atopic dermatitis, allergic contact dermatitis, Netherton syndrome, ichthyosis, post-injury skin barrier / permeability restoration, pruritus, skin cancer, itchiness, pigmentation associated with melanosis, and pigmentation associated with vitiligo.

[0279] In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as cancer. Examples of cancers that can be treated as provided herein include solid tumors, for example, solid tumors in which bone marrow cells (monocytes, macrophages, dendritic cells, granulocytes, neutrophils, microglia (in the CNS), or other innate immune cells) infiltrate the tumor microenvironment. Examples of such cancers that can be treated with the pharmaceutical compositions provided herein include, but are not limited to, glioblastoma, head and neck cancer, kidney cancer (e.g., clear cell carcinoma), pancreatic cancer, gastric cancer, and breast cancer. Other cancers include, but are not limited to, bone cancer, ovarian cancer, prostate cancer, sarcoma, colorectal cancer, lung cancer, melanoma, bladder cancer, liver cancer, and uterine cancer. In some embodiments, the cancer is a hematopoietic cancer such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer may be an early-stage cancer or a late-stage cancer. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site originating from one of the above types of cancer. In some embodiments, the cancer is a PAR-2 ​​positive cancer. In some embodiments, the cancer is a cancer with increased PAR-2 ​​(e.g., increased PAR-2 ​​mRNA and / or increased PAR-2 ​​protein).

[0280] In some embodiments, the pharmaceutical compositions provided herein are used to alleviate pain. Examples of pain include, but are not limited to, cancer pain, arthralgia, chemotherapy-induced peripheral neuropathy pain, migraine pain, toothache, bladder pain, pancreatitis pain, irritable bowel syndrome-related pain, visceral pain, osteoarthritis-related pain, rheumatoid arthritis-related pain, and spinal cord injury pain.

[0281] In some embodiments, the pharmaceutical compositions provided herein are used to treat orofacial granulomatosis.

[0282] In some embodiments, the pharmaceutical compositions provided herein are used to treat inflammatory conditions in patients. In certain embodiments, the inflammatory condition is a pathology associated with rheumatoid arthritis, osteoarthritis, inflammatory-induced visceral hypersensitivity, periodontal disease, or acute coronavirus infection.

[0283] VI. Use and Method In various embodiments, this specification provides in vitro and in vivo methods using anti-human PAR-2 ​​antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, as described herein. In one embodiment, a method is provided for inhibiting ligand-mediated PAR-2 ​​activation, comprising the step of contacting PAR-2 ​​with an anti-human PAR-2 ​​antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof. In another embodiment, a method is provided for inhibiting the binding of a PAR-2 ​​activating ligand to PAR-2, comprising the step of blocking the ligand-binding site of PAR-2 ​​with an anti-human PAR-2 ​​antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof. In yet another embodiment, a method is provided for inhibiting the binding of PAR-2 ​​to a soluble PAR-1 or soluble PAR-2 ​​ligand generated from protease activity, comprising the step of blocking the ligand-binding site of PAR-2 ​​with an anti-human PAR-2 ​​antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof. In another embodiment, a method is provided for inhibiting the transactivation of PAR-2 ​​by PAR-1, comprising the step of blocking the ligand-binding site of PAR-2 ​​with an anti-human PAR-2 ​​antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof.

[0284] In another embodiment, a method is provided for antagonizing the activation of PAR-2 ​​by a PAR-2 ​​activating ligand, comprising the step of contacting PAR-2 ​​with an anti-human PAR-2 ​​antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof, in the presence of one or more of its ligands. Exemplary ligands include, for example, the soluble PAR-2 ​​activating ligands SLIGKV, SLIGRL, 2-Froyl-LIGRLO, PAR-2 ​​anchoring ligands, or PAR-1 anchoring ligands.

[0285] VI(a). Use and methods of therapy In one embodiment, a method is provided for inhibiting the activation of PAR-2 ​​in vivo in a subject requiring it (e.g., a human subject), comprising the step of administering to the subject an anti-human PAR-2 ​​antibody or its antigen-binding fragment or a pharmaceutical composition described herein. In some embodiments, a method is provided for inhibiting the activation of PAR-2 ​​by a PAR-2 ​​activating ligand in vivo in a subject requiring it (e.g., a human subject), comprising the step of administering to the subject an anti-human PAR-2 ​​antibody or its antigen-binding fragment or a pharmaceutical composition described herein. In certain embodiments, the PAR-2 ​​activating ligand is a soluble PAR-2 ​​activating ligand, a PAR-2 ​​anchoring ligand, or a PAR-1 anchoring ligand.

[0286] PAR-2 ​​activity has been suggested to be involved in or associated with several diseases and conditions, including inflammatory diseases, pain, gastrointestinal conditions, neurological disorders, and cardiovascular disorders (see, for example, Linder et al., J. Immunol. 165: pp. 6504-6510 (2000); Vergnolle et al., Nature Medicine 7: pp. 821-826 (2001); Cenac et al., J. Olin. Investigation 117: pp. 636-647 (2007); Vergnolle, British J. Pharmacol. 141: pp. 1264-1274 (2004); Knight et al., J. Allergy Clin. Immunol. 108: pp. 797-803 (2001); Schmidlin et al., J. Immunol. 169: pp. 5315-5321 (2002)). Antibodies that bind to PAR-2 ​​have the ability to antagonize PAR-2 ​​activity in vivo. Therefore, anti-PAR-2 ​​antibodies are potentially useful for the treatment and / or improvement of various disease conditions. See also U.S. Patent Application Publication No. 2011 / 0059095.

[0287] In some embodiments, this specification provides methods for treating diseases or conditions associated with increased PAR-2 ​​expression, and / or diseases or conditions that can be alleviated by blocking the interaction between a PAR-2 ​​activating ligand and the extracellular domain of PAR-2 ​​and / or blocking PAR-2 ​​activation by the PAR-2 ​​activating ligand, such as respiratory diseases, skin diseases, cancer, inflammatory conditions, orofacial granulomatosis, and pain associated with various diseases or conditions. Such methods may include administering an anti-human PAR-2 ​​antibody or its antigen-binding fragment or a pharmaceutical composition thereof as described herein to a patient in need (e.g., a human patient).

[0288] VI(a)(1). Airway diseases In some embodiments, methods for treating respiratory tract diseases are provided herein. Such methods may include administering an anti-human PAR-2 ​​antibody or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, as described herein, to a patient in need (e.g., a human patient).

[0289] In some embodiments, the patient has symptoms of a respiratory tract disease, and the anti-human PAR-2 ​​antibody or its antigen-binding fragment or the pharmaceutical composition thereof described herein is administered to treat the respiratory tract disease. In some embodiments, the patient is at risk of developing a respiratory tract disease, and the anti-human PAR-2 ​​antibody, antigen-binding fragment, or pharmaceutical composition is administered to reduce the risk of respiratory tract disease, to delay its onset, or to prevent it.

[0290] Examples of airway diseases that can be treated as provided herein include, but are not limited to, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, pulmonary arterial hypertension, acute respiratory distress syndrome (ARDS), respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic pneumonia, bronchitis, allergic bronchitis, bronchiectasis, tuberculosis, hypersensitivity pneumonitis, asthma-like disorders, sarcoid disorders, reactive airway disease (or dysfunction) syndromes, comosmosis, interstitial lung disease, eosinophilic syndrome, rhinitis, sinusitis, and parasitic lung diseases, as well as airway hypersensitivity associated with virus-induced conditions (e.g., respiratory syncytial virus (RSV), parainfluenza virus (PIV), rhinovirus (RV), and adenovirus).

[0291] asthma Asthma is a chronic inflammatory disease of the airways. Chronic inflammation is associated with airway hyperresponsiveness (an excessive constrictive response to specific triggers such as viruses, allergens, and exercise), which leads to recurrent episodes of wheezing, shortness of breath, chest tightness, and / or cough, which may vary in duration and intensity. Symptomatic episodes are generally associated with widespread but variable intrapulmonary airflow obstruction, which can usually be reversed spontaneously or with appropriate asthma treatment, such as rapid-acting bronchodilators. See, for example, Quirt J. et al., Allergy Asthma Clin Immunol., Vol. 14 (Augmented 2): p. 50 (2018).

[0292] PAR-2 ​​expression is observed in multiple airway cell types in asthma patients, such as epithelium (Knight, DA et al., J Allergy Clin Immunol, Vol. 108: pp. 797-803 (2001)), smooth muscle (Aubier, M. et al., J Allergy Clin Immunol, Vol. 138: pp. 729-739 (2016)), fibroblasts (Akers, IA et al., Am J Physiol Lung Cell Mol Physiol, Vol. 278: pp. L193-201 (2000)), and endothelial cells, as well as eosinophils, neutrophils (Miike, S. et al., J Immunol, Vol. 167: pp. 6615-6622 (2001)), dendritic cells, mast cells, and monocytes (Palikhe, N. et al., PLoS PAR-2 ​​activation is increased in innate immune cells, including those mentioned in One., Vol. 10 (No. 12): e0144500 p. (2015). PAR-2 ​​activation stimulates the release of various inflammatory mediators, particularly thymic interstitial lymphopoietin (TSLP) (Kouzaki, H. et al., J Immunol, Vol. 183: pp. 1427-1434 (2009)), as well as mucus secretion (Lee, HJ et al., PLoS One, Vol. 7: e43188 p. (2012)), and increases mucin secretion in bronchial epithelial cells (Lin, K. et al., Int J Biochem Cell Biol, Vol. 40: pp. 1379-1388 (2008)). Furthermore, PAR-2 ​​activation stimulates the proliferation and migration of fibroblasts and airway smooth muscle cells (Berger, P. et al., J Appl Physiol, Vol. 91, pp. 1372-1379 (2001); Bagher, M. et al., Cell Communication and Signaling, Vol. 16: p. 59 (2018)). PAR-2 ​​can be activated by exogenous proteases present in common allergens (Kawabata, A. & Kawao, N., J Pharmacol Sci, Vol. 97: pp. 20-24 (2005)) or by endogenous proteases released in response to asthma-inducing factors (Cocks, TM et al., Nature, Vol. 398: pp. 156-160 (1999)).PAR-2 ​​expression in airway cells correlates with asthma severity (Knight, DA et al., J Allergy Clin Immunol, Vol. 108: pp. 797-803 (2001); Aubier, M. et al., J Allergy Clin Immunol, Vol. 138: pp. 729-739 (2016); Palikhe, N. et al., PLoS One, Vol. 10 (No. 12): e0144500 (2015)).

[0293] Ovalbumin (OVA)-induced experimental asthma was significantly reduced in PAR-2- / - mice and significantly increased in mice engineered to overexpress PAR-2 ​​(Schmidlin, F. et al., J Immunol, Vol. 169: pp. 5315-5321 (2002)). This could be improved by administering anti-PAR-2 ​​antibodies or PAR-2 ​​blocking peptides (Asaduzzaman, M. et al., Clin Exp Allergy, Vol. 45: pp. 1844-1855 (2015)). Furthermore, PAR-2 ​​blockade with small molecule inhibitors significantly improved cockroach dung pellet-induced experimental asthma in mice (Nadeem, A. et al., Immunology, Vol. 145: pp. 391-403 (2015)).

[0294] In some embodiments, administration of an anti-human PAR-2 ​​antibody or its antigen-binding fragment, or a pharmaceutical composition thereof, provided herein, can prevent, reduce the risk of, and / or treat asthma and related conditions such as eosinophilic esophagitis. In some embodiments, administration of an anti-human PAR-2 ​​antibody, antigen-binding fragment, or pharmaceutical composition can modulate one or more PAR-2 ​​activities in an individual with asthma.

[0295] Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) encompasses a diverse range of clinical syndromes that share the common characteristic of restricted expiratory airflow. The American Thoracic Society defines COPD in terms of chronic bronchitis and emphysema. Chronic bronchitis is characterized by clinical manifestations of excessive cough and sputum production, while emphysema refers to chronic dyspnea resulting from enlargement of air spaces and destruction of lung tissue. The GOLD Initiative defines COPD as “a medical condition characterized by airflow limitation that is not entirely reversible.” Airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to harmful particles or gases. Asthma is also characterized by airflow obstruction and inflammation, but in addition, asthma involves hypersensitivity of the airways to irritants. Therefore, asthma is distinguished from COPD by the reversibility of its dysfunction. See, for example, Devine JF, Am Health Drug Benefits, Vol. 1 (No. 7): pp. 34-42 (2008).

[0296] Although PAR-2 ​​expression is not thought to increase in COPD patients (Cocks, TM & Moffatt, JD, Pulm Pharmacol Ther, Vol. 14: pp. 183-191 (2001); Miotto, D. et al., Thorax, Vol. 57: pp. 146-151 (2002)), exposure to tobacco smoke may enhance neutrophil elastase-induced IL-8 production by human bronchial epithelial cells (Lee, KH et al., Experimental & Molecular Medicine, Vol. 50: p. 79 (2018)). Furthermore, neutrophil elastase has been shown to induce the release of MUC5AC, an asthma / COPD-related mucin, from human lung epithelial cells upon PAR-2 ​​activation (Zhou, J. et al., Mol Cell Biochem, Vol. 377: pp. 75-85 (2013)). PAR-2-mediated fibroblast proliferation and extracellular matrix deposition may contribute to COPD-associated pulmonary fibrosis (Akers, IA et al., Am J Physiol Lung Cell Mol Physiol, Vol. 278: pp. L193-201 (2000)).

[0297] In some embodiments, administration of an anti-human PAR-2 ​​antibody or its antigen-binding fragment, or a pharmaceutical composition thereof, provided herein, can prevent COPD, reduce its risk, and / or treat it. In some embodiments, administration of an anti-human PAR-2 ​​antibody, antigen-binding fragment, or pharmaceutical composition can modulate one or more PAR-2 ​​activities in an individual having COPD.

[0298] Idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic, progressive scarring of the lungs and pathological features of typical interstitial pneumonia. The current paradigm suggests that a key initiating factor is alveolar epithelial cell damage. See, for example, Barratt et al., J Clin Med., vol. 7(no. 8):201 (2018).

[0299] PAR-2 ​​expression is increased in the lung epithelium of IPF patients (Borensztajn, K. et al., Am J Pathol, Vol. 177: pp. 2753-2764 (2010)). PAR-2 ​​expression levels correlate with disease severity (Wygrecka, M. et al., Am J Respir Crit Care Med, Vol. 183: pp. 1703-1714 (2011)) and clinical features such as honeycomb formation observed on chest computed tomography (CT) scans (Park, YS et al., Respiratory Medicine, Vol. 107: pp. 256-262 (2013)). PAR-2- / - mice are resistant to the induction of experimental pulmonary fibrosis (Borensztajn, K. et al., Am J Pathol, Vol. 177: pp. 2753-2764 (2010)). Furthermore, treatment with PAR-2 ​​blocking peptides improved experimental pulmonary fibrosis, whether administered prophylactically or therapeutically (Lin, C. et al., Mol Med, Vol. 21: pp. 576-583 (2015)).

[0300] In some embodiments, administration of an anti-human PAR-2 ​​antibody or its antigen-binding fragment, or a pharmaceutical composition thereof, provided herein, can prevent IPF, reduce its risk, and / or treat it. In some embodiments, administration of an anti-human PAR-2 ​​antibody, antigen-binding fragment, or pharmaceutical composition can modulate one or more PAR-2 ​​activities in an individual having IPF.

[0301] VI(a)(2). Skin diseases In some embodiments, methods for treating skin diseases are provided herein. Such methods may include administering an anti-human PAR-2 ​​antibody or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, as described herein, to a patient in need (e.g., a human patient).

[0302] In some embodiments, the patient has symptoms of a skin disease, and the anti-human PAR-2 ​​antibody or its antigen-binding fragment or the pharmaceutical composition thereof described herein is administered to treat the skin disease. In some embodiments, the patient is at risk of developing a skin disease, and the anti-human PAR-2 ​​antibody, antigen-binding fragment or pharmaceutical composition is administered to reduce the risk of developing a skin disease, delay its onset, or prevent it.

[0303] Examples of skin conditions that can be treated as provided herein include barrier dysfunction conditions such as atopic dermatitis (e.g., Andersen et al., Pain, 2017, Vol. 158: pp. 1780-1791 (2017)), allergic contact dermatitis, Netherton syndrome (e.g., Hovnanian A., Cell Tissue Research, Vol. 351: pp. 289-300 (2013); see Briot et al., Journal of Investigative Dermatology, Vol. 130: pp. 2736-2742 (2010)), ichthyosis (e.g., see Frateschi et al., Nat Commun. Vol. 18 (No. 2): p. 161 (2011)), and post-injury skin barrier / permeability restoration (e.g., Hachem et al., Journal of Investigative Dermatology) Examples of skin conditions include, but are not limited to, those associated with melanosis (see Vol. 126: pp. 2074-2086 (2006)), pruritus (see, e.g., Frateschi et al., Nat Commun. Vol. 18 (No. 2): p. 161 (2011); Andersen et al., Pain, Vol. 158: pp. 1780-1791 (2017)), skin cancer (see, e.g., Henehan et al., Experimental Dermatology, Vol. 28: pp. 877-885 (2019)), itching of the skin, pigmentation associated with melanosis, and pigmentation associated with vitiligo (see, e.g., Henehan et al., Experimental Dermatology, Vol. 28: pp. 877-885 (2019)).

[0304] PAR-2 ​​is expressed in various cell types of human skin and increases during inflammation (Steinhoff, M. et al., Exp Dermatol, Vol. 8: pp. 282-294 (1999)), and is thought to play a role in barrier maintenance, inflammation, and itching (Lee, SE et al., Yonsei Med J, Vol. 51: pp. 808-822 (2010)). PAR-2+ mast cells are significantly increased in the skin of patients with atopic dermatitis and have been associated with the development of chronic pruritus (Steinhoff, ibid. (1999)). PAR-2 ​​polymorphisms have been identified as risk factors for atopic dermatitis and are associated with increased serum IgE and eosinophil counts (Lee, JH et al., J Allergy Clin Immunol, Vol. 128: pp. 1326-1334 (2011)). Disease-related polymorphisms have also been identified in PAR-2 ​​activating proteases (Vasilopoulos, Y. et al., J Invest Dermatol, Vol. 123: pp. 62-66 (2004); Chien, YH et al., Clinical Reviews in Allergy & Immunology, Vol. 33: pp. 178-190 (2007)). Experimental allergic dermatitis is significantly reduced in PAR-2- / - mice (Kawagoe, J. et al., Jpn J Pharmacol, Vol. 88: pp. 77-84 (2002)). PAR-2 ​​expression has also been linked to periodontal disease, a condition that can be treated with PAR-2 ​​antagonists (see, for example, International Publication No. 2010 / 132954).

[0305] VI(a)(3). Cancer In some embodiments, methods for treating cancer are provided herein. Methods for treating cancer may include administering an anti-human PAR-2 ​​antibody or its antigen-binding fragment or a pharmaceutical composition thereof as described herein to a patient in need (e.g., a human patient). In some embodiments, methods for treating cancer are provided herein, wherein the cancer is a solid tumor. Examples of solid tumors include solid tumors in which bone marrow cells (monocytes, macrophages, dendritic cells, granulocytes, neutrophils, microglia (in the CNS), or other innate immune cells) infiltrate the tumor microenvironment. Examples of such cancers that can be treated as provided herein include, but are not limited to, glioblastoma, head and neck cancer, kidney cancer (e.g., clear cell carcinoma), pancreatic cancer, and breast cancer. Other cancers include, but are not limited to, ovarian cancer, sarcoma, colorectal cancer, lung cancer, melanoma, bladder cancer, liver cancer, and uterine cancer.

[0306] In some embodiments, cancers treated by the method of the Disclosure may include, but are not limited to, hematopoietic carcinomas such as leukemia, lymphoma, or myeloma. In some embodiments, cancers treated by the method of the Disclosure may be early-stage or late-stage. In some embodiments, the cancer may be a primary tumor. In some embodiments, the cancer may be a metastatic tumor at a second site originating from one of the above types of cancers.

[0307] In some embodiments, the cancer to be treated by the methods of the Disclosure is a PAR-2 ​​positive cancer. In some embodiments, the cancer to be treated by the methods of the Disclosure is a cancer with increased PAR-2 ​​(e.g., increased PAR-2 ​​mRNA and / or increased PAR-2 ​​protein). Success in cancer treatment may include, for example, a reduction in tumor burden, or a reduction in the rate of metastasis, or a reduction in tumor invasiveness, or a reduction in the rate of tumor growth.

[0308] PAR-2 ​​is overexpressed in various cancers and is associated with malignancy, progression, and poor prognosis (Schaffner, F. & Ruf, W., Arterioscler Thromb Vasc Biol Vol. 29: pp. 1999-2004 (2009)). PAR-2 ​​signaling is observed in several cell lines: stomach (Miyata, S. et al., J Biol Chem Vol. 275: pp. 4592-4598 (2000)), colon (Ducroc, R. et al., Life Sci, Vol. 70: pp. 1359-1367 (2002)), breast (Matej, R. et al., Physiol Res Vol. 56: pp. 475-484 (2007)), glioblastoma (Dutra-Oliveira, A. et al., Biochem Biophys Res Commun, Vol. 421: pp. 221-227 (2012)), melanoma (Kempkes, C. et al., J Invest Dermatol, Vol. 132: pp. 375-384 (2012)), and prostate (Wilson, SR et al., prostate It has been reported that PAR-2 ​​enhances proliferation in the breast (Ge, L. et al., J Biol Chem Vol. 279: pp. 55419-55424 (2004)), and the colon (Zhou, B. et al., Oncol Rep, Vol. 25: pp. 503-511 (2011)). Furthermore, blocking PAR-2 ​​signaling suppressed proliferation in a pancreatic cancer xenograft model (Iwaki, K. et al., Int J Cancer, Vol. 122: pp. 658-663 (2008)). PAR-2- / - mice showed delayed disease onset and reduced metastasis in a breast cancer model (Schaffner, F. et al., Blood, Vol. 116: pp. 6106-6113 (2010)). Furthermore, blocking PAR-2 ​​signaling inhibited the proliferation and migration of malignant glioblastoma cell lines (Gessler, F. et al., Neuroscience Vol. 165: pp. 1312-1322 (2010)) and hepatocellular carcinoma cell lines (Kaufmann, R. et al., Carcinogenesis Vol. 30: pp. 1487-1496 (2009)) in vitro.

[0309] PAR-2 ​​may at least partially promote cancer invasion and metastasis by facilitating tumor cell migration, angiogenesis, and interactions with host vascular cells, including platelets, fibroblasts, and endothelial cells lining the inside of blood vessels. It is hypothesized that inhibiting PAR-2 ​​can inhibit these processes. See, for example, Wojtukiewicz et al., Cancer Metastasis Rev, vol. 34: pp. 775-796 (2015).

[0310] In some embodiments, an anti-human PAR-2 ​​antibody or its antigen-binding fragment or a pharmaceutical composition thereof is administered in combination with radiotherapy and / or chemotherapeutic agents.

[0311] VI(a)(4). Pain associated with various diseases and / or conditions In some embodiments, anti-human PAR-2 ​​antibodies or their antigen-binding fragments or pharmaceutical compositions thereof are used to relieve pain. In some embodiments, a patient has symptoms of a disease, and the anti-human PAR-2 ​​antibodies or their antigen-binding fragments or pharmaceutical compositions thereof described herein are administered to relieve pain associated with the disease and / or condition.

[0312] Where used herein, “pain reduction” or “pain relief” means reducing the level of pain experienced by the subject. Pain relief can be evaluated as described in Younger et al., Curr Pain Headache Rep., Vol. 13(No. 1): pp. 39-43 (2009).

[0313] Examples of pain include cancer pain, arthralgia, chemotherapy-induced peripheral neuropathy pain, toothache (see, e.g., Ito, M. et al., Mol Pain Vol. 13: pp. 1-17 (2017)), bladder pain, pancreatitis pain (see, e.g., Sharma, A. et al., Am J Physiol Gastrointest Liver Physiol, Vol. 288 (No. 2): G388-95 (2005)), irritable bowel syndrome-related pain (see, e.g., Suckow et al., Mol Pain Vol. 5: p. 54 (2009); see Xu, W. et al., Evid Based Complement Alternat Med Vol. 2018: pp. 7048584 (2018)), visceral pain (see, e.g., Vergnolle, N., Br J Pharmacol, Vol. 141 (No. 8): pp. 1264-1274 (2004); Cenac, Examples include, but are not limited to, N., Curr Neuropharmacol, Vol. 11, pp. 598-605 (2013), osteoarthritis-related pain, rheumatoid arthritis-related pain, spinal cord injury pain (see, for example, Yoon, H. et al., Glia, Vol. 65 (No. 12): pp. 2070-2086 (2017)), and migraine pain.

[0314] migraine pain The involvement of PAR-2 ​​in migraine has been demonstrated in rodent models. Activation of dural PAR-2 ​​causes focal vasodilation (Dux, M. et al., Neuroscience, Vol. 161: pp. 887-894 (2009)), resulting in migraine-like behavioral responses in wild-type mice that are not present in PAR-2- / - mice, and can be blocked by either sumatriptan or a PAR-2 ​​peptide antagonist (Hassler, SN et al., Cephalalgia: An International Journal of Headache, Vol. 39: pp. 111-122 (2019)). PAR-2 ​​activation in neurons and / or mast cells is also considered to promote migraine-like pain in animal models.

[0315] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment or the pharmaceutical composition thereof described herein is administered to alleviate migraine pain or to reduce the incidence, severity, or duration of migraine pain.

[0316] Joint pain PAR-2 ​​is overexpressed in chondrocytes of patients with osteoarthritis (OA) (Xiang, Y. et al., Osteoarthritis Cartilage, Vol. 14: pp. 1163-1173 (2006)) and rheumatoid arthritis (RA) (Busso, N. et al., Arthritis Rheum, Vol. 56: pp. 101-107 (2007)). PAR-2 ​​protein levels are positively correlated with the severity of synovitis in RA and OA patients (Tindell, AG et al., Rheumatol Int, Vol. 32 (No. 10): pp. 3077-3086 (2012)). Furthermore, in monoiodacetate (MIA)-induced (Muley, M. et al., Journal of neuroinflammation, Vol. 14: p. 168 (2017)) and surgically induced (Huesa, C. et al., Ann Rheum Dis, Vol. 75: pp. 1989-1997 (2016)) osteoarthritis models, PAR-2- / - mice showed a significant reduction in synovitis, a decrease in nociceptive behavior, and an improvement in weight bearing. See also Ferrell, WR et al., J Clin Invest, Vol. 111 (No. 1): pp. 35-41 (2003); Huesa, C. et al., Ann Rheum Dis, Vol. 75 (No. 11): pp. 1989-1997 (2016).

[0317] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate joint pain.

[0318] Chemotherapy-induced peripheral neuropathy Painful neuropathy is a common side effect of cancer chemotherapy. The etiology is unknown, and involvement of multiple systems, including the possible role of PAR-2, has been suggested (Flatters, SJ et al., British Journal of Anaesthesia, Vol. 119: pp. 737-749 (2017)). In experimental models of chemotherapy-induced pain, PAR-2 ​​blocking peptides reversed mechanical allodynia and thermal hyperalgesia induced by paclitaxel (Chen, Y. et al., Neuroscience Vol. 193: pp. 440-451 (2011)), mechanical pain and cold sensitivity induced by oxaliplatin (Chen, K. et al., J Neurol Sci Vol. 352: pp. 62-67 (2015); Tian, ​​L. et al., Transl Neurosci Vol. 6: pp. 111-116 (2015)), and bortezomib (Wang, Q. et al., Journal of biological regulators and homeostatic agents, Vol. 31: pp. 977-983 (2017)).

[0319] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate pain associated with chemotherapy-induced peripheral neuropathy.

[0320] Cancer pain Cancer often causes pain, which is one of the major factors affecting quality of life. Pain may be directly caused by the cancer itself or may be due to nerve damage. See, for example, Portenoy, R. et al., Pain, Vol. 81: pp. 129-134 (1999); Lam, D. et al., Pain, Vol. 156 (No. 5): pp. 923-930 (2015); McCulloch, K. et al., Front Endocrinol (Lausanne), Vol. 9: p. 257 (2018); Morgan, CR et al., J Orofac Pain, Vol. 23 (No. 3): pp. 265-267 (2009).

[0321] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate cancer pain.

[0322] Acute and chronic cancer pain Serine proteases have been identified as possible nociceptive mediators. The membrane-immobilized serine protease TMPRSS2 is significantly upregulated in cancer patients, and its expression levels correlate with the patient's pain severity. TMPRSS2 can activate PAR-2, and TMPRSS2-induced mechanoalgesia is linked to PAR-2 - / - It is not present in mice (Lam, DK et al., Pain, Vol. 156: pp. 923-930 (2015)). Serine proteases are released from head and neck cancers (Lam, DK et al., Pain, Vol. 149: pp. 263-272 (2010)). The supernatant of head and neck cancer cell cultures induces mechanical allodynia in wild-type mice, but PAR-2 - / - It is not induced in mice (Lam, DK et al., Pain vol. 149: pp. 263-272 (2010)). Furthermore, in a mouse model of chemoinducible head and neck cancer, cancer-induced chronic allodynia is associated with PAR-2 - / - It was completely absent in mice (Lam, DK et al., J Neurosci Vol. 32: pp. 14178-14183 (2012)).

[0323] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate acute and chronic cancer pain.

[0324] Bone cancer pain In a tumor cell induction model of bone cancer, pain behavior correlates with the upregulation of PAR-2 ​​in the sciatic nerve and dorsal root ganglia. Pain behavior was not observed in PAR-2- / - mice and could be reversed in wild-type mice by intrathecal administration of a PAR-2 ​​blocking peptide (Liu, S. et al., European Journal of Pain, Vol. 18: pp. 326-337 (2014)). In a rat bone cancer model, PAR-2 ​​blockade was found to increase the analgesic effect of morphine (Bao, Y. et al., Reg Anesth Pain Med, Vol. 40: pp. 158-165 (2015)).

[0325] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate bone cancer pain.

[0326] Pancreatic cancer pain The number of perineurial mast cells increases in pancreatic cancer patients with cancer-associated pain (Demir, IE et al., PLoS One Vol. 8: e60529 (2013)), and PAR-2+ neurons increase in pancreatic cancer (Zhu, J. et al., Oncotarget Vol. 8: pp. 61810-61823 (2017)). The interaction between mast cells and PAR-2+ neurons has been suggested to be involved in the development of neuropathic pain (Sakamoto, A. et al., Pharmacol Res, Vol. 105: pp. 84-92 (2016)). The supernatant of pancreatic cancer cell cultures induced pain behavior in rats, which was alleviated by treatment with PAR-2 ​​blocking peptides. Pain behavior in a nude mouse orthotopic pancreatic cancer model was also reduced by treatment with PAR-2 ​​blocking peptides (Zhu, J. et al., Oncotarget Vol. 8: pp. 61810-61823 (2017)).

[0327] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate pancreatic cancer pain.

[0328] bladder pain Intravesical infusion of PAR-2 ​​activating peptides into the bladder induced prostanoid-dependent hyperalgesia in mice (Tsubota, M. et al., J Pharmacol Sci, Vol. 136: pp. 46-49 (2018)), and intrathecal administration of PAR-2 ​​blocking peptides reduced bladder activity and pain in a mouse model of cystitis (Chen, D. et al., Transl Neurosci, Vol. 7: pp. 133-138 (2016)).

[0329] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to relieve bladder pain.

[0330] Irritable bowel syndrome (IBS) PAR-2 ​​and tryptase expression were increased in biopsies derived from IBS patients (Liang, WJ et al., Gut and Liver, Vol. 10: pp. 382-390 (2016)), and increased proteolytic activity was observed in the culture supernatant of biopsies from IBS patients compared to healthy controls (Cenac, N. et al., J Clin Invest, Vol. 117: pp. 636-647 (2007)). When administered intracolonically, these supernatants induced visceral hyperalgesia and allodynia in wild-type mice, but not in PAR-2- / - mice. In endosomes, PAR-2 ​​has been suggested to be involved in persistent pain in IBS (Jimenez-Vargas, NN et al., Proc Natl Acad Sci USA, Vol. 115, pp. E7438-E7447 (2018)).

[0331] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate pain associated with IBS.

[0332] Pancreatitis PAR-2 ​​is expressed by most nociceptive neurons in the thoracic dorsal root ganglia of rats (Hoogerwerf, WA et al., Gastroenterology Vol. 127: pp. 883-891 (2004)), and is upregulated in the dorsal root ganglia of rats with experimental chronic pancreatitis (Zhang, W. et al., Pancreas Vol. 40: pp. 300-307 (2011)). Infusion of PAR-2 ​​activating peptide into the rat pancreatic duct induced pain behavior (Hoogerwerf, WA et al., Gastroenterology Vol. 127: pp. 883-891 (2004)). PAR-2 ​​has been suggested to function upstream of both TRPA1 (Terada, Y. et al., J Pharmacol Sci, Vol. 123: pp. 284-287 (2013)) and TRPV1 (Nishimura, S. et al., Life Sci, Vol. 87: pp. 643-650 (2010)) in pancreatic pain.

[0333] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate pain associated with pancreatitis.

[0334] V(a)(5). Other diseases and conditions Examples of rheumatic disorders that can be treated by the methods disclosed herein include, but are not limited to, adult and juvenile rheumatoid arthritis, scleroderma, systemic lupus erythematosus, lupus-like syndrome, undifferentiated connective tissue disease, gout, osteoarthritis, polymyalgia rheumatica, seronegative spondyloarthropathy (including ankylosing spondylitis), Reiter's disease, psoriatic arthritis, and chronic Lyme arthritis.

[0335] Examples of additional diseases or conditions that can be treated by the methods of this disclosure include, but are not limited to, fibrosis, arthritis, Still's disease and uveitis associated with rheumatoid arthritis, orofacial granulomatosis (e.g., Ketabchi et al., Oral Diseases, Vol. 13: pp. 419-425 (2007)), Guillain-Barré disease, type 1 diabetes, Graves' disease, Addison's disease, Raynaud's phenomenon (including Raynaud's disease and Raynaud's syndrome), autoimmune hepatitis, GVHD (graft-versus-host disease), and diseases resulting in inflammation of voluntary and other muscles (including dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleimyomatosis).

[0336] Fibrosis PAR-2 ​​has been suggested to be involved in the development of pulmonary fibrosis (Wygrecka, M. et al., Am J Respir Crit Care Med, Vol. 183: pp. 703-1714 (2011)), cutaneous fibrosis (Cevikbas, F. et al., Exp Dermatol, Vol. 20: pp. 69-71 (2011)), renal fibrosis (Liu, H. et al., Inflamm Res, Vol. 59: pp. 551-559 (2010)), and cardiomyopathy (Murray, DB et al., J Cell Commun Signal, Vol. 6: pp. 45-51 (2012)). PAR-2 ​​levels are elevated in idiopathic pulmonary hypertension, and blocking PAR-2 ​​signaling reverses experimental pulmonary hypertension in mice (Kwapiszewska, G. et al., Circ Res, Vol. 110: pp. 1179-1191 (2012)). PAR-2 - / - Mice are protected from CCl4-induced liver fibrosis (Knight, V. et al., Hepatology, Vol. 55: pp. 879-887 (2012)).

[0337] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, described herein is administered to alleviate pain associated with fibrosis.

[0338] arthritis PAR-2 ​​is overexpressed in chondrocytes of patients with osteoarthritis (OA) (Xiang, Y. et al., Osteoarthritis Cartilage, Vol. 14: pp. 1163-1173 (2006)) and rheumatoid arthritis (RA) (Busso, N. et al., Arthritis Rheum, Vol. 56: pp. 101-107 (2007)). PAR-2 ​​protein levels are positively correlated with the severity of synovitis in RA and OA patients, and inflammation also correlates with PAR-2 ​​levels in OA patients (Tindell, AG et al., Rheumatol Int, Vol. 32 (No. 10): pp. 3077-3086 (2012)). PAR-2 ​​expression in circulating CD14+ and CD3+ cells is elevated in RA patients (Crill, A. et al., Ann Rheum Dis, Vol. 71: pp. 1049-1054 (2012)). The release of inflammatory mediators from cultured RA synovial cells was significantly reduced in the presence of a PAR-2 ​​antagonist (Kelso, EB et al., Arthritis Rheum, Vol. 56: pp. 765-771 (2007)). Both adjuvant-induced chronic arthritis and surgically induced osteoarthritis are associated with PAR-2 - / - In mice, it is significantly reduced (Ferrell, WR et al., J Clin Invest, vol. 111: pp. 35-41 (2003); Amiable, N. et al., J Rheumatol, vol. 38: pp. 911-920 (2011)).

[0339] In some embodiments, the anti-human PAR-2 ​​antibody or its antigen-binding fragment or the pharmaceutical composition thereof described herein is administered to alleviate pain associated with arthritis.

[0340] VI(b). Administration and medication The anti-human PAR-2 ​​antibodies or their antigen-binding fragments provided herein, or the pharmaceutical compositions thereof provided herein, may be administered by any preferred means, including parenteral, intrapulmonary, intranasal, intratumoral, intrafocal, intracerebrospinal, intracranial, intraspinal, synovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusions include intramuscular, intravenous administration as a bolus or by continuous infusion over a period of time, intra-arterial, intra-articular, intraperitoneal, or subcutaneous administration. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous.

[0341] The appropriate dosage and administration regimen for the anti-human PAR-2 ​​antibody or its antigen-binding fragment provided herein, or the pharmaceutical composition thereof, will depend on the disease to be treated, the severity and course of the disease, the route of administration, and other factors, when used alone or in combination with one or more other additional therapeutic agents.

[0342] In some embodiments, this specification provides antibodies or antigen-binding fragments thereof, or pharmaceutical compositions, for use as pharmaceuticals.

[0343] In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions for use in methods for treating airway diseases. In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions for use in methods for treating a target airway disease, comprising the step of administering to a target an effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein.

[0344] In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions for use in methods for treating skin diseases. In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions for use in methods for treating a target skin disease, comprising the step of administering an effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein to a target.

[0345] In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein for use in methods for treating cancer. In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein for use in methods for treating a target cancer, comprising the step of administering to a target an effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein.

[0346] In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions for use in methods for reducing pain associated with various diseases and / or conditions. In some embodiments, this specification provides antibodies or antigen-binding fragments thereof or pharmaceutical compositions for use in methods for reducing pain associated with various diseases and / or conditions of a subject, comprising the step of administering an effective amount of the antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein to a subject.

[0347] In some embodiments, the antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein are administered to adults once a month, once every two weeks, once a week, twice a week, three times a week, or more frequently, to treat diseases or conditions associated with increased PAR-2 ​​expression and / or diseases or conditions that can be alleviated by antagonizing the activation of PAR-2 ​​by PAR-2 ​​activating ligands (e.g., respiratory diseases, skin diseases, cancer, inflammatory conditions, orofacial granulomatosis, and pain associated with various diseases or conditions). In some embodiments, one or more “loading doses” with shorter dosing intervals and / or higher dosing levels can be provided to more rapidly raise the antibody concentration to a therapeutically effective level, and then longer dosing intervals can be used to maintain the antibody concentration at or near the therapeutically effective level.

[0348] When administered by injection, the effective amount of the antibody or its antigen-binding fragment or pharmaceutical composition provided herein may range from about 1 mg / m² to about 20 mg / m² per adult dose. Alternatively, a fixed dose may be administered, which may range from about 5 mg / dose to about 600 mg / dose, or from about 5 mg / dose to about 2400 mg / dose. In some embodiments, the dose is 15, 30, 60, 180, 500, 1200, or 2400 mg. In some embodiments, one range of the quantitative dose is about 20 mg / dose to about 30 mg / dose.

[0349] In some embodiments, a fixed dose of 20–600 mg / dose or 25 mg / dose is administered repeatedly by injection. When a route of administration other than injection is used, the dose is appropriately adjusted according to standard medical practice. An example of a therapy regimen involves injecting a dose of about 20–600 mg or 20–30 mg of the antibody or its antigen-binding fragment or pharmaceutical composition provided herein one to three times per week for a period of at least three weeks, although longer treatment periods may be necessary to induce the desired degree of improvement. For pediatric subjects (4–17 years), one exemplary preferred regimen involves subcutaneous injection of about 0.4 mg / kg to about 25 mg / kg of the antibody or its antigen-binding fragment or pharmaceutical composition provided herein, administered two or three times per week.

[0350] In some embodiments, the methods provided herein involve subcutaneous injection of about 0.5 mg to about 10 mg of the antibody or its antigen-binding fragment or pharmaceutical composition provided herein, administered once or twice a week. Some embodiments relate to pulmonary administration (e.g., by nebulizer) of about 3 mg or more of the antibody or its antigen-binding fragment or pharmaceutical composition provided herein, administered once a week.

[0351] Examples of therapeutic regimens provided herein include subcutaneous injection of an antibody or its antigen-binding fragment or pharmaceutical composition provided herein, administered once weekly at a dose of approximately 1.5 mg to approximately 3 mg, for the treatment of diseases or conditions associated with increased PAR-2 ​​expression and / or diseases or conditions that can be alleviated by antagonizing the activation of PAR-2 ​​by a PAR-2 ​​activating ligand (e.g., respiratory diseases, skin diseases, cancer, orofacial granulomatosis, inflammatory conditions, and pain associated with various diseases or conditions). The once-weekly administration of the antibody or its antigen-binding fragment or pharmaceutical composition provided herein is continued until the desired result is achieved, for example, until the symptoms of the subject subside. Treatment may be restarted as needed, or a maintenance dose may be administered instead.

[0352] Other examples of therapeutic regimens provided herein involve subcutaneous or intravenous administration of an antibody or its antigen-binding fragment or pharmaceutical composition provided herein in doses of approximately 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg per kilogram of body weight (mg / kg) of the subject. In some embodiments, the dose is approximately 0.5 mg / kg. The dose may be administered to the subject once, or more than once at certain intervals, for example, once a week, three times a month, twice a month, once a month, once every two months, once every three months, once every six months, or once a year. The duration of treatment, as well as any changes in the dosage and / or frequency of treatment, may be modified or altered during the course of treatment to meet the specific needs of the patient.

[0353] VI(c) Combination Therapy In some embodiments, the Disclosure provides anti-human PAR-2 ​​antibodies or antigen-binding fragments thereof provided herein, or pharmaceutical compositions thereof provided herein, as well as methods for treating a subject with one or more other therapeutic agents.

[0354] In some embodiments, the method includes the step of administering one or more PAR-2 ​​antagonists described herein and one or more other therapeutic agents (e.g., therapeutic or palliative agents). If the method involves administering more than one therapeutic agent to a subject, the order, timing, frequency, concentration, and volume of administration are limited only by medical requirements and limitations of the therapeutic agents, i.e., two therapeutic agents may be administered to a subject, for example, simultaneously, sequentially, alternately, or according to any other regimen.

[0355] In certain embodiments, this method includes, but is not limited to, one or more PAR-2 ​​antagonists described herein and one or more other therapeutic agents for chronic obstructive pulmonary disease, including, for example, short-acting bronchodilators (e.g., albuterol, ipratropium, revalbuterol, or a combination thereof); long-acting bronchodilators (e.g., aclidinium, alformoterol, formoterol, indacaterol, tiotropium, salmeterol, umeclidinium, or a combination thereof); and inhaled steroids (e.g., flutica The process includes administering a combination inhalant (e.g., fluticasone or budesonide); fluticasone, umeclidinium, and vilanterol; formoterol and budesonide; salmeterol and fluticasone; aclidinium and formoterol; albuterol and ipratropium; formoterol and glycopyrrolate; glycopyrrolate and indacaterol; orodaterol and tiotropium; or umeclidinium and vilanterol); a phosphodiesterase-4 inhibitor; theophylline; and an antibiotic.

[0356] In certain embodiments, the method includes administering one or more PAR-2 ​​antagonists described herein along with one or more other therapeutic agents for asthma, including, for example, inhaled corticosteroids (e.g., fluticasone propionate, budesonide, ciclesonide, beclomethasone, mometasone, and fluticasone furoate); leukotriene modifiers (e.g., montelukast, zafirlukast, and diloiton); combination inhalants (fluticasone-salmeterol, budesonide-formoterol, formoterol-mometasone, and fluticasone furoate-vilanterol); theophylline; short-acting beta-agonists (e.g., albuterol and revalbuterol); anticholinergics (ipratropium and tiotropium); and oral and intravenous corticosteroids (prednisone and methylprednisolone).

[0357] In certain embodiments, the method involves administering one or more of the PAR-2 ​​antagonists described herein, along with one or more other therapeutic agents for idiopathic pulmonary fibrosis, including, but not limited to, pirfenidone and nintedanib.

[0358] In certain embodiments, the method includes administering one or more PAR-2 ​​antagonists described herein, as well as one or more other therapeutic agents for pulmonary hypertension, including but not limited to vasodilators (e.g., epoprostenol), guanylate cyclase (GSC) stimulants (e.g., riociguat), endothelin receptor antagonists (e.g., bosentan, macitentan, and ambrisentan), sildenafil, tadalafil, high-dose calcium channel blockers (amlodipine, diltiazem, and nifedipine), anticoagulants (e.g., warfarin), digoxin, and diuretics.

[0359] In certain embodiments, such combination therapies achieve synergistic or additive effects, for example, by attacking multiple sites or molecular targets of a tumor. Types of combination therapies that can be used in conjunction with the present disclosure include inhibiting or activating multiple nodes in a single disease-related pathway, multiple pathways in target cells, and multiple cell types within target tissue (as necessary).

[0360] VI(d). Detection and diagnostic use The anti-human PAR-2 ​​antibodies or their antigen-binding fragments described herein (see, for example, Section II) can be used to assay the level of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) in a biological sample using classic methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labeling such as glucose oxidase; iodine ( 125 I, 121 I), carbon ( 14 C), sulfur (35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Examples of labels include radioisotopes such as Tc; luminescence labels such as luminol; fluorescent labels such as fluorescein and rhodamine; and biotin. Such labels can be used to label the antibodies or antigen-binding fragments described herein. Alternatively, a secondary antibody or antigen-binding fragment that recognizes the anti-human PAR-2 ​​antibody or antigen-binding fragment described herein can be labeled and used in combination with the anti-human PAR-2 ​​antibody or antigen-binding fragment to detect PAR-2 ​​protein levels (e.g., human PAR-2 ​​protein).

[0361] Assays on the expression levels of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) are intended to involve qualitatively or quantitatively measuring or estimating the level of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) in a first biological sample, either directly (e.g., by determining or estimating an absolute protein level) or relatively (e.g., by comparing it to disease-related protein levels in a second biological sample). The expression level of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) in the first biological sample can be measured or estimated and compared to a reference PAR-2 ​​protein (e.g., human PAR-2 ​​protein) level, which is determined by taking a second biological sample from an individual without the disorder or by averaging the levels of a population of individuals without the disorder. As will be understood in the art, once the “reference” PAR-2 ​​protein (e.g., human PAR-2 ​​protein) level is known, it can be repeatedly used as a reference for comparison.

[0362] As used herein, the term “biological sample” refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells capable of expressing the PAR-2 ​​protein (e.g., human PAR-2 ​​protein). Methods for obtaining tissue biopsies and bodily fluids from animals (e.g., humans) are well known in the art.

[0363] The anti-human PAR-2 ​​antibodies described herein can be used in prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications, which are well known to those skilled in the art and are standard practice. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro determination and evaluation of immune system status and / or immune response can be used to predict, diagnose, and monitor patient samples, for example, those found to have or suspected to have pain associated with respiratory diseases, skin diseases, cancer, and various diseases or conditions.

[0364] The anti-human PAR-2 ​​antibodies and their antigen-binding fragments described herein may carry detectable or functional labels. When fluorescent labels are used, specific binding members can be identified and quantified using currently available microscopy and fluorescence-activated cell sorting analysis (FACS), or a combination of both methods known in the art. The anti-human PAR-2 ​​antibodies or their antigen-binding fragments described herein may carry fluorescent labels. Examples of fluorescent labels include, for example, reactive probes and conjugate probes, such as aminocoumarin, fluorescein, and Texas red, Alexa fluorescent dyes, Cy dyes, and DyLight dyes. The anti-human PAR-2 ​​antibodies are isotopes. 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 The label may be supported with a radioactive label such as Re. When a radioactive label is used, the specific binding of the anti-human PAR-2 ​​antibody or antigen-binding fragment to the PAR-2 ​​protein (e.g., human PAR-2 ​​protein) can be identified and quantified using currently available counting procedures known in the art. If the label is an enzyme, detection can be achieved by any of the currently used colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric, or gasometric methods known in the art. This can be achieved by contacting the sample or control sample with the anti-human PAR-2 ​​antibody or its antigen-binding fragment under conditions that allow for the formation of a complex between the antibody or its antigen-binding fragment and the PAR-2 ​​protein (e.g., human PAR-2 ​​protein). Any complex formed between the antibody or its antigen-binding fragment and the PAR-2 ​​protein (e.g., human PAR-2 ​​protein) is detected and compared in the sample and the control. In light of the specific binding of the antibodies or their antigen-binding fragments described herein to human PAR-2, the expression of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) on the cell surface can be specifically detected using the antibodies or their antigen-binding fragments. The antibodies or their antigen-binding fragments described herein can also be used to purify PAR-2 ​​protein (e.g., human PAR-2 ​​protein) by immunoaffinity purification.

[0365] This specification also includes assay systems that can be prepared in the form of test kits for quantitative analysis of the degree of presence of PAR-2 ​​protein (e.g., human PAR-2 ​​protein). The system or test kit may include a labeled component, such as a labeled antibody or antigen-binding fragment, and one or more additional immunochemical reagents. For example, see Section VII below for details of the kits.

[0366] In some embodiments, this specification provides a method for in vitro detection of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) in a sample, comprising the step of contacting the sample with an antibody or an antigen-binding fragment thereof. In some embodiments, this specification provides the use of an antibody or an antigen-binding fragment thereof provided herein for in vitro detection of PAR-2 ​​protein (e.g., human PAR-2 ​​protein) in a sample. In some embodiments, this specification provides an antibody or an antigen-binding fragment thereof provided herein for use in detecting PAR-2 ​​protein (e.g., human PAR-2 ​​protein) in or in a sample obtained from a subject. In some embodiments, this specification provides an antibody or an antigen-binding fragment thereof provided herein for use as a diagnostic agent. In some embodiments, the antibody includes a detectable label.

[0367] VII. Kit This specification provides kits comprising one or more antibodies or antigen-binding fragments thereof as described herein. In some embodiments, this specification provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof as described herein. Optionally, such containers may be accompanied by a notice in the form prescribed by the governmental authority regulating the manufacture, use, or sale of a pharmaceutical or biological product, the notice reflecting the authority's approval for manufacture, use, or sale for human administration.

[0368] This specification also provides kits that can be used in detection methods. In some embodiments, the kit comprises, in one or more containers, an antibody or its antigen-binding fragment described herein, a purified antibody or its antigen-binding fragment. In some embodiments, the kit described herein comprises substantially isolated PAR-2 ​​protein (e.g., human PAR-2 ​​protein) that can be used as a control. In some embodiments, the kit described herein further comprises a control antibody or its antigen-binding fragment that does not react with PAR-2 ​​protein (e.g., human PAR-2 ​​protein). In some embodiments, the kit described herein comprises one or more elements for detecting the binding of an antibody or its antigen-binding fragment to PAR-2 ​​protein (e.g., human PAR-2 ​​protein) (for example, the antibody or its antigen-binding fragment may be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody or its antigen-binding fragment that recognizes the first antibody or its antigen-binding fragment may be conjugated to a detectable substrate). In some embodiments, the kits provided herein may include recombinantly produced or chemically synthesized PAR-2 ​​protein (e.g., human PAR-2 ​​protein). The PAR-2 ​​protein provided in the kit (e.g., human PAR-2 ​​protein) may be attached to a solid support. In some embodiments, the detection means of the kit described above includes a solid support to which the PAR-2 ​​protein (e.g., human PAR-2 ​​protein) is attached. Such kits may also include an unattached reporter-labeled anti-human antibody or its antigen-binding fragment, or an anti-mouse / rat antibody or its antigen-binding fragment. In certain embodiments, the binding of the antibody or its antigen-binding fragment to the PAR-2 ​​protein (e.g., human PAR-2 ​​protein) can be detected by the binding of the reporter-labeled antibody or its antigen-binding fragment. [Examples]

[0369] VIII. Examples The examples in this section (i.e., Section VIII) are provided as examples only and not as limitations.

[0370] (Example 1) Production of selective anti-PAR-2 ​​antibodies To generate anti-PAR-2 ​​antibodies, genetic immunization was performed in PAR-2 ​​knockout (KO) mice and rats. The polynucleotide sequence encoding human PAR-2 ​​(SEQ ID NO: 1) was cloned into a mammalian expression vector and used for immunization of PAR-2 ​​KO mice and rats.

[0371] F2RL1(PAR-2) knockout mice were generated by inserting a construct containing hygromycin and neomycin selector genes into the endogenous F2RL1 locus in mice. The target construct was electroporated into RW4 mouse embryonic stem (ES) cells. ES cells positive for the construct were injected into C57 mouse blastocysts. The resulting chimeric males were backcrossed with females, and the offspring were screened for germline transmission of the vector. Positive offspring were outcrossed to generate homozygous knockout mice. F2RL1(PAR-2) knockout rats were generated using standard techniques. The absence of the F2RL1 message in animals was confirmed by quantitative PCR.

[0372] After 4 to 12 immunization cycles, serum anti-PAR-2 ​​titers were measured for each animal using 3T3 cells transfected with human PAR-2. Animals with positive serum titers were used for monoclonal anti-PAR-2 ​​antibody production.

[0373] Antibody-secreting plasma and memory B cells were prepared from B cell-containing tissues (spleen, lymph nodes, bone marrow), and 3T3 cells transfected as the target source were used to screen single cells in high-efficiency microreactors (e.g., as described in U.S. Patents 2016 / 0252495, 9188593, and 10087408) for the secretion of antibodies that are selective for PAR-2 ​​but not for PAR-1 ("PAR-2 ​​selective antibodies"). Binding of the secreted antibodies to the target was detected using a fluorescently labeled secondary antibody. The screening assay included carboxyfluorescein succinimimidyl ester-labeled PAR-1 expressing cell lines, which allowed for the identification of antibodies that cross-react with PAR-1 or other non-PAR-2 ​​cellular components. Such antibodies were excluded from further consideration.

[0374] Over 1500 cells producing PAR-2 ​​selective antibodies were identified. Single cells expressing PAR-2 ​​selective antibodies were harvested, lysed, and cellular mRNA was isolated for cDNA synthesis using reverse transcriptase. Heavy and light chain antibody sequences were amplified from each antibody-expressing cell using primer sequences complementary to the rodent antibody variable region. The polynucleotide sequences encoding each antibody heavy and light chain variable region can be elucidated using next-generation sequencing technologies such as the Illumina MiSeq Next Generation Sequencer, or as described in U.S. Patent No. 9188593.

[0375] Sequence sequencing of the polynucleotides encoding each of these PAR-2 ​​selective antibodies identified at least 421 unique pairs of antibody heavy and light chain sequences. These paired heavy and light chains encoded by these sequence pairs were expressed as full-length human IgG4 isotype antibodies, and their PAR-2 ​​binding and selectivity were evaluated by flow cytometry. Specifically, Expi293F cells were transiently transfected with a mammalian expression plasmid encoding human PAR-2 ​​(e.g., SEQ ID NO: 28 or amino acids 26-397 of SEQ ID NO: 28), human PAR-1 (SEQ ID NO: 47), or a chimeric molecule ("Nt-PAR-2") (SEQ ID NO: 48) consisting of human PAR-2 ​​(e.g., SEQ ID NO: 28 or amino acids 26-397 of SEQ ID NO: 28), or N-terminal residues (residues 1-74) of PAR-2 ​​fused to PAR-1 residues 102-425. Cells were used for flow cytometry analysis 36-48 hours after transfection. By using Nt-PAR-2 ​​as a target, antibodies that bind to the N-terminal residue of PAR-2 ​​(containing a protease cleavage site on PAR-2) could be distinguished from antibodies that bind to other extracellular sites on PAR-2, such as the PAR-2 ​​activating ligand binding site. Expi293F cells transiently expressing one of the different target proteins were labeled for 10 minutes with a single fluorophore encoder dye (Intellicyt) of different intensities at dilutions of 1 / 250 for high intensity and / or 1 / 1500 for medium intensity, enabling simultaneous analysis of multiple cell populations. After washing, the cells were mixed with unlabeled cell types in the same ratio and analyzed for antibody binding.

[0376] Cells, 2x10 5Cells were seeded in 96-well plates at a concentration of 1 / well. After washing with phosphate-buffered saline (PBS), primary antibody (anti-PAR-2 ​​antibody) was added to each well at a concentration of 10 μg / mL in 50 μL of FACS buffer (0.5% w / v BSA in PBS) and incubated for 10–15 minutes. After washing, secondary antibody (anti-human Fc-FITC) was added to each well at a dilution of 1:200 in 50 μL for 10–15 minutes. After washing, the cells were resuspended in fluorescence-activated cell sorting (FACS) buffer. Flow cytometry analysis was performed on an Intellicyt iQue screener. Gates around cells of different encoder dye-positive intensities were drawn, and these populations were analyzed separately for binding to each other with the primary antibody and for unlabeled cells.

[0377] Table 5 lists antibodies that were expressed at detectable levels as determined by flow cytometry analysis, and antibodies that bound to human PAR-2 ​​(SEQ ID NO: 28) but not to human PAR-1 (SEQ ID NO: 47). Human PAR-1:

[0378] [ka]

[0379] Antibodies that were positive for human PAR-2 ​​binding and negative for PAR-1 binding were also screened to exclude antibodies bound to the N-terminus of PAR-2, where the anchoring ligand and protease cleavage site are located. This screening was performed using a chimeric construct consisting of the N-terminus of human PAR-2 ​​along with the remainder of the human PAR1 and Nt-PAR-2 ​​(SEQ ID NO: 48) proteins. Anti-PAR-2 ​​antibodies that did not bind to the N-terminus of PAR-2 ​​were tested for function in cell-based assays. Nt-PAR-2:

[0380] [ka]

[0381] [Table 5A]

[0382] [Table 5B]

[0383] [Table 5C]

[0384] (Example 2) Identification of antagonist anti-PAR-2 ​​antibodies 2.1 Screening of antagonist anti-PAR-2 ​​antibodies that inhibit β-arrestin signaling To screen for antagonist anti-PAR-2 ​​antibodies that inhibit β-arrestin signaling, various antibodies identified as selective for the extracellular loop of human PAR-2 ​​(positive for binding to PAR-2, but negative for binding to PAR-1 and Nt-PAR-2) were screened for their ability to antagonize SLIGKV-induced PAR-2 ​​β-arrestin activity. Specifically, Tango® F2RL1-blaU2OS cells (Thermo Fisher Scientific) were subjected to 4x10⁶ assays in 128 μL of assay buffer (1% dialyzed FBS, 0.1 mM non-essential amino acids, 25 mM HEPES (pH 7.3), 100 U / mL penicillin, 100 U / mL streptomycin, and phenol red-free DMEM). 4Cells were seeded in a 96-well black transparent-bottom plate at a concentration of cells / well. Starting at approximately 666 nM, the antibody was added to the cells in 16 μL / well assay buffer supplemented with 0.5% DMSO, using 10 serial dilutions (including a 0 μM antibody dot) obtained by serial dilution to 1 / 4. Each concentration was tested on the cells in 2 or 3 replicates. After incubation at 37°C / 5% CO2 for 30 min, the soluble PAR-2 ​​agonist SLIGKV (Sigma) (SEQ ID NO: 46) was added to the cells at a concentration of 1 or 2 μM (depending on the experiment) in 16 μL / well assay buffer containing 0.5% DMSO. The plate was incubated at 37°C / 5% CO2 for 16 hours. After loading the cell-permeable LiveBLAzer™ FRET B / G substrate (Life Technologies) with a final volume of 32 μL / well using the manufacturer's protocol, the plates were incubated at room temperature in a dark room for 2 hours. Cell fluorescence was then measured on a Molecular Devices Flex Station 3 plate reader (excited at 410 nm and emitting at both 458 nm and 522 nm, 7 readings / well). After subtracting background using cell-free wells, the curve for the blue / green fluorescence emission ratio was fitted to the log of the molar concentration of the antibody using nonlinear regression (using a 4-parameter logistic curve fitting).

[0385] Table 6 lists the efficacy of 34 selected anti-PAR-2 ​​extracellular loop selective antibodies in inhibiting PAR-2 ​​signaling in PAR-2 ​​β-arrestin cell assays (mean values ​​are shown when multiple independent assays were performed).

[0386] [Table 6]

[0387] 2.2 Screening of anti-PAR-2 ​​extracellular loop selective antibodies with broad antagonist activity To screen for anti-PAR-2 ​​extracellular loop selective antibodies with broad antagonist activity, eight anti-PAR-2 ​​extracellular loop selective antibodies were selected for further evaluation of their ability to prevent trypsin-activated PAR-2 ​​signaling-induced calcium flux. The Chemiscreen Human PAR-2 ​​receptor calcium-optimized stable CHEM-1 cell line (Eurofins) was used for the PAR-2 ​​calcium flux assay. CHEM-1 PAR-2 ​​cells were fed 7.5 x 10⁶ cells in 100 μL of serum-reduced basal medium (1% heat-inactivated FBS, 0.1 mM non-essential amino acids, 10 mM HEPES (pH 7.3), DMEM high-glucose medium (4.5 g / L D-glucose)). 4 Cells were seeded at a rate of one cell / well in a 96-well black transparent-bottom plate. The plate was incubated at 37°C / 5% CO2 for 20–24 hours. After removing the culture medium, the cells were washed in 100 μL / well of assay medium (1% w / v BSA in HBSS containing 20 mM HEPES) and removed by aspiration. Fluo4NW dye (Life Technologies), reconstituted in 2.5 mM probenecid in assay buffer, was added to the cells at a rate of 100 μL / well. The plate was incubated in a dark room at 37°C / 5% CO2 for 30 minutes. The antibody was then added to the cells in 50 μL / well of assay medium as a 10-point serial dilution (containing 0 μM antibody), starting at approximately 666 nM and sequentially diluted to 1 / 3. Each concentration was tested on the cells in two or three replicates. After incubation in a dark room at room temperature for 30 minutes, the agonist trypsin (Sigma) was administered using Molecular Devices' FLIPR Tetra (200 μL dispensing height, 75 μL / s dispensing rate) to the specified EC (Emission Control). 80 The agonist was added to cells in 50 μL / well of assay buffer at a specific concentration. Cell fluorescence was measured immediately after agonist addition using FLIPR Tetra (excitation at 470–495, emission at 515–575, with a 20% increase in excitation intensity, an 80-second increase, and variable exposure time). Nonlinear regression was used to fit curves for Max-min fluorescence against the logarithm of the antibody molar concentration (using a 4-parameter logistic curve fitting).

[0388] Table 7 lists the efficacy of anti-PAR-2 ​​extracellular loop selective antibodies in inhibiting PAR-2 ​​signaling in calcium flux cell assays (mean values ​​are shown when multiple independent assays were performed). These assays were performed using trypsin EC80 as the PAR-2 ​​activator.

[0389] [Table 7]

[0390] Using the PAR-2 ​​calcium flux assay, three broad groups of antibodies were identified: antibodies Ab84, Ab87, and Ab225 failed to inhibit trypsin-induced calcium flux; antibodies Ab1, Ab20, Ab39, and Ab77 showed low-efficacy inhibition; and antibody 309 was able to completely inhibit calcium flux with an IC50 value of approximately 6.7 nM.

[0391] 2.3 Screening for antibodies that inhibit inflammatory cytokine release from PAR-2 ​​activated A549 cells Inhibition of PAR-2-mediated IL-6 and IL-8 cytokine release from human A549 epithelial-like cells was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA). Adherent A549 cells were placed in 100 μl / well in Ham's F-12K nutrient medium (supplemented with 10% fetal bovine serum) in 2x10⁶ wells of a flat-bottomed 96-well plate. 4 Cells were seeded in wells and incubated overnight at 37°C / 5%CO2. After removing the medium and washing once with 200 μl / well of 1x phosphate-buffered saline (PBS), the cells were serum-starved by incubation for 24 hours at 37°C / 5%CO2 with 100 μl / well of serum-free medium. After 24 hours, half-logarithmic serial dilutions of antibody starting at 666.7 nM and 100 μM EC2 were used. 50SLIGKV at a concentration of 200 μl / well was added to the cells and incubated at 37°C / 5% CO2 for 24 hours. After 24 hours of incubation, the supernatant was collected and IL-6 and IL-8 cytokine levels were assessed using a commercially available IL-6 and IL-8 ELISA kit, and absorbance at 450 nm (subtracting the 570 nm wavelength) was assessed using a SpectraMax absorbance reader. After subtracting background, PAR-2-mediated IL-6 and IL-8 expression levels were interpolated from a standard curve and plotted with the antibody concentration plotted against the IL-6 and IL-8 concentrations using a 4-parameter logistic curve fitting. Ab309 was more potent than Ab87 in inhibiting the release of IL-6 and IL-8 from A549 cells.

[0392] (Example 3) Humanization of potent anti-PAR-2 ​​extracellular loop selective antibodies Humanization of the potent anti-PAR-2 ​​extracellular loop selective antibody 309 (Ab309) was performed by transplanting a complementation-determining region (CDR) derived from the variable region of a non-human "donor" antibody into a human "acceptor" variable region framework. The following CDR residues were used to define the CDRs: AbM nomenclature heavy chain CDR1 (residues H26, H27, H28, H29, H30, H31, H32, H33, H34 and H35 as defined by the Kabat numbering scheme); Kabat-based heavy chain CDR2 (residues H50, H51, H52, H53, H54, H55, H56, H57, H58, H59 and H60 as defined by the Kabat numbering scheme, excluding the last 5 amino acids); Kabat-based heavy chain CDR3 (residues H95, H96, H97, H98 as defined by the Kabat numbering scheme, H99, H100, H100A, H100B, H100C, H101 and H102); and light chain CDRs by Kabat (as defined by the Kabat numbering scheme, CDR1 residues L24, L25, L26, L27, L28, L29, L30, L31, L32, L33 and L34; CDR2 residues L50, L51, L52, L53, L54, L55 and L56; and CDR3 residues L89, L90, L91, L92, L93, L94, L96 and L97).

[0393] CDRs derived from the variable region of the heavy chain of antibody 309 were transplanted into the human antibody variable region as shown in Figure 1A. CDRs derived from the variable region of the light chain of antibody 309 were transplanted into the human antibody variable region as shown in Figure 1B. Antibodies with high identity to the human germline sequence were preferred.

[0394] The variable regions of humanized antibodies were converted to recombinant IgG4 and expressed. Specifically, the variable heavy and light chains of each antibody were back-translated and codon-optimized. Polynucleotides encoding the antibody variable region sequences were synthesized by gene synthesis. All heavy chain variable regions were subcloned into mammalian expression vectors encoding human IgG4 using standard restriction enzyme cloning (throughout this specification, the term "IgG4" refers to human IgG4 including the S228P substitution and terminal lysine deletion (K447Δ) (residue numbering by Euro index)) (SEQ ID NO: 34). The sequences encoding the variable light chains were subcloned into mammalian expression vectors encoding the human kappa constant region (SEQ ID NO: 37).

[0395] Simultaneous transfection of heavy and light chain plasmids was performed in Expi293F cells using the manufacturer's protocol (ThermoFisher). Cell culture supernatant was collected by centrifugation, and antibodies were captured on a protein A resin-containing column. Antibody elution was performed using either 100 mM acetic acid, 100 mM arginine HCl, or 5 mM histidine at pH 3.5, and the resulting eluent was neutralized to pH 7-8 using a basic buffer. The antibodies were desalted by gel filtration in either 10 mM histidine HCl, 100 mM arginine HCl at pH 6, or PBS at pH 6, and concentrated using an Amicon Ultra-15 centrifugation filter unit (Merck Millipore) if necessary.

[0396] Cell lines expressing human PAR-1, human PAR-2, or a chimera ("Nt-PAR-2") consisting of the N-terminal residues (residues 1-74) of PAR-2 ​​fused to residues 102-425 of PAR-1 were generated by electroporation of mammalian expression vectors encoding the corresponding proteins (human PAR-2 ​​(e.g., SEQ ID NO: 28 or amino acids 26-397 of SEQ ID NO: 28), human PAR-1 (SEQ ID NO: 47), Nt-PAR-2 ​​(SEQ ID NO: 48)) into C6 rat or 3T3 cell lines, under the selection of stable antibiotic marker genes. The selectivity of purified humanized antibodies was evaluated using flow cytometry with human PAR-2, PAR-1, and Nt-PAR-2 ​​in transfected cells.

[0397] A subset of these antibodies (Ab309 (parental mouse antibody), Ab309-4e, Ab-309-6e, Ab309-11e, Ab309-12e, Ab309-4i, Ab309-6i, Ab309-11i, and Ab309-12i) were evaluated for inhibition of PAR-2 ​​signaling in β-arrestin and / or calcium flux assays as described in Examples 2.1 and 2.2 above. The inhibitory efficacy of humanized anti-PAR-2 ​​extracellular loop selective antibodies (Ab309, Ab309-4e, Ab309-6e, Ab309-11e, Ab309-12e, Ab309-4i, Ab309-6i, Ab309-11i, and Ab309-12i) is listed in Table 8 (mean values ​​are shown when multiple independent assays were performed).

[0398] [Table 8]

[0399] Ab309-4e, Ab-309-6e, Ab309-11e, and Ab309-12e were similar in their ability to block β-arrestin signaling in this assay and similar in activity to Ab309.

[0400] Of the four antibodies tested, Ab309-4e and 309-12e showed the most potent inhibition of trypsin-activated calcium flux or beta-arrestin activity, demonstrating efficacy equivalent to that of the parent non-optimized antibody Ab309.

[0401] (Example 4) Optimization of humanized anti-PAR-2 ​​extracellular loop selective antibodies For further development, we selected the humanized anti-PAR-2 ​​extracellular loop selective antibody 309-4e and constructed a group of variants to minimize potential immunogenicity and manufacturing burden while enhancing efficacy.

[0402] Attempts to minimize potential immunogenicity included replacing rodent sequences with sequences based on human germline sequences where possible, and modifying sequences predicted in silico when bound to human MHC class II molecules. Several software programs, such as EpiBase (Lonza) or EpiVax, are available to perform this analysis.

[0403] We identified residues or sequence motifs associated with undesirable post-translational modifications such as glycosylation, aspartate isomerization, deamidation, and oxidation, and constructed various variants by creating different amino acid substitutions with the selected residues. Attempts to improve antibody efficacy included creating substitutions with each CDR residue predicted to be solvent-exposed ...

Claims

1. It specifically binds to human protease-activated receptor 2 (PAR-2), (a) Sequence numbers 10, 11, 12, 16, 17, and 18, respectively; (b) Sequence IDs 7, 8, 9, 13, 14, and 15, respectively; (c) Sequence numbers 10, 11, 12, 16, 19, and 18, respectively; (d) Sequence IDs 10, 11, 12, 16, 29, and 18, respectively; or (e) Sequence numbers 10, 11, 12, 16, 22, and 18 respectively An isolated antibody or its antigen-binding fragment comprising the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3 sequences, and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences.

2. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3 sequences and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of sequence numbers 10, 11, 12, 16, 17, and 18, respectively.

3. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3 sequences and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of sequence numbers 7, 8, 9, 13, 14, and 15, respectively.

4. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3 sequences and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of sequence numbers 10, 11, 12, 16, 19, and 18, respectively.

5. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3 sequences and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of sequence numbers 10, 11, 12, 16, 29, and 18, respectively.

6. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, VH CDR3 sequences and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of sequence numbers 10, 11, 12, 16, 22, and 18, respectively.

7. (a) Sequence IDs 20 and 23, respectively (b) Sequence IDs 21 and 24, respectively (c) Sequence IDs 21 and 25 respectively, (d) Sequence IDs 21 and 26, respectively, or (e) Sequence IDs 21 and 27 The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL) containing the amino acid sequence.

8. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain variable region (VH) and a light chain variable region (VL), respectively, containing the amino acid sequences of SEQ ID NOs. 20 and 23.

9. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain variable region (VH) and a light chain variable region (VL), respectively, containing the amino acid sequences of SEQ ID NOs. 21 and 24.

10. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain variable region (VH) and a light chain variable region (VL), respectively, containing the amino acid sequences of SEQ ID NOs. 21 and 25.

11. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain variable region (VH) and a light chain variable region (VL), respectively, containing the amino acid sequences of SEQ ID NOs. 21 and 26.

12. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain variable region (VH) and a light chain variable region (VL), respectively, containing the amino acid sequences of SEQ ID NOs. 21 and 27.

13. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region and a light chain constant region.

14. The heavy chain constant region is human IgG 1 IgG 2 IgG 3 , and IgG 4 The antibody or antigen-binding fragment thereof according to claim 13, which is an isotype selected from the group consisting of isotypes.

15. The heavy chain constant region is human IgG 4 Human IgG containing the heavy chain constant region, or S228P substitution (according to EU numbering) and / or terminal lysine deletion (K447Δ) (according to EU numbering). 4 The antibody or antigen-binding fragment thereof according to claim 14, which is a heavy chain constant region.

16. Human IgG 4 The antibody or antigen-binding fragment thereof according to claim 15, wherein the heavy chain constant region comprises an S228P substitution (according to EU numbering) or a terminal lysine deletion (K447Δ) (according to EU numbering).

17. The antibody or antigen-binding fragment thereof according to claim 13, wherein the light chain constant region is the human IgGκ light chain constant region.

18. An antibody or antigen-binding fragment thereof according to claim 1, having a binding affinity (K -10 ) for human PAR-2 of 4×10 -9 M to 1×10 D M.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in claim 1, and a pharmaceutically acceptable excipient.

20. A method for inhibiting the activation of PAR-2 ​​by a PAR-2 ​​activating ligand in vitro, comprising the step of blocking ligand binding to PAR-2 ​​using the antibody or antigen-binding fragment thereof described in claim 1.

21. An isolated polynucleotide comprising a first nucleic acid molecule encoding the heavy chain variable region (VH) or heavy chain of an antibody or its antigen-binding fragment, and a second nucleic acid molecule encoding the light chain variable region (VL) or light chain of an antibody or its antigen-binding fragment, wherein the first nucleic acid molecule encodes the VH of SEQ ID NO: 20 and the second nucleic acid molecule encodes the VL of SEQ ID NO: 23, or the first nucleic acid molecule encodes the VH of SEQ ID NO: 21 and the second nucleic acid molecule encodes the VL of SEQ ID NO: 24, 25, 26, or 27.

22. An isolated vector comprising the polynucleotide described in claim 21.

23. A host cell containing the polynucleotide described in claim 21.

24. A method for producing an antibody or antigen-binding fragment thereof that binds to human PAR-2, comprising the step of culturing a host cell as described in claim 23 such that a nucleic acid molecule is expressed and an antibody or antigen-binding fragment thereof is produced, further comprising the step of isolating the antibody or antigen-binding fragment thereof from the culture.

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