Anti-par2 antibodies and uses thereof

Abstract

To provide novel anti-inflammatory pain therapeutics.SOLUTION: The present disclosure provides antibodies and antigen-binding fragments capable of binding PAR2. In some embodiments, the anti-PAR2 antibodies or antigen-binding fragments thereof bind PAR2 in a pH-dependent manner. The disclosure further provides methods for making and using the antibodies and antigen-binding fragments. The antibodies of the disclosure are useful, inter alia, for inhibiting PAR2-mediated signaling and for treating diseases and disorders caused by or related to PAR2 activity and / or PAR2 signaling.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 472,462, filed Mar. 16, 2017, and U.S. Provisional Patent Application No. 62 / 637,766, filed Mar. 2, 2018. The foregoing applications are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. A copy of the ASCII created on Mar. 8, 2018, is named 1848081 - 0002 - 093 - WO1_SL.txt and is 351,668 bytes in size.

Background Art

[0003] Chronic pain is a condition that can affect anyone and imposes a burden on patients, the healthcare system, and the economy. Approximately 100 million people in the United States suffer from chronic pain, and the annual incremental cost of healthcare due to pain, including medical expenses and the economic costs of lost time and lost wages, is estimated to be between $560 billion and $635 billion (Institute of Medicine of The National Academies, 2011). Further, in a survey of chronic pain patients, more than half had little or no control over their pain (Non - Patent Document 1). Pain can be caused by a variety of conditions and diseases, from cancer to diabetes and arthritis, and can be classified into categories of nociceptive, neuropathic, and mixed pain. Nociceptive pain is defined by the stimulation of nerve fibers (e.g., by thermal, mechanical, or chemical stimuli), while neuropathic pain is pain caused by a variety of causes such as nerve damage, nerve disease, and importantly, inflammation. Thus, inflammation, which is a process by which the organism mobilizes immune cells and releases immune factors at the site of injury or infection, can be a process that helps in both injury repair and the cause of pain.

[0004] Many treatments for pain inhibit inflammation. Two general classes of anti-inflammatory pain medications are steroidal anti-inflammatory agents and non-steroidal anti-inflammatory drugs (NSAIDs). Steroidal anti-inflammatory agents typically suppress prostaglandins and leukotrienes, which are products of inflammation. Such agents are reliable and powerful, but carry a risk of serious side effects including, for example, decreased bone density, weight fluctuations, immune system suppression, and growth / puberty abnormalities (Non-Patent Document 2; Non-Patent Document 3). NSAIDs inhibit cyclooxygenase-1 and / or 2 (COX-1 and / or COX-2), which catalyze the reaction from arachidonic acid to prostaglandins. Chronic pain and inflammation require long-term treatment, and long-term inhibition of the COX enzymes can cause gastrointestinal problems such as stomach bleeding and ulcers. Considering the risks associated with such anti-inflammatory pain treatments, alternative approaches for treating pain are needed.

[0005] G protein-coupled receptors (GPCRs) are a family of membrane proteins that share a common structural motif of seven transmembrane domains with an N-terminal extracellular domain and a C-terminal intracellular domain linked together (Non-Patent Document 4). GPCRs sense extracellular signals such as photons, hormones, chemokines, etc., and activate intracellular G proteins. There are many families of GPCRs, such as the Frizzled, rhodopsin, secretin, adhesion, and protease-activated receptor (PAR) families (Non-Patent Document 5; Non-Patent Document 6). Although various GPCR families share overall structural features, they exhibit different functions, bind to different ligands, and are activated by different mechanisms. Activation of the PAR family of GPCRs is associated with inflammation and nociception (Non-Patent Document 7).

[0006] Four PAR receptors have been identified: PAR1, PAR2, PAR3, and PAR4 (Non-Patent Document 8; Non-Patent Document 7). PAR2 activation has been shown to enhance inflammation and nociception, making its inhibition an attractive target for anti-inflammatory pain therapy. Unlike other GPCRs, PARs are activated by proteolytic cleavage of the extracellular domain protein that presents an N-terminal sequence functioning as a tethered agonist. PAR2 is specifically cleaved and activated by trypsin and tryptase.

[0007] PAR2 expression has been detected in vascularized tissues, airways, osteoblasts, cardiovascular tissues, keratinocytes, exocrine glands, leukocytes, mast cells, intestinal epithelium, kidneys, neurons, pancreas, and various smooth muscle types (Non-Patent Document 8 cited above). PAR2 has also been associated with various diseases or conditions related to neurogenic inflammation, nociception, and pain transmission. PAR2 can be activated by several host- and pathogen-derived serine proteases (e.g., trypsin, mast cell tryptase, tissue kallikrein, or members of the blood coagulation cascades TF-FVIIa and FVa-FXa).

[0008] Monoclonal antibodies have been shown to be useful for various therapeutic applications, and many antibody therapeutics are currently on the market (Non-Patent Document 9; Non-Patent Document 10). The most common type of antibody in blood circulation is immunoglobulin G (IgG). The usefulness of IgG antibodies for therapeutic purposes depends on several factors, including the specificity of the antibody for its target, the strength of its binding to the target, and the degree of efficiency with which the antibody can be produced and the speed at which the antibody disappears from the serum (serum half-life of the antibody). The serum half-life of an antibody is generally regulated by FcRn (neonatal Fc receptor), which binds to the Fc domain of immunoglobulin G (IgG). In vivo, IgG is thought to be taken up nonspecifically by fluid-phase pinocytosis (Non-Patent Document 11). When IgG binds to FcRn within the endosome, it is recognized and directed to recycling endosomes, away from lysosomes and degradation, and back to the cell surface. The recycling rate of IgG is estimated to be only 44% catabolism rate, and antibody therapeutics can still be depleted within just a few days after administration (Non-Patent Document 12).

[0009] Pain associated with inflammation is often a chronic condition. It is desirable to minimize the dosage and frequency of administration of therapeutic molecules. Therefore, new anti-inflammatory pain therapeutics are needed. Standard monoclonal antibodies are attractive candidates, but in some cases, they can be limited by their serum half-life. Therefore, alternative treatments may be desirable.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Summary of the Invention

Means for Solving the Problems

[0011] The present disclosure provides antibodies that bind to PAR2. The antibodies of the present disclosure are useful, inter alia, for inhibiting PAR2-mediated signal transduction and for treating diseases and disorders caused by or associated with PAR2 activity and / or PAR2 signal transduction.

[0012] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to PAR2 and has a higher affinity at pH 7.4 than at pH 6.0. In some embodiments, the antibody or antigen-binding fragment binds to PAR2 with an affinity that is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher at pH 7.4 than at pH 6.0. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 3; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 4; and iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 5; and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 8; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 9; and iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 10; the amino acid substitutions, deletions, or insertions reduce the binding affinity of the antibody or antigen-binding fragment thereof for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50, or 10 times or less compared to an antibody or antigen-binding fragment having a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 7 when tested at a pH of 7.4 in a PAR2 binding assay. In some embodiments, the amino acid substitutions, deletions, or insertions include conservative substitutions.In some embodiments, the antibody or antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, insertions, or deletions in the VH CDR as compared to the CDR amino acid sequences present in the sequence of SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, insertions, or deletions in the VL CDR as compared to the CDR amino acid sequences present in the sequence of SEQ ID NO: 7. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions with histidine.

[0013] In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 17 of SEQ ID NO: 4 (e.g., positions 4, 5, and 7 to 17); and iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 8 of SEQ ID NO: 5; and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9; and iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 14 of SEQ ID NO: 10. In some embodiments, the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3, ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 13, ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 14, iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 15, and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 18, ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 19, iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 20. In some embodiments, histidine is present at the amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO: 4; and histidine is present at the amino acid positions corresponding to positions 2 and 3 of SEQ ID NO: 5.In some embodiments, histidine is present at the amino acid position corresponding to position 5 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 7 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 8 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 12 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 15 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 16 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 17 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid positions corresponding to positions 5 and 8 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid positions corresponding to positions 5, 8, 12, and 16 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO: 4. In some embodiments, histidine is present at the amino acid position corresponding to position 2 of SEQ ID NO: 5. In some embodiments, histidine is present at the amino acid position corresponding to position 3 of SEQ ID NO: 5. In some embodiments, histidine is present at the amino acid positions corresponding to positions 2 and 3 of SEQ ID NO: 5. In some embodiments, histidine is present at the amino acid position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, histidine is present at the amino acid position corresponding to position 5 of SEQ ID NO: 10. In some embodiments, histidine is present at the amino acid position corresponding to position 6 of SEQ ID NO: 10. In some embodiments, histidine is present at the amino acid position corresponding to position 14 of SEQ ID NO: 10.In some embodiments, VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, 544, 554, 564, 574, 584, 594, 604, 614, 624, 634, 644, 654, 664, 674, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774, 784, 794, and 811-818. In some embodiments, VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, and 819-820.In some embodiments, VL-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, and 800. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 14; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15; and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 811; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 819, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 814; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 820, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 816; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 818; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH comprises a framework region that is at least 90% identical to each of SEQ ID NOs: 803-806.In some embodiments, VH comprises a framework region that is at least 95% identical to each of SEQ ID NOs: 803 - 806. In some embodiments, VH comprises a framework region corresponding to SEQ ID NOs: 803 - 806. In some embodiments, VL comprises a framework region that is at least 90% identical to each of SEQ ID NOs: 807 - 810. In some embodiments, VL comprises a framework region that is at least 95% identical to each of SEQ ID NOs: 807 - 810. In some embodiments, VL comprises a framework region corresponding to SEQ ID NOs: 807 - 810. In some embodiments, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, and 792.In some embodiments, VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797. In some embodiments, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 12, and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 12, and VL comprises the amino acid sequence corresponding to SEQ ID NO: 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 821, and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 824, and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 827, and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 831, and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is a scFv. In some embodiments, the antigen-binding fragment is a Fab’. In some embodiments, the antibody or antigen-binding fragment is an antibody.In some embodiments, the antibody is. It is a monoclonal antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment is humanized. In some embodiments, the antibody or antigen-binding fragment is human. In some embodiments, VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841. In some embodiments, VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841.In some embodiments, VH is encoded by a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841. In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. In some embodiments, VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, VH is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11. In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16.In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, VL is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment binds to PAR2. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction of trypsin, tryptase, and / or matriptase with PAR2. In some embodiments, the antibody or antigen-binding fragment inhibits the cleavage of PAR2 by trypsin, tryptase, and / or matriptase. In some embodiments, the antibody or antigen-binding fragment inhibits the cleavage of the extracellular domain of PAR2. In some embodiments, the antibody or antigen-binding fragment inhibits the exposure of the tether ligand. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction of the tether ligand with the second transmembrane loop of PAR2. In some embodiments, the antibody or antigen-binding fragment binds to PAR2 with a higher affinity at pH 7.4 than at pH 6.0. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 100 nM when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 7.4 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 50 nM when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 7.4 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 40 nM when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 7.4 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 greater than 500 nM when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 6.0 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 greater than 1000 nM when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 6.0 in a PAR2 binding assay.In some embodiments, the antibody or antigen-binding fragment has an IC50 greater than 1100 nM when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 more than 20-fold lower at pH 7.4 than at pH 6.0 when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 more than 25-fold lower at pH 7.4 than at pH 6.0 when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 more than 30-fold lower at pH 7.4 than at pH 6.0 when competing with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 3.0×10 -10 M in a calcium influx assay in human A549 cells. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 1.5×10 -10 M in a calcium influx assay in human A549 cells. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 7.0×10 -10 M in a calcium influx assay in rat KNRK cells. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 5.5×10 -10 M in a calcium influx assay in rat KNRK cells. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 7.0×10 -11 M in a calcium influx assay in cynomolgus cynom-K1 cells. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than 5.0×10 -11Has an IC50 of less than. In some embodiments, the antibody or antigen-binding fragment disappears from the serum of the treated patient more slowly than an antibody or antigen-binding fragment lacking a histidine modification. In some embodiments, the optionally present histidine or histidines, when tested at a pH of 7.4 in a PAR2 binding assay, reduces the binding affinity of the antibody or antigen-binding fragment for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50 or 10-fold compared to an antibody or antigen-binding fragment having a VH with the amino acid sequence of SEQ ID NO: 2 and a VL with the amino acid sequence of SEQ ID NO: 7. -11 Has an IC50 of less than. In some embodiments, the antibody or antigen-binding fragment disappears from the serum of the treated patient more slowly than an antibody or antigen-binding fragment lacking a histidine modification. In some embodiments, the optionally present histidine or histidines, when tested at a pH of 7.4 in a PAR2 binding assay, reduces the binding affinity of the antibody or antigen-binding fragment for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50 or 10-fold compared to an antibody or antigen-binding fragment having a VH with the amino acid sequence of SEQ ID NO: 2 and a VL with the amino acid sequence of SEQ ID NO: 7. -11 Has an IC50 of less than. In some embodiments, the antibody or antigen-binding fragment disappears from the serum of the treated patient more slowly than an antibody or antigen-binding fragment lacking a histidine modification. In some embodiments, the optionally present histidine or histidines, when tested at a pH of 7.4 in a PAR2 binding assay, reduces the binding affinity of the antibody or antigen-binding fragment for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50 or 10-fold compared to an antibody or antigen-binding fragment having a VH with the amino acid sequence of SEQ ID NO: 2 and a VL with the amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, the present disclosure provides a nucleic acid capable of expressing any of the antibodies or antigen-binding fragments disclosed herein. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841.In some embodiments, the nucleic acid comprises any one nucleotide sequence of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841. In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. In some embodiments, the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 16.

[0015] In some embodiments, the disclosure provides a vector comprising any of the nucleic acids disclosed herein. In some embodiments, the disclosure provides a set of vectors comprising any one or more of the nucleic acids disclosed herein.

[0016] In some embodiments, the disclosure provides a host cell comprising any one or more of the vectors disclosed herein.

[0017] In some embodiments, the disclosure provides a composition comprising a pharma- ceutically acceptable carrier and any of the antibodies or antigen-binding fragments disclosed herein.

[0018] In some embodiments, the present disclosure provides a lyophilized composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0019] In some embodiments, the disclosure provides a reconstituted lyophilized composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, in some embodiments, the composition is formulated for administration by lozenge, spray, oral administration, delayed or sustained release, transmucosal administration, syrup, mucoadhesive, buccal preparation, mucoadhesive tablet, topical administration, parenteral administration, injection, subcutaneous administration, oral solution, rectal administration, buccal administration, or transdermal administration.

[0020] In some embodiments, the present disclosure provides a kit comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, or any of the compositions disclosed herein.

[0021] In some embodiments, the present disclosure provides a method for treating pain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the antibodies or antigen-binding fragments disclosed herein. In some embodiments, the present disclosure provides a method for treating pain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the compositions disclosed herein. In some embodiments, the pain is selected from the group consisting of nociceptive pain, neuropathic pain, and mixed pain. In some embodiments, the pain is associated with headache, chronic headache, migraine, cancer, viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease, liver disease, multiple sclerosis, spinal cord injury, postherpetic neuralgia, diabetic neuropathy, low back pain, inflammatory heart disease, kidney disease, gastritis, gingivitis, periodontal disease, asthma, chronic obstructive pulmonary disease, autoimmune disease, irritable bowel syndrome, fibromyalgia, lower limb pain, restless legs syndrome, diabetic neuropathy, allergic conditions, surgical procedures, acute or chronic physical injuries, fractures or contusions, spinal cord injuries, inflammatory diseases, non-inflammatory neuropathic pain conditions or functional nociceptive pain conditions, or combinations thereof. In some embodiments, the pain is osteoarthritis pain. In some embodiments, the subject is human.

[0022] In some embodiments, the present disclosure provides a method for making any one of the antibodies or antigen-binding fragments disclosed herein, the method comprising expressing any one of the nucleic acids disclosed herein in cultured cells and purifying the antibody or antigen-binding fragment. In some embodiments, the present disclosure provides the following: [Item 1] An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH: i) has the amino acid sequence of SEQ ID NO: 3, but optionally has 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 3 in VH-CDR1; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions are optionally present in the sequence of SEQ ID NO: 4; and iii) including a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions are optionally present in the sequence of SEQ ID NO: 5; and said VL being: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions are optionally present in the sequence of SEQ ID NO: 8; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions are optionally present in the sequence of SEQ ID NO: 9; and iii) including a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions are optionally present in the sequence of SEQ ID NO: 10; wherein when tested at a pH of 7.4 in a PAR2 binding assay, the amino acid substitutions, deletions, or insertions reduce the binding affinity of said antibody or antigen-binding fragment thereof for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50, or 10-fold or less compared to an antibody or antigen-binding fragment having a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 7. [Item 2] The antibody or antigen-binding fragment thereof according to item 1 above, wherein the amino acid substitutions, deletions, or insertions include conservative substitutions. [Item 3] An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH is: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 17 of SEQ ID NO: 4; and iii) having the amino acid sequence of SEQ ID NO: 5, and comprising a VH-CDR3 in which histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 8 of SEQ ID NO: 5; and said VL being: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9; and iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, and comprising histidine optionally present at any one or more of the amino acid positions corresponding to positions 1 to 14 of SEQ ID NO: 10, an antibody or an antigen-binding fragment thereof. [Item 4] Said VH being: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3, ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, iii) comprising a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, and said VL being: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, iii) comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, the antibody or an antigen-binding fragment thereof according to Item 3 above. [Item 5] Said VH being: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 13, ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 14, iii) comprising a VH-CDR3 having the amino acid sequence of SEQ ID NO: 15, and said VL being: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 18, ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 19, iii) comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 20, the antibody or an antigen-binding fragment thereof according to Item 3 above. [Item 6] The histidine is present at amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO: 4; and the histidine is present at amino acid positions corresponding to positions 2 and 3 of SEQ ID NO: 5, the antibody or antigen-binding fragment thereof according to item 3 above. [Item 7] The histidine is present at the amino acid position corresponding to position 5 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 3 or items 5 to 6 above. [Item 8] The histidine is present at the amino acid position corresponding to position 7 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 3 or items 5 to 7 above. [Item 9] The histidine is present at the amino acid position corresponding to position 8 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 3 or items 5 to 7 above. [Item 10] The histidine is present at the amino acid position corresponding to position 12 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 1 or items 5 to 7 above. [Item 11] The histidine is present at the amino acid position corresponding to position 15 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 3 or items 5 to 10 above. [Item 12] The histidine is present at the amino acid position corresponding to position 16 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 3 or items 5 to 11 above. [Item 13] The histidine is present at the amino acid position corresponding to position 17 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 1 or items 5 to 11 above. [Item 14] The histidine is present at amino acid positions corresponding to positions 5 and 8 of SEQ ID NO: 4, the antibody or antigen-binding fragment thereof according to any one of item 3 or items 5 to 11 above. [Item 15] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 11 above, wherein histidine is present at amino acid positions corresponding to positions 5, 8, 12, and 16 of SEQ ID NO: 4. [Item 16] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 11 above, wherein histidine is present at amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO: 4. [Item 17] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 16 above, wherein histidine is present at the amino acid position corresponding to position 2 of SEQ ID NO: 5. [Item 18] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 17 above, wherein histidine is present at the amino acid position corresponding to position 3 of SEQ ID NO: 5. [Item 19] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 18 above, wherein histidine is present at amino acid positions corresponding to positions 2 and 3 of SEQ ID NO: 5. [Item 20] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 19 above, wherein histidine is present at the amino acid position corresponding to position 1 of SEQ ID NO: 10. [Item 21] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 20 above, wherein histidine is present at the amino acid position corresponding to position 5 of SEQ ID NO: 10. [Item 22] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 21 above, wherein histidine is present at the amino acid position corresponding to position 6 of SEQ ID NO: 10. [Item 23] The antibody or antigen-binding fragment thereof according to any one of items 3 or 5 to 22 above, wherein histidine is present at the amino acid position corresponding to position 14 of SEQ ID NO: 10. [Item 24] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, 544, 554, 564, 574, 584, 594, 604, 614, 624, 634, 644, 654, 664, 674, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774, 784, 794, and 811-818. [Item 25] The antibody or antigen-binding fragment thereof according to item 3 or 24 above, wherein the VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, and 819-820. [Item 26] The antibody or antigen-binding fragment thereof according to any one of items 3, 24, or 25 above, wherein the VL-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, and 800. [Item 27] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 14; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 20. [Item 28] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 811; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 819; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. [Item 29] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 814; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 820; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. [Item 30] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 816; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. [Item 31] The antibody or antigen-binding fragment thereof according to Item 3 above, wherein the VH-CDR2 contains an amino acid sequence corresponding to SEQ ID NO: 818; the VH-CDR3 contains an amino acid sequence corresponding to SEQ ID NO: 15, and the VL-CDR3 contains an amino acid sequence corresponding to SEQ ID NO: 10 or 20. [Item 32] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 31 above, wherein the VH contains a framework region that is at least 90% identical to each of SEQ ID NOs: 803 to 806. [Item 33] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 31 above, wherein the VH contains a framework region that is at least 95% identical to each of SEQ ID NOs: 803 to 806. [Item 34] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 31 above, wherein the VH contains a framework region corresponding to SEQ ID NOs: 803 to 806. [Item 35] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 34 above, wherein the VL contains a framework region that is at least 90% identical to each of SEQ ID NOs: 807 to 810. [Item 36] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 34 above, wherein the VL contains a framework region that is at least 95% identical to each of SEQ ID NOs: 807 to 810. [Item 37] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 34 above, wherein the VL contains a framework region corresponding to SEQ ID NOs: 807 to 810. [Item 38] The VH contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, 792 and 821 - 831, and the antibody or antigen - binding fragment thereof described in item 3 above. [Item 39] The VL contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797, and the antibody or antigen - binding fragment thereof described in item 3 above. [Item 40] The VH contains an amino acid sequence corresponding to SEQ ID NO: 821, and the VL contains an amino acid sequence corresponding to SEQ ID NO: 7 or 17, and the antibody or antigen - binding fragment thereof described in item 3 above. [Item 41] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH contains an amino acid sequence corresponding to SEQ ID NO: 824, and the VL contains an amino acid sequence corresponding to SEQ ID NO: 7 or 17. [Item 42] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH contains an amino acid sequence corresponding to SEQ ID NO: 827, and the VL contains an amino acid sequence corresponding to SEQ ID NO: 7 or 17. [Item 43] The antibody or antigen-binding fragment thereof according to item 3 above, wherein the VH contains an amino acid sequence corresponding to SEQ ID NO: 831, and the VL contains an amino acid sequence corresponding to SEQ ID NO: 7 or 17. [Item 44] The antibody or antigen-binding fragment thereof according to any one of items 3 to 39 above, wherein the VH contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 12, and the VL contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 17. [Item 45] The antibody or antigen-binding fragment thereof according to any one of items 1 to 44 above, wherein the antibody or antigen-binding fragment is an antigen-binding fragment. [Item 46] The antibody or antigen-binding fragment thereof according to item 45 above, wherein the antigen-binding fragment is scFv. [Item 47] The antibody or antigen-binding fragment thereof according to item 45 above, wherein the antigen-binding fragment is Fab’. [Item 48] The antibody or antigen-binding fragment thereof according to any one of items 1 to 44 above, wherein the antibody or antigen-binding fragment is an antibody. [Item 49] The antibody or antigen-binding fragment thereof according to item 48 above, wherein the antibody is a monoclonal antibody. [Item 50] The antibody or antigen-binding fragment thereof according to item 48 or 49 above, wherein the antibody is an IgG antibody. [Item 51] The antibody or antigen-binding fragment according to any one of items 1 to 50 above, wherein the antibody or antigen-binding fragment is humanized. [Item 52] The antibody or antigen-binding fragment according to any one of items 1 to 50 above, wherein the antibody or antigen-binding fragment is human. [Item 53] The antibody or antigen-binding fragment according to any one of items 1 to 52 above, wherein the VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 to 841. [Item 54] The antibody or antigen-binding fragment according to item 53 above, wherein the VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 to 841. [Item 55] The VH is the antibody or antigen-binding fragment according to Item 53 above, encoded by a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and any one of 833 - 841. [Item 56] The VL is the antibody or antigen-binding fragment according to any one of Items 1 - 55 above, encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. [Item 57] The antibody or antigen-binding fragment according to item 56 above, wherein the VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. [Item 58] The antibody or antigen-binding fragment according to item 56 above, wherein the VL is encoded by a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. [Item 59] The antibody or antigen-binding fragment according to any one of items 1 to 58 above, wherein the VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 11. [Item 60] The antibody or antigen-binding fragment according to item 59 above, wherein the VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 11. [Item 61] The antibody or antigen-binding fragment according to item 59 above, wherein the VH is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11. [Item 62] The antibody or antigen-binding fragment according to any one of items 1 to 61 above, wherein the VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16. [Item 63] The antibody or antigen-binding fragment according to item 62 above, wherein the VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16. [Item 64] The antibody or antigen-binding fragment according to item 62 above, wherein the VL is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 16. [Item 65] The antibody or antigen-binding fragment according to any one of items 1 to 64 above, wherein the antibody or antigen-binding fragment binds to PAR2. [Item 66] The antibody or antigen-binding fragment according to any one of items 1 to 65 above, wherein the antibody or antigen-binding fragment inhibits the interaction of trypsin, tryptase, and / or matriptase with PAR2. [Item 67] The antibody or antigen-binding fragment according to any one of items 1 to 66 above, wherein the antibody or antigen-binding fragment inhibits the cleavage of PAR2 by trypsin, tryptase, and / or matriptase. [Item 68] The antibody or antigen-binding fragment according to any one of items 1 to 67 above, wherein the antibody or antigen-binding fragment inhibits the cleavage of the extracellular domain of PAR2. [Item 59] The antibody or antigen-binding fragment according to any one of items 1 to 68 above, wherein the antibody or antigen-binding fragment inhibits the exposure of the tether ligand. [Item 70] The antibody or antigen-binding fragment according to any one of items 1 to 69 above, wherein the antibody or antigen-binding fragment inhibits the interaction of the tether ligand with the second transmembrane loop of PAR2. [Item 71] The antibody or antigen-binding fragment according to any one of Items 1 to 70 above, wherein the antibody or antigen-binding fragment binds to PAR2 with a higher affinity at pH 7.4 than at pH 6.0. [Item 72] The antibody or antigen-binding fragment according to any one of Items 1 to 70 above, wherein the antibody or antigen-binding fragment has a K of less than about 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 650 pM, 600 pM, 500 pM, 200 pM, 100 pM or 50 pM D and binds to PAR2 at pH 7.4. [Item 73] The antibody or antigen-binding fragment according to Item 72 above, wherein the antibody or antigen-binding fragment has a K of less than about 700 pM D and binds to PAR2 at pH 7.4. [Item 74] The antibody or antigen-binding fragment according to any one of Items 1 to 70 above, wherein the antibody or antigen-binding fragment has a K greater than about 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM, or 100 nM D and binds to PAR2 at pH 6.0. [Item 75] The antibody or antigen-binding fragment according to any one of Items 1 to 70 above, wherein the antibody or antigen-binding fragment has a K greater than about 30 nM D and binds to PAR2 at pH 6.0. [Item 76] The antibody or antigen-binding fragment according to any one of Items 1 to 75 above, wherein when the antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 at pH 7.4 in a PAR2 binding assay, it has an IC of less than 100 nM 50 . [Item 77] The antibody or antigen-binding fragment according to any one of Items 1 to 75 above, wherein when the antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 at pH 7.4 in a PAR2 binding assay, it has an IC of less than 50 nM 50An antibody or antigen-binding fragment according to any one of items 1 to 76 above, having [Item 78] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 at pH 7.4 in a PAR2 binding assay, an IC of less than 40 nM 50 An antibody or antigen-binding fragment according to any one of items 1 to 77 above, having [Item 79] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 at pH 6.0 in a PAR2 binding assay, an IC greater than 500 nM 50 An antibody or antigen-binding fragment according to any one of items 1 to 78 above, having [Item 80] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 at pH 6.0 in a PAR2 binding assay, an IC greater than 1000 nM 50 An antibody or antigen-binding fragment according to any one of items 1 to 79 above, having [Item 81] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 at pH 6.0 in a PAR2 binding assay, an IC greater than 1100 nM 50 An antibody or antigen-binding fragment according to any one of items 1 to 80 above, having [Item 82] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 in a PAR2 binding assay, an IC more than 20-fold lower at pH 6.0 than at pH 7.4 50 An antibody or antigen-binding fragment according to any one of items 1 to 81 above, having [Item 83] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 in a PAR2 binding assay, an IC more than 25-fold lower at pH 6.0 than at pH 7.450 An antibody or antigen-binding fragment according to any one of items 1 to 82 above, having [Item 84] When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3 to 5 and 8 to 10 in a PAR2 binding assay, an IC that is more than 30-fold lower at pH 7.4 than at pH 6.0 50 An antibody or antigen-binding fragment according to any one of items 1 to 83 above, having [Item 85] When the antibody or antigen-binding fragment has an IC of less than 3.0×10 -10 M in a calcium influx assay in human A549 cells 50 An antibody or antigen-binding fragment according to any one of items 1 to 84 above, having [Item 86] When the antibody or antigen-binding fragment has an IC of less than 1.5×10 -10 M in a calcium influx assay in human A549 cells 50 An antibody or antigen-binding fragment according to any one of items 1 to 85 above, having [Item 87] When the antibody or antigen-binding fragment has an IC of less than 7.0×10 -10 M in a calcium influx assay in rat KNRK cells 50 An antibody or antigen-binding fragment according to any one of items 1 to 86 above, having [Item 88] When the antibody or antigen-binding fragment has an IC of less than 5.5×10 -10 M in a calcium influx assay in rat KNRK cells 50 An antibody or antigen-binding fragment according to any one of items 1 to 87 above, having [Item 89] When the antibody or antigen-binding fragment has an IC of less than 7.0×10 -11 M in a calcium influx assay in cynomolgus monkey CYNOM-K1 cells 50 An antibody or antigen-binding fragment according to any one of items 1 to 88 above, having [Item 90] The antibody or antigen-binding fragment has an IC in a calcium influx assay in cynomolgus monkey CYNOM-K1 cells of less than 5.0×10 -11 M, and is the antibody or antigen-binding fragment according to any one of items 1 to 89 above. 50 [Item 91] The antibody or antigen-binding fragment has an IC in a calcium influx assay in mouse LL / 2 cells of less than 6.0×10 -11 M, and is the antibody or antigen-binding fragment according to any one of items 1 to 90 above. 50 [Item 92] The antibody or antigen-binding fragment has an IC in a calcium influx assay in mouse LL / 2 cells of less than 4.0×10 -11 M, and is the antibody or antigen-binding fragment according to any one of items 1 to 91 above. 50 [Item 93] The antibody or antigen-binding fragment is slower than an antibody or antigen-binding fragment lacking histidine modification and disappears from the serum of the treated patient, and is the antibody or antigen-binding fragment according to any one of items 92 above. [Item 94] When the optionally present histidine or histidines are tested at a pH of 7.4 in a PAR2 binding assay, the binding affinity of the antibody or its antigen-binding fragment for human PAR2 is 1000, 800, 700, 500, 400, 300, 200, 100, 50 or 10 times lower than that of an antibody or antigen-binding fragment having a VH with the amino acid sequence of SEQ ID NO: 2 and a VL with the amino acid sequence of SEQ ID NO: 7, and is the antibody or its antigen-binding fragment according to any one of items 3 or 5 to 93 above. [Item 95] A nucleic acid capable of expressing the antibody or antigen-binding fragment according to any one of items 1 to 94 above. [Item 96] ​​​A nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841. [Item 97] The nucleic acid according to Item 96 above, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841. [Item 98] The nucleic acid according to item 96 above, wherein the nucleic acid contains any one of the nucleotide sequences of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 to 841. [Item 99] A nucleic acid containing a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. [Item 100] The nucleic acid according to item 99 above, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. [Item 101] The nucleic acid according to item 99 above, wherein the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. [Item 102] A nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 11. [Item 103] The nucleic acid according to item 102 above, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 11. [Item 104] The nucleic acid according to item 102 above, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 11. [Item 105] A nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16. [Item 106] The nucleic acid according to item 105 above, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16. [Item 107] The nucleic acid according to item 105 above, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 16. [Item 108] A vector comprising the nucleic acid according to any one of items 95 to 107 above. [Item 109] a) A nucleic acid according to any one of items 96 to 98 or 102 to 104 above; and b) A set of vectors comprising a nucleic acid according to any one of items 99 to 101 and 105 to 107 above. [Item 110] A host cell comprising one or more of the vectors according to item 108 or 109 above. [Item 111] A composition comprising a pharmaceutically acceptable carrier and an antibody or antigen-binding fragment according to any one of items 1 to 94 above. [Item 112] A lyophilized composition comprising an antibody or antigen-binding fragment thereof according to any one of items 1 to 94 above. [Item 113] A reconstituted lyophilized composition comprising an antibody or antigen-binding fragment thereof according to any one of items 1 to 94 above. [Item 114] The composition according to any one of items 111 to 113 above, wherein the composition is formulated for administration by lozenge, aerosol, oral administration, delayed release or sustained release, transmucosal administration, syrup, mucoadhesion, buccal formulation, mucoadhesive tablet, topical administration, parenteral administration, injection, subcutaneous administration, oral solution, rectal administration, buccal administration or transdermal administration. [Item 115] A kit comprising an antibody or antigen-binding fragment according to any one of items 1 to 94 above or a composition according to any one of items 111 to 114 above. [Item 116] A method for treating pain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of items 1 to 94 above. [Item 117] A method for treating pain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of items 111 to 114 above. [Item 118] The method according to item 116 or 117 above, wherein the pain is selected from the group consisting of nociceptive pain, neuropathic pain, and mixed pain. [Item 119] The pain is headache, chronic headache, migraine, cancer, viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease, liver disease, multiple sclerosis, spinal cord injury, postherpetic neuralgia, diabetic neuropathy, low back pain, inflammatory heart disease, kidney disease, gastritis, gingivitis, periodontal disease, asthma, chronic obstructive pulmonary disease, autoimmune disease, irritable bowel syndrome, fibromyalgia, lower limb pain, restless legs syndrome, diabetic neuropathy, allergic conditions, surgical procedures, acute or chronic physical injuries, fractures or contusions, spinal cord injuries, inflammatory diseases, non-inflammatory neuropathic pain conditions or functional nociceptive pain conditions, or a combination thereof. The method according to any one of items 116 or 117 above. [Item 120] The method according to item 119 above, wherein the pain is osteoarthritis pain. [Item 121] The method according to any one of items 116 to 120 above, wherein the subject is a human. [Item 122] A method for producing an antibody or antigen-binding fragment according to any one of items 1 to 94 above, the method comprising the steps of expressing a nucleic acid according to any one of items 95 to 106 above in cultured cells and purifying the antibody or antigen-binding fragment.

[0023] The patent or patent application filed contains at least one drawing made in color. A copy of the patent or patent application publication having a colored drawing will be provided by the patent office upon request and payment of the necessary fees.

Brief Description of the Drawings

[0024]

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BRIEF DESCRIPTION OF THE INVENTION

[0025] Before describing this disclosure, it is to be understood that this disclosure is not limited to the specific methods and experimental conditions described, and that such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0027] Any methods and materials similar or equivalent to those described herein can be used in the practice and testing of this disclosure, but the preferred methods and materials are described herein.

[0028] A. Definitions As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0029] In this specification, amino acids can generally be referred to by either the well-known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can likewise be referred to by the generally accepted single-letter codes.

[0030] As used herein, it is convenient to point out that "and / or" should be construed to mean a specific disclosure of each of the two designated features or components, with or without the other. For example, "A and / or B" is to be construed as (i) A, (ii) B, and (iii) the specific disclosure of each of A and B, as if each were individually recited herein.

[0031] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to polymers of naturally occurring amino acids and polymers of non-naturally occurring amino acids, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids.

[0032] As used herein, phrases such as "protease-activated receptor 2", "PAR2", etc. refer to a human PAR2 protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the amino acid sequences of SEQ ID NO: 801, or a biologically active fragment thereof.

[0033] The term "tethered ligand" refers to the region of the N-terminal portion of PAR2 that binds to and activates PAR2 itself. In some embodiments, the tethered ligand portion of PAR2 is not exposed until a protease (e.g., thrombin or trypsin) proteolytically cleaves a portion of the PAR2 enzyme. In some embodiments, the tethered ligand corresponds to a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the amino acid sequences of SEQ ID NO: 802.

[0034] As used herein, "antibodies that bind to PAR2", "anti-PAR2 antibodies", etc. include antibodies that bind to membrane-bound PAR2 or fragments thereof, and antigen-binding fragments thereof. In some embodiments, the anti-PAR2 antibody or antigen-binding fragment thereof binds to the tethered ligand portion of PAR2.

[0035] B. Antibodies and Antigen-Binding Fragments Thereof As used herein, "an antibody or antigen-binding fragment of the present disclosure" refers to any one or more of the antibodies and antigen-binding fragments provided herein. The antibodies and antigen-binding fragments of the present disclosure include a heavy chain (VH) containing a heavy chain variable domain and a light chain (VL) containing a light chain variable domain. The VH domain includes three CDRs, such as any of the CDRs provided herein and defined or identified by the Chotia, Kabat, or IMGT system. These CDRs are typically interspersed with framework regions (FRs) and together comprise the VH domain. Similarly, the VL includes three CDRs, such as any of the CDRs provided herein and defined by the Chotia, Kabat, or IMGT system. These CDRs are typically interspersed with framework regions (FRs) and together comprise the VL domain. The FR regions, such as FR1, FR2, FR3, and / or FR4, can likewise be defined or identified by the Chotia, Kabat, or IMGT system. Throughout this application, when a CDR is present and is identified or defined by the Chothia, Kabat, or IMGT system, it means that the CDR follows these systems (e.g., Chothia CDR, Kabat CDR, or IMGT CDR). Any of these terms can be used to indicate whether a Chothia CDR, Kabat CDR, or IMGT CDR is being referenced.

[0036] As used herein, the term "antibody" also includes antigen-binding fragments of full-length antibody molecules. As used herein, terms such as "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody" include any naturally occurring, enzymatically obtained, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody may be obtained from full-length antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion or recombinant genetic engineering techniques including manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated by chemical or molecular biological techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, introduce codons, create cysteine residues, modify, add, or delete amino acids, and the like.

[0037] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to PAR2 and has a higher affinity at pH 7.4 than at pH 6.0. In some embodiments, the antibody or antigen-binding fragment has an affinity at pH 7.4 that is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than at pH 6.0 and binds to PAR2. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 3; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 4; and iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 5; and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 8; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 9; and iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 10; the amino acid substitutions, deletions, or insertions reduce the binding affinity of the antibody or antigen-binding fragment thereof for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50, 10, or 5 times or less compared to an antibody or antigen-binding fragment having a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 7 when tested at a pH of 7.4 in a PAR2 binding assay. In some embodiments, the amino acid substitutions, deletions, or insertions include conservative substitutions.In some embodiments, the antibody or antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, insertions, or deletions in the VH CDR as compared to the CDR amino acid sequences present in the sequence of SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, insertions, or deletions in the VL CDR as compared to the CDR amino acid sequences present in the sequence of SEQ ID NO: 7. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions with histidine.

[0038] In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 13, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 13; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 14, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 14; and iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 15, but optionally having 1, 2, 3, 4, or 15 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 15; and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 18, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 18; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 19, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 19; and iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 20, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 20; the amino acid substitutions, deletions, or insertions reduce the binding affinity of the antibody or antigen-binding fragment thereof for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50, 10, or 5-fold or less, when tested at pH 7.4 in a PAR2 binding assay, compared to an antibody or antigen-binding fragment having a VH having the amino acid sequence of SEQ ID NO: 12 and a VL having the amino acid sequence of SEQ ID NO: 17. In some embodiments, the amino acid substitutions, deletions, or insertions include conservative substitutions. In some embodiments, the antibody or antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, insertions, or deletions in the VH CDR compared to the CDR amino acid sequences present in the sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, insertions, or deletions in the VL CDR compared to the CDR amino acid sequences present in the sequence of SEQ ID NO: 17.In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 13, ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 14, iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 15, and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 18, ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 19, iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions or deletions are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions with histidine.

[0039] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VH domain comprises at least one, two, or all three of the CDRs of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, and 792 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems). In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VH domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, and 792 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems).In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VH domain comprises at least one, two, or all three of the CDRs of the amino acid sequence set forth in SEQ ID NO: 2 or 12 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems). In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VL domain comprises at least one, two, or all three of the CDRs of the amino acid sequence set forth in any one of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems).In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VL domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems). In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VL domain comprises at least one, two, or all three of the CDRs of the amino acid sequence set forth in SEQ ID NO: 7 or 17 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems).In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VH domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, and 792 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems), and the VL domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems).In some embodiments, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; the VH domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 2 or 12 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems), and the VL domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 7 or 17 (e.g., when measured using any of the Chothia, IMGT, or Kabat systems).

[0040] Once the nucleotide sequence encoding such an antibody is determined, chimeric or humanized antibodies can be produced by recombinant methods. The nucleic acid encoding the antibody is generally known in the art and is introduced into and expressed in host cells using the materials and procedures described herein. In some embodiments, VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841. In some embodiments, VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or 11.In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796. In some embodiments, VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6 or 16.

[0041] The present disclosure includes anti-PAR2 antibodies that bind to PAR2 and antigen-binding fragments thereof. In some embodiments, the antibody is a neutralizing and / or blocking anti-PAR2 antibody or antigen-binding fragment. As used herein, a "neutralizing" or "blocking" antibody or antigen-binding fragment is one in which binding to PAR2: (i) interferes with the interaction between PAR2 and proteases (e.g., trypsin, tryptase, and / or matriptase); (ii) inhibits cleavage of PAR2 by proteases; (iii) inhibits PAR2 signaling or PAR2 activation; and / or (iv) results in inhibition of at least one biological function of PAR2. In some embodiments, the antibody or antigen-binding fragment of the present disclosure inhibits PAR2 activation. In some embodiments, the antibody or antigen-binding fragment inhibits the conversion of inactive uncleaved PAR2 to active cleaved PAR2. In some embodiments, the antibody or antigen-binding fragment inhibits the exposure of the tether ligand. In some embodiments, the antibody or antigen-binding fragment inhibits activation of the PAR2 receptor by its tether ligand. In some embodiments, the antibody or antigen-binding fragment inhibits the binding of the tether ligand to the second transmembrane domain of PAR2. The inhibition provided by an anti-PAR2 neutralizing or blocking antibody need not be complete so long as it is detectable using an appropriate assay. In some embodiments, the antibody or its antigen-binding fragment inhibits PAR2 activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to uninhibited active PAR2. Some examples of assays for detecting the activity of representative anti-PAR2 antibodies or antigen-binding fragments are described in the illustrative section. Those skilled in the art are familiar with additional anti-PAR2 antibody activity assays.

[0042] In certain embodiments, any of the antibodies or antigen-binding fragments disclosed herein interfere with the interaction between PAR2 and a protease. In some embodiments, the protease is trypsin. In some embodiments, the protease is neutrophil elastase. In some embodiments, the protease is neutrophil proteinase 3. In some embodiments, the protease is mast cell tryptase. In some embodiments, the protease is tissue factor / factor VIIa / factor Xa. In some embodiments, the protease is a kallikrein-related peptidase. In some embodiments, the protease is membrane-bound serine protease-1 / matriptase 1. In some embodiments, the protease is a parasite cysteine protease. In some embodiments, the anti-PAR2 antibody or antigen-binding fragment has an IC 50 value of less than about 15 nM and blocks the interaction between PAR2 and a protease (e.g., trypsin) in vitro. In certain embodiments, the antibody or antigen-binding fragment of the disclosure has an IC 50 value of less than about 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 500 pM, 400 pM, 200 pM, 100 pM, 50 pM, 5 pM, 1 pM, or 0.1 pM and blocks the interaction between PAR2 and a protease (e.g., trypsin) in vitro at pH 7.4. In certain embodiments, the antibody or antigen-binding fragment of the disclosure has an IC 50 value greater than about 300 nM, 500 nM, 750 nM, 1000 nM, 1100 nM, or 1200 nM and blocks the interaction between PAR2 and a protease (e.g., trypsin) in vitro at pH 6.0. In certain embodiments, the IC 50 of the anti-PAR2 antibody or fragment thereof is measured in an epitope competition assay such as the epitope competition assay described in the illustrative section provided herein. In some embodiments, the IC 50is measured in a cell titer assay. In some embodiments, the cell titer assay utilizes human cells (e.g., A549 cells), rat cells (e.g., KNRK cells), cynomolgus monkey cells (e.g., CYNOM-K1 cells), or mouse cells (e.g., LL / 2 cells). In some embodiments, the cell titer assay utilizes a calcium influx assay (e.g., the calcium influx assay described in the illustrative section provided herein). In some embodiments, the antibody or antigen-binding fragment has an IC 50 less than 1 nM, 500 pM, 400 pM, 200 pM, 100 pM, 50 pM, 10 pM, 5 pM, 1 pM, or 0.1 pM and inhibits calcium influx in the calcium influx assay. In some embodiments, the antibody or antigen-binding fragment prevents abnormal activation of PAR2 by trypsin. In some embodiments, the antibody or antigen-binding fragment inhibits / reduces inflammatory pain.

[0043] In certain embodiments, any of the antibodies or antigen-binding fragments disclosed herein prevent the interaction between PAR2 and a protease (e.g., trypsin). In some embodiments, the antibody prevents a protease (e.g., trypsin) from binding to, cleaving, and / or activating PAR2. The present disclosure provides anti-PAR2 antibodies and antigen-binding fragments thereof that bind to PAR2 molecules with high affinity at physiological extracellular pH (i.e., pH 7.4). In some embodiments, the antibody and antigen-binding fragment of the antibody have a K D less than about 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 650 pM, 600 pM, 500 pM, 200 pM, 100 pM, or 50 pM and bind to PAR2 at pH 7.4 (e.g., at 25°C or 37°C). In some embodiments, the antibody and antigen-binding fragment of the antibody have a K DIt has and binds to PAR2 at a weakly acidic pH (such as pH 6.0) (for example, at 25°C or 37°C). In some embodiments, the weakly acidic pH is the pH of the endosomal compartment. In some embodiments, K D can be measured according to current standard methods, such as using surface plasmon resonance (SPR) or quartz crystal microbalance (QCM).

[0044] The present disclosure also includes anti-PAR2 antibodies and antigen-binding fragments thereof that specifically bind to PAR2 and have a dissociation half-life (t1 / 2) exceeding about 1.5 minutes, 1.75 minutes, 2 minutes, 2.5 minutes, 3 minutes, 5 minutes, 10 minutes, 20 minutes, or 30 minutes, as measured using an assay such as surface plasmon resonance at 25°C or 37°C at pH 7.4. In some embodiments, the anti-PAR2 antibody and antigen-binding fragment thereof bind to PAR2 and have a dissociation half-life (t1 / 2) of less than about 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 13 seconds, 7 seconds, 5 seconds, or 3 seconds, as measured using an assay such as surface plasmon resonance at 25°C or 37°C at a weakly acidic pH (such as pH 6). In some embodiments, the weakly acidic pH is the pH of the endosomal compartment. In some embodiments, K D can be measured according to current standard methods, such as using surface plasmon resonance (SPR) or quartz crystal microbalance (QCM).

[0045] The antibodies or antigen-binding fragments of the present disclosure may have one or more of the aforementioned biological characteristics, or any combination thereof. Other biological characteristics of the antibodies of the present disclosure will be apparent to those skilled in the art from the disclosure of the present disclosure, including the illustrative sections provided herein.

[0046] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using a default gap weight. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative amino acid substitutions compared to a reference sequence (e.g., any of the amino acid sequences of SEQ ID NO: 2, 7, 12, or 17). "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted to correct upward for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0047] Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0048] Depending on the amino acid sequences of their heavy-chain constant domains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these are further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0049] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) Fab’ fragments; (iii) F(ab’)2 fragments; (iv) Fd fragments; (v) Fv fragments; (vi) single-chain Fv (scFv) molecules; (vii) dAb fragments; and (viii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or limited FR3-CDR3-FR4 peptides. Other modified molecules, such as domain-specific antibodies, single-domain antibodies, camelid antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), adnectins, small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the term “antigen-binding fragment” as used herein.

[0050] An antigen-binding fragment of an antibody will typically comprise at least one variable domain (e.g., at least one of VH or VL). The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR adjacent to or in-frame with one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains can be positioned in any suitable arrangement relative to each other. For example, the variable regions can be dimeric and can contain VH-VH, VH-VL or VL-VL dimers. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0051] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any arrangement of variable and constant domains that includes any of the above exemplary arrangements, the variable and constant domains may be directly linked to each other or may be linked by an entire or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. In some embodiments, the hinge region includes a glycine-serine linker.

[0052] Furthermore, the antigen-binding fragments of the antibodies of the present disclosure can include homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above that associate non-covalently with each other and / or with one or more monomeric VH or VL domains (e.g., by disulfide bonds).

[0053] Similar to full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will generally comprise at least two different variable domains, where each variable domain is capable of specifically binding to a distinct antigen or a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the present disclosure using conventional techniques available in the art.

[0054] In certain embodiments of the present disclosure, the anti-PAR2 antibodies of the present disclosure are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or somatic mutations in vivo), for example, in the CDRs, and in some embodiments in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0055] The antibodies of the present disclosure can be, in some embodiments, recombinant human antibodies. As used herein, the term "recombinant human antibody" refers to all human antibodies made, expressed, produced or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from recombinants, combinatorial human antibody libraries (described further below), antibodies isolated from an animal (e.g., a mouse) into which human immunoglobulin genes have been transfected (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies made, expressed, produced or isolated by any other means including splicing to other DNA sequences of human immunoglobulin gene sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if an animal into which human Ig sequences have been transfected is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may be sequences that do not naturally occur within the human antibody germline repertoire in vivo.

[0056] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of about 150-160 kDa in which the dimer is maintained by interchain heavy-chain disulfide bonds. In the second form, the dimer is not linked by interchain disulfide bonds, and a molecule of about 75-80 kDa is formed and composed of covalently linked light and heavy chains (half-antibodies). These forms were extremely difficult to separate even after affinity purification.

[0057] The frequency of occurrence of the second form in various intact IgG isotypes is due to structural differences related to the hinge region isotype of the antibody, among other things. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to levels normally observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure contemplates antibodies having one or more mutations in the hinge, CH2, or CH3 region, which may be desirable, for example, to improve the yield of the desired antibody form during production.

[0058] The antibodies of the present disclosure can be isolated antibodies or isolated antigen-binding fragments. As used herein, "isolated antibody" or "isolated antigen-binding fragment" means an antibody or antigen-binding fragment that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody or antigen-binding fragment that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" or "isolated antigen-binding fragment" for the purposes of the present disclosure. Isolated antibodies also include antibodies in vivo in situ within recombinant cells. An isolated antibody or antigen-binding fragment is an antibody or antigen-binding fragment that has been subjected to at least one purification or isolation step. According to certain embodiments, the isolated antibody or antigen-binding fragment may be substantially free of other cellular materials and / or chemical substances.

[0059] The anti-PAR2 antibodies or antigen-binding fragments disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies are derived. This disclosure includes antibodies and antigen-binding fragments derived from any of the amino acid sequences disclosed herein, where one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody or antigen-binding fragment is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are reverted to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues, such as only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3, are reverted to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally derived). In some embodiments, VH framework region 1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 803. In some embodiments, VH framework region 2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 804.In some embodiments, the VH framework region 3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 805. In some embodiments, the VH framework region 4 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 806. In some embodiments, the VL framework region 1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 807. In some embodiments, the VL framework region 2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 808. In some embodiments, the VL framework region 3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 809. In some embodiments, the VL framework region 4 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 810. In some embodiments, the VH framework comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions as compared to any one of the reference sequences of SEQ ID NOs: 803 - 806. In some embodiments, the VL framework comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions as compared to any one of the reference sequences of SEQ ID NOs: 807 - 810.

[0060] Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions. For example, certain individual residues may be mutated to the corresponding residues of a particular germline sequence, while certain other residues that are different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced (where appropriate) biological properties as an antagonist or agonist, reduced immunogenicity, and the like. Antibodies and antigen-binding fragments obtained by this general approach are encompassed within the present disclosure.

[0061] The present disclosure also includes anti-PAR2 antibodies comprising variants of any of the amino acid sequences of VH, VL, and / or CDR disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-PAR2 antibodies having VH, VL, and / or CDR amino acid sequences having 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitution relative to any of the amino acid sequences of VH, VL, and / or CDR disclosed herein.

[0062] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. One antigen can have two or more epitopes. Thus, different antibodies can bind to different regions of an antigen and can have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are brought about by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epitopes are those brought about by adjacent amino acid residues in a polypeptide chain. In certain situations, an epitope can include a sugar moiety, a phosphoryl group, or a sulfonyl group on the antigen.

[0063] It should be noted that any part of the antibody or antigen-binding fragment of the present disclosure may be similarly modified with an epitope tag, a PEG moiety, etc. Further, the antibody or antigen-binding fragment may contain two or more epitope tags such as two epitope tags, or may contain zero epitope tags.

[0064] When referring to a nucleic acid or a fragment thereof, the terms "substantial identity" or "substantially identical" mean that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as described below, when measured by any well-known algorithm for sequence identity such as FASTA, BLAST or Gap, there is nucleotide sequence identity at at least about 95%, and more preferably about 96%, 97%, 98% or 99% of the nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in some instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0065] Sequence similarity for a polypeptide, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications including conservative amino acid substitutions to align similar sequences. For example, GCG software includes programs such as GAP and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from different species, or between a wild-type protein and its mutant. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using the program FASTA, which is part of GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the percent sequence identity of the most overlapping region between the alignment and query sequences and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing numerous sequences from various organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402. In some embodiments, sequences are compared using the EMBOSS Needle pairwise sequence alignment.

[0066] In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is a scFv. In some embodiments, the antigen-binding fragment is a Fab’. In some embodiments, the antibody or antigen-binding fragment is an antibody. In some embodiments, the antibody is a monoclonal antibody.

[0067] With the development of monoclonal antibodies, antibodies have become useful and have attracted interest as pharmaceuticals. Monoclonal antibodies are produced using any method that produces antibody molecules by a continuous cell line in culture. Examples of suitable methods for producing monoclonal antibodies include the hybridoma method of Kohler et al. (1975, Nature 256:495-497) and the human B cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; and Brodeur et al., 1987, Monoclonal Antibody Production Techniques and Applications, (Marcel Dekker, Inc., New York), pp. 51-63). In many cases, hybridomas are used to produce initial antibodies of mouse or rodent origin. This initial antibody may then be modified using recombinant techniques or the like to produce rodent variants, chimeric antibodies, and humanized antibodies. There are other methods for producing the initial antibody, and such methods are known in the art. However, regardless of the method used to produce the initial antibody or further variants of the initial antibody, any given antibody of non-human origin can be modified to increase its human likeness.

[0068] The antibodies or antigen-binding fragments of the present disclosure can be produced by screening a combinatorial library for antibodies having a desired activity. For example, various methods for making phage display libraries and screening such libraries for antibodies having desired binding properties are known in the art. Such methods are generally described in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001). For example, one method for generating an antibody of interest is by use of a phage antibody library as described in Lee et al., J. Mol. Biol. (2004), 340(5):1073-93.

[0069] In principle, synthetic antibody clones are selected by screening a phage library containing phages that display various fragments of the antibody variable region (Fv) fused to a phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments that can bind to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones of the library. The binding clones can then be eluted from the antigen and further enriched by additional antigen adsorption / elution cycles. Any of the antibodies of the present disclosure can be obtained by designing a suitable antigen screening procedure for selecting the phage clone of interest, followed by constructing a full-length antibody clone using the Fv sequence and a suitable constant region (Fc) sequence from the phage clone of interest, as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. It may be advantageous to increase the human-likeness of non-human antibodies to make them more suitable for use in human subjects and human cells, regardless of whether for diagnostic, therapeutic, or research purposes. The antibody may be modified for use as a therapeutic agent. Examples of such antibodies (including antibody fragments) include chimeric antibodies, humanized antibodies, and fully human antibodies. Numerous methods for making chimeric antibodies, humanized antibodies, and human antibodies exist in the art. In the context of the present disclosure, an antibody is considered humanized if at least one of the VH domain or the VL domain is humanized. Further, compared to the parental non-human (e.g., mouse) antibody, the VH domain or the VL domain is humanized if at least a portion of the amino acid sequence of at least one of the FR regions has been modified such that the amino acid sequence of that portion corresponds to that of a human antibody or a human consensus sequence.In certain embodiments, at least one, two, three, or four FR regions of the VH domain and / or at least one, two, three, or four FR regions of the VL domain are (in whole or in part) modified such that their sequences are more closely related to human sequences. For any of the foregoing in certain embodiments, a humanized antibody fragment can be provided in the context of a human or non-human light chain constant region and / or heavy chain constant region (e.g., including one or more of the domains of CL and CH1, hinge, CH2, and / or CH3). In certain embodiments, when present, the humanized antibody or antigen-binding fragment of the present disclosure is provided within the context of a human light chain constant domain and / or heavy chain constant domain. Antibodies and antibody-binding fragments that combine any of the humanized light chain variable domains and / or heavy chain variable domains disclosed herein are exemplary of the antibodies and antigen-binding fragments of the present disclosure. In some embodiments, the antibody or antigen-binding fragment is humanized. In some embodiments, the antibody or antigen-binding fragment is chimeric. In some embodiments, the antibody or antigen-binding fragment is human.

[0070] According to certain embodiments of the present disclosure, there are provided anti-PAR2 antibodies comprising an Fc domain comprising one or more mutations that enhance or decrease the binding of the antibody to the FcRn receptor, for example, at acidic pH as compared to neutral pH. For example, the present disclosure includes anti-PAR2 antibodies comprising mutations in the CH2 or CH3 region of the Fc domain, wherein the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome having a pH range of about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); positions 250 and 428 (e.g., L or F); positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include the modifications of 428L (e.g., M428L) and 434S (e.g., N434S); the modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F); the modifications of 433K (e.g., H433K) and position 434 (e.g., 434Y); the modifications of positions 252, 254, and 256 (e.g., 252Y, 254T, and 256E); the modifications of 250Q and 428L (e.g., T250Q and M428L); and modifications at positions 307 and / or 308 (e.g., 308F or 308P). In yet another embodiment, the modifications include the modifications of 265A (e.g., D265A) and / or 297A (e.g., D297A). All possible combinations of the aforementioned Fc domain mutations, as well as other mutations within the antibody variable domains disclosed herein, are contemplated to be within the scope of the present disclosure. In some embodiments, the antibody comprises the triple mutation L234F / L235E / P331S (“TM”). TM results in a significant reduction in the binding activity of the human IgG1 molecule to human C1q, CD64, CD32A, and CD16.See, for example, Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 64:700-704 (2008). Antibodies having an increased half-life can also be made by modifying amino acid residues that have been identified as being involved in the interaction between Fc and the FcRn receptor. For example, introduction of the triple mutation M252Y / S254T / T256E (“YTE”) into the CH2 domain of a human immunoglobulin G (IgG) molecule results in an increase in their binding to the human neonatal Fc receptor (FcRn). See U.S. Patent No. 7,083,784, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the antibody comprises the YTE modification.

[0071] According to certain embodiments of the present disclosure, provided are anti-PAR2 antibodies comprising one or more mutations in the VH and / or VL domains that enhance or decrease the binding of the antibody to PAR2, for example, at an acidic pH as compared to neutral pH. For example, the present disclosure includes anti-PAR2 antibodies comprising mutations in the CDR2 (SEQ ID NO: 4) or CDR3 (SEQ ID NO: 5) region of the VH domain and / or the CDR3 (SEQ ID NO: 10) of the VL domain, wherein the mutations replace one or more amino acids with histidine and reduce the affinity of the VH and / or VL domains for PAR2 in an acidic environment (e.g., in an endosome having a pH range of about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such VH modifications include, for example, modifications at amino acid positions 4, 5, 7, 8, 10, 11, 12, 14, 15, 16, and 17 of CDR2 (SEQ ID NO: 4) and amino acid positions 1, 2, 4, 5, and 7 of CDR3 (SEQ ID NO: 5). Non-limiting examples of such VL modifications include, for example, modifications at positions 1, 2, 4, 5, 6, 7, 8, 9, 12, and 14 of CDR3 (SEQ ID NO: 10). In yet another embodiment, VH comprises modifications at positions 5, 8, 12, 16, and 17 of CDR2 (SEQ ID NO: 4) and positions 2 and 3 of CDR3 (SEQ ID NO: 5). All possible combinations of the aforementioned VH and VL domain mutations, as well as other mutations within the Fc domain disclosed herein, are considered to be within the scope of the present disclosure.

[0072] In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 17 of SEQ ID NO: 4 (e.g., positions 4, 5, and 7 to 17); and iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 8 of SEQ ID NO: 5; and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8; ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9; and iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 14 of SEQ ID NO: 10. In some embodiments, the VH comprises: i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3, ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, and the VL comprises: i) a VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, ii) a VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, iii) a VL-CDR3 having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or its antigen fragment has histidine at any one or more of the amino acid positions corresponding to positions 7, 8, 12, 15, 16, or 17 of SEQ ID NO: 4. In some embodiments, the antibody or its antigen fragment has histidine at any one or more of the amino acid positions corresponding to positions 2 or 3 of SEQ ID NO: 5. In some embodiments, the antibody or its antigen fragment has histidine at any one or more of the amino acid positions corresponding to positions 1, 5, 6, or 14 of SEQ ID NO: 10. In some embodiments, histidine is present at the amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO: 4; and histidine is present at the amino acid positions corresponding to positions 2 and 3 of SEQ ID NO: 5.In some embodiments, VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, 544, 554, 564, 574, 584, 594, 604, 614, 624, 634, 644, 654, 664, 674, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774, 784, 794, and 811-818. In some embodiments, VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, and 819-820.In some embodiments, VL-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 and 800. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 14; wherein VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 811; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 819, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 814; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 820, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 816; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20. In some embodiments, VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 818; VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15, and VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20.In some embodiments, VH comprises a framework region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 803-806, respectively. In some embodiments, VL comprises a framework region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 807-810, respectively. In some embodiments, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the sequences selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, and 792.In some embodiments, VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the sequences selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 12 and VL comprises the amino acid sequence corresponding to SEQ ID NO: 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 821 and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 824 and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 827 and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17. In some embodiments, VH comprises the amino acid sequence corresponding to SEQ ID NO: 831 and VL comprises the amino acid sequence corresponding to SEQ ID NO: 7 or 17.

[0073] The present disclosure also includes anti-PAR2 antibodies comprising a chimeric heavy chain constant (CH) region, where the chimeric CH region comprises segments derived from CH regions of two or more immunoglobulin isotypes. For example, the antibodies of the present disclosure may comprise a chimeric CH region comprising a portion or all of the CH2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule in combination with a portion or all of the CH3 domain derived from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, the antibodies of the present disclosure comprise a chimeric CH region having a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0074] According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Antibodies comprising the chimeric CH regions described herein may exhibit modified Fc effector functions in certain embodiments without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., U.S. Patent Application Publication No. 2015-0203591A1).

[0075] The present disclosure includes anti-PAR2 antibodies or antigen-binding fragments that interact with one or more amino acids of PAR2. The epitope to which the antibody can bind can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of PAR2. Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) of PAR2.

[0076] Using various techniques known to those skilled in the art, it is possible to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Representative techniques include, for example, conventional cross - blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine - scanning mutagenesis, peptide - blot analysis (Reineke, 2004, Methods Mol Biol 248:443 - 463), and peptide - cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be employed (Tomer, 2000, Protein Science 9:487 - 496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium and then binding an antibody to the deuterium - labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen - deuterium exchange to occur at all residues except those protected (and remaining deuterium - labeled) by the antibody. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium - labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267 (2):252 - 259; Engen and Smith (2001) Anal.Chem.73:256A - 265A.

[0077] The present disclosure further includes an anti-PAR2 antibody or an antigen-binding fragment thereof that binds to the same epitope as any of the antibodies or antigen-binding fragments described herein (e.g., an antibody or antigen-binding fragment comprising the amino acid sequences of SEQ ID NOs: 12 and 17). Similarly, the present disclosure also includes an anti-PAR2 antibody or antigen-binding fragment that competes with any of the antibodies or antigen-binding fragments described herein (e.g., an antibody or antigen-binding fragment comprising the amino acid sequences of SEQ ID NOs: 12 and 17) with respect to binding to PAR2. One of ordinary skill in the art can readily determine whether an antibody binds to the same epitope as a reference anti-PAR2 antibody or competes with it for binding by using conventional methods known in the art and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-PAR2 antibody of the present disclosure, the reference antibody is bound to the PAR2 protein. Next, the ability of the test antibody to bind to the PAR2 molecule is evaluated. If the test antibody can bind to PAR2 after the binding with the reference anti-PAR2 antibody is saturated, it can be concluded that the test antibody binds to an epitope different from the reference anti-PAR2 antibody. On the other hand, if the test antibody cannot bind to the PAR2 molecule after the binding with the reference anti-PAR2 antibody is saturated, the test antibody may bind to the same epitope as the epitope bound by the reference anti-PAR2 antibody of the present disclosure. Next, additional conventional experiments (e.g., peptide mutagenesis and binding analysis) are performed to confirm whether the observed lack of binding of the test antibody is due to actually binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) is involved in the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art. According to certain embodiments of the present disclosure, for example, if one antibody at a 1-, 5-, 10-, 20-, or 100-fold excess inhibits the binding of the other by at least 50%, preferably 75%, 90%, or 99% as measured in a competitive binding assay, the two antibodies bind to the same (or overlapping) epitope.(See, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).

[0078] Alternatively, two antibodies are considered to bind to the same epitope if, essentially, all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of the other, the two antibodies are considered to have "overlapping epitopes".

[0079] To determine whether an antibody competes (or cross-competes) with a reference anti-PAR2 antibody for binding, the binding method described above is carried out in two ways: In the first approach, the reference antibody is bound to the PAR2 protein under saturation conditions, and then the binding of the test antibody to the PAR2 molecule is evaluated. In the second approach, the test antibody is bound to the PAR2 molecule under saturation conditions, and then the binding of the reference antibody to the PAR2 molecule is evaluated. In both approaches, if only the first (saturated) antibody can bind to the PAR2 molecule, the test antibody and the reference antibody are concluded to compete for binding to PAR2. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0080] Methods for making monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known method can be used in the context of the present disclosure to make human antibodies that specifically bind to human PAR2.

[0081] Using the VelocImmune™ technology, for example, or any other known method for making fully human monoclonal antibodies, first isolate a chimeric antibody with high affinity for PAR2 having human variable regions and murine constant regions. Characterize the antibody and select for desired characteristics including affinity, selectivity, epitope, etc. as described in the experimental section below. If desired, replace the murine constant region with a desired human constant region, for example, wild type or modified IgG1 or IgG4, to make a fully human anti-PAR2 antibody. The constant region selected may vary according to the particular use, but the characteristics of high affinity antigen binding and target specificity reside in the variable regions. In certain instances, the fully human anti-PAR2 antibody is isolated directly from antigen positive B cells.

[0082] The anti-PAR2 antibodies and antibody fragments of the present disclosure can vary from those of the antibodies described, but include proteins having an amino acid sequence that retains the ability to bind to PAR2 (e.g., SEQ ID NO: 801), or in some embodiments more specifically, the tether ligand of PAR2 (e.g., SEQ ID NO: 802). Such variant antibodies or antibody fragments include one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the antibodies described. Similarly, the DNA sequences encoding the anti-PAR2 antibodies of the present disclosure include one or more additions, deletions, or substitutions of nucleotides when compared to the sequences described, but include sequences that encode anti-PAR2 antibodies or antibody fragments that are essentially biologically equivalent to the anti-PAR2 antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid and DNA sequences are described above.

[0083] Two antibodies or antigen-binding fragments are considered to be biologically equivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives such that when they are administered at the same molar dose under similar experimental conditions, either single or multiple administrations, there is no significant difference in the rate and extent of their absorption. Some antibodies or antigen-binding fragments are considered to be equivalents or pharmaceutical alternatives even if their rates of absorption are not equivalent, but the extent of their absorption is equivalent, and yet such differences in absorption rate are intentional, reflected in the labeling, and, for example, not essential for achieving effective in vivo drug concentrations in long-term use and not considered medically significant for the particular drug being tested, and thus may be considered biologically equivalent.

[0084] In some embodiments, two antibodies or antigen-binding fragments are biologically equivalent if there are no clinically meaningful differences in their safety, purity, and titer.

[0085] In some embodiments, two antibodies or antigen-binding fragments are biologically equivalent if they can be switched one or more times without an increased risk of adverse effects, including a clinically significant change in immunogenicity or attenuation of efficacy, as compared to continued therapy without switching the reference product and the biological product for the patient.

[0086] In some embodiments, two antibodies or antigen-binding fragments are biologically equivalent if they act by a common mechanism of action for the conditions under which both are used, to the extent such mechanism of action is known.

[0087] Biological equivalence can be demonstrated by methods in vivo and in vitro. As measurements of biological equivalence, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolite in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro tests that correlate with and are a reasonable prediction of in vivo bioavailability data in humans; (c) in vivo tests in humans or other mammals that measure the appropriate acute pharmacological effect of an antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or biological equivalence of an antibody.

[0088] Biologically equivalent variants of the anti-PAR2 antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of intramolecular disulfide bridges that are unnecessary or inappropriate upon refolding. In other situations, biologically equivalent antibodies or antigen-binding fragments can include anti-PAR2 antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody or antigen-binding fragment, such as mutations that eliminate or remove glycosylation.

[0089] According to certain embodiments, the present disclosure provides anti-PAR2 antibodies or antigen-binding fragments that bind to human PAR2 but not to PAR2 from other species. The present disclosure also includes anti-PAR2 antibodies that bind to human PAR2 and bind to PAR2 from one or more non-human species. For example, the anti-PAR2 antibodies of the present disclosure may bind to human PAR2 and, optionally, may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee PAR2. According to certain embodiments, the antibody or antigen-binding fragment binds to PAR2 in human A549 cells, rat KNRK cells, cynomolgus monkey CYNOM-K1 cells, or mouse LL / 2 cells.

[0090] The present disclosure encompasses anti-PAR2 monoclonal antibodies ( "immunoconjugates") conjugated to therapeutic components such as cytotoxins, chemotherapeutic agents, immunosuppressive agents, or radioisotopes. Examples of suitable cytotoxic and chemotherapeutic agents for forming immunoconjugates are known in the art (see, e.g., WO 05 / 103081).

[0091] In some embodiments, the antibodies of the disclosure can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of one target polypeptide or can contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-PAR2 antibodies or antigen-binding fragments of the disclosure can be bound or co-expressed with another functional molecule, such as another peptide or protein. For example, the antibody or its antigen-binding fragment can be functionally bound to one or more other molecular entities, such as another antibody or antigen-binding fragment for generating a bispecific or multispecific antibody having a second binding specificity (e.g., by chemical linkage, gene fusion, non-covalent association, or other modalities). For example, the disclosure includes bispecific antibodies where one arm of the immunoglobulin is specific for human PAR2 or a fragment thereof and the other arm of the immunoglobulin is specific for a second therapeutic target or conjugated to a therapeutic component.

[0092] Exemplary bispecific antibody or antigen-binding fragment formats that can be used in the context of the present disclosure include the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, where the first and second Ig CH3 domains differ from each other by at least one amino acid, and the difference in at least one amino acid reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody without an amino acid difference. In one embodiment, the first Ig CH3 domain binds to protein A, and the second Ig CH3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 further includes the Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); for IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). Variant forms of the above bispecific antibody formats are considered to be within the scope of the present disclosure.

[0093] Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-Fv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab<2> bispecific format (e.g., for confirmation of the aforementioned formats, see Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies or antigen-binding fragments can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that self-assemble into multimeric complexes with a defined composition, valence, and shape. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012].)

[0094] C. Nucleic Acids and Expression Systems In some embodiments, the present disclosure provides a nucleic acid capable of expressing any of the antibodies of the antigen-binding fragments disclosed herein. The nucleic acid can be a single-stranded or double-stranded DNA or RNA molecule. In further embodiments, the nucleic acid sequence of the antibody or antigen-binding fragment can be an isolated nucleic acid sequence, a recombinant nucleic acid sequence, and / or a nucleic acid sequence fused to a heterologous nucleotide sequence, or a nucleic acid sequence in a DNA library. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, and / or 791.In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and / or 796. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, 6, 11 and / or 16.

[0095] In certain embodiments, the nucleic acid encoding the antibody or antigen-binding fragment also comprises a nucleotide sequence that hybridizes under highly stringent conditions to the nucleotide sequence of the polynucleotide encoding any of the above antibodies or antigen-binding fragments, or the complementary sequence thereof. In some embodiments, the nucleic acid hybridizes under highly stringent conditions to a polynucleotide encoding an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, and 792.In some embodiments, the nucleic acid hybridizes under highly stringent conditions to a polynucleotide encoding an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797. Those skilled in the art will readily appreciate that the appropriate stringency conditions for promoting DNA hybridization can be varied. For example, after performing hybridization with 6.0× sodium chloride / sodium citrate (SSC) at about 45° C., washing with 2.0× SSC at 50° C. can be performed. For example, the salt concentration in the washing step can be selected from low stringency of about 2.0× SSC at 50° C. to high stringency of about 0.2× SSC at 50° C. In addition, the temperature of the washing step can be increased from low stringency conditions at room temperature (about 22° C.) to high stringency conditions at about 65° C. Even if both the temperature and the salt are changed, other variable factors can be changed while the temperature or the salt concentration is kept constant. In one embodiment, the present disclosure provides a nucleic acid that hybridizes under low stringency conditions of washing with 2× SSC at room temperature after 6× SSC at room temperature.

[0096] Isolated nucleic acids that differ from nucleic acids encoding an antibody or antigen-binding fragment thereof due to the degeneracy of the genetic code are also within the scope of the present disclosure. For example, some amino acids are represented by two or more triplets. Codons that specify the same amino acid, i.e., synonyms (e.g., CAU and CAC are synonyms for histidine), can result in "silent" mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that lead to changes in the amino acid sequence of the target protein are expected to exist among mammalian cells. Those skilled in the art will understand that these mutations of one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein can exist among individuals of a given species due to natural allelic variations. Any and all such nucleotide variants and the resulting amino acid polymorphisms are within the scope of the present disclosure.

[0097] In some embodiments, the present disclosure provides a vector comprising any of the nucleic acids disclosed herein. In some embodiments, the present disclosure provides a host cell comprising any of the vectors disclosed herein.

[0098] Regardless of whether the antibodies of the present disclosure are full-length antibodies or antigen-binding fragments, the antibodies and antigen-binding fragments of the present disclosure can be recombinantly expressed in cell lines. In these embodiments, a sequence encoding a particular antibody or antigen-binding fragment can be used for transformation of a suitable host cell, such as a mammalian host cell or a yeast host cell. According to these embodiments, any known method for introducing a polynucleotide into a host cell can be used to perform the transformation, including, for example, packaging of the polynucleotide into a virus (or within a viral vector) and using the virus (or vector) or transduction of the host cell by transfection procedures known in the art. Generally, the transformation procedure used can depend on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, and direct microinjection of DNA into the nucleus.

[0099] According to certain embodiments of the present disclosure, a nucleic acid molecule encoding an amino acid sequence of a heavy chain constant region (all or part), a heavy chain variable region of the present disclosure, a light chain constant region, or a light chain variable region of the present disclosure is inserted into a suitable expression vector using standard ligation techniques. In preferred embodiments, a heavy chain constant region or a light chain constant region is added to the C-terminus of a suitable variable region and ligated to the expression vector. The vector is typically selected to be functional in the particular host cell being used (i.e., the vector is compatible with the host cell machinery such that gene amplification and / or gene expression can occur). For an overview of expression vectors, see Goeddel (ed.), 1990, Meth. Enzymol. Vol. 185, Academic Press, N.Y. In the context of antibody expression, both the heavy and light chains may be expressed from the same vector (e.g., from the same or different promoters present on the same vector), or the heavy and light chains may be expressed from different vectors. In certain embodiments, the heavy and light chains are transfected into the same host cell and expressed from different vectors that are co-expressed. Irrespective of whether the heavy and light chains are expressed from the same or different vectors in the same host cell, the chains are then able to associate to form an antibody (or antibody fragment, depending on the heavy and light chain portions being expressed).

[0100] Typically, an expression vector used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences are collectively referred to as "adjacent sequences" in certain embodiments and will generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including a donor splice site and an acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding the polypeptide to be expressed, and a selectable marker sequence. These vector components are well known and there are numerous commonly available vectors that can be selected and used for protein expression. The vector can be easily selected based on the desired host cell and application.

[0101] The origin of replication is usually part of a commercially available prokaryotic expression vector and its origin serves to amplify the vector in the host cell. If the selected vector does not contain an origin of replication site, it can be chemically synthesized based on known sequences and ligated to the vector. For example, the origin of replication derived from plasmid pBR322 (New England Biolabs, Beverly, Mass) is suitable for most Gram-negative bacteria, and origins of various viruses (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not necessary for mammalian expression vectors (e.g., the SV40 origin is often utilized only because it also contains the viral early promoter).

[0102] The expression vectors and cloning vectors of the present disclosure will typically contain a promoter that is recognized by the host organism and operably linked to a molecule encoding a heavy chain and / or a light chain. The promoter is a non-transcribed sequence that is located upstream (i.e., on the 5' side) of the start codon of the structural gene (generally within about 100 to 1000 bp) and controls the transcription of the structural gene. Promoters are typically classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters elicit an increase in the level of transcription from DNA under their control in response to some change in culture conditions such as the presence or absence of nutrients or a change in temperature. On the other hand, constitutive promoters elicit the production of a continuous gene product; that is, they control gene expression little or not at all. A number of promoters recognized by various potential host cells are well known. Suitable promoters are operably linked to DNA encoding a heavy chain or a light chain that includes the antibody or antigen-binding fragment of the present disclosure. In certain embodiments, the same promoter is used for both the heavy chain and the light chain. In other embodiments, different promoters (present on the same or different vectors) are used for each.

[0103] Promoters suitable for use with yeast hosts are also well known in the art. It is advantageous to use a yeast enhancer together with the yeast promoter. Promoters suitable for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40), but are not limited thereto. Other suitable mammalian promoters include heterologous mammalian promoters such as the heat shock promoter and the actin promoter.

[0104] Additional promoters of interest include: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-10); the CMV promoter; the promoter contained in the long 3' terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-45); the control sequence of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25), but are not limited thereto. The following animal transcriptional regulatory regions that exhibit tissue specificity and are utilized in transgenic animals are also of interest: the elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); the insulin gene regulatory region active in pancreatic β cells (Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene regulatory region active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-44); the mouse mammary tumor virus regulatory region active in testicular cells, mammary cells, lymphoid cells and mast cells (Leder et al., 1986, Cell 45:485-95); the albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-76); the α-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-48; Hammer et al., 1987, Science 235:53-58); the α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-71); the β-globin gene regulatory region active in myeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene regulatory region active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-86); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234:1372-78).

[0105] The vector may also contain enhancer sequences for increasing the transcription of DNA encoding the light or heavy chain.

[0106] The expression vectors of the present disclosure can be constructed from starting vectors such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not pre-existing in the vector, they may be obtained individually and ligated to the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

[0107] A vector is constructed, and after inserting a nucleic acid molecule encoding a light chain or a heavy chain or a light chain and a heavy chain containing the antibody or antigen-binding fragment of the present disclosure into an appropriate site of the vector, the complete vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the expression vector into the selected host cell can be carried out by well-known methods including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell used. These methods and other suitable methods are well known to those skilled in the art.

[0108] When cultured under appropriate conditions, the host cell can synthesize the antibody or antigen-binding fragment of the present disclosure and then recover it from the culture medium (when the host cell secretes it into the medium), or directly recover it from the host cell that produces it (when it is not secreted). The selection of an appropriate host cell will depend on various factors such as the desired expression level, the polypeptide modifications (such as glycosylation or phosphorylation) desired or required for activity, and the ease of folding into a biologically active molecule.

[0109] Mammalian cell lines available as host cells for expression are well known in the art and include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and numerous other cell lines, including but not limited to, numerous immortalized cell lines available from the American Type Culture Collection (A.T.C.C.). In another embodiment, a cell line derived from the B cell lineage that does not produce its own antibodies but has the ability to produce and secrete heterologous antibodies (e.g., mouse myeloma cell lines NS0 and SP2 / 0) may be selected. In other embodiments, cells other than mammalian cells, such as yeast cell lines (e.g., Pichia), are used.

[0110] In certain embodiments, the cell line stably expresses the antibodies or antigen-binding fragments of the present disclosure. In other embodiments, the cells transiently express the antibodies or antigen-binding fragments of the present disclosure.

[0111] D. Therapeutic Formulations and Administration The present disclosure provides pharmaceutical compositions comprising the anti-PAR2 antibodies or antigen-binding fragments thereof of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated using suitable carriers, excipients, and other agents that provide improved transfer, delivery, and resistance. A number of suitable formulations can be found in the formularies known to all pharmacists: Remingtons Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA, etc.), anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also "Compendium of excipients for parenteral formulations" by Powell et al., PDA (1998) J Pharm Sci Technol 52:238-311.

[0112] The dosage of the antibody administered to a patient can vary depending on the patient's age and physique, target disease, pathological condition, route of administration, etc. Preferred dosages are usually calculated according to body weight or body surface area. Depending on the severity of the pathological condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering the anti-PAR2 antibody or antigen-binding fragment can be determined empirically. For example, the patient's progression can be monitored by periodic evaluation and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0113] Various delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, and can be used to administer the pharmaceutical compositions of the present disclosure (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intrathecal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelium or inner layer of the mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered in combination with other biologically active agents. Administration may be systemic or local.

[0114] In some embodiments, the antibodies and antigen-binding fragments thereof are useful in the treatment of conditions and disorders associated with the central nervous system and particularly the brain. When administering a macromolecule such as an antibody or antigen-binding fragment to the subject's brain, various factors must be considered, namely, the ability of the antibody or antigen-binding fragment to cross the blood-brain barrier (BBB). Those skilled in the art are aware of methods for administering such macromolecules to the brain. For example, in some embodiments, the antibody or antigen-binding fragment is covalently modified with one or more cationic polyamines such as hexamethylenediamine or tetramethylenediamine to increase the likelihood of the antibody or antigen-binding fragment migrating intracellularly across the BBB. In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment that targets PAR2 and also targets a receptor that facilitates transport across the BBB (e.g., the transferrin receptor, insulin receptor, and TMEM30A). In some embodiments, the antibody or antigen-binding fragment is conjugated to an agent that targets a receptor that facilitates transport across the BBB (e.g., the transferrin receptor, insulin receptor, and TMEM30A). In some embodiments, the BBB is transiently disrupted before or during administration of the antibody or fragment. In some embodiments, the BBB is transiently disrupted by ultrasound, radiation, biochemical treatment (e.g., by a K Ca receptor agonist such as NS-1619), or by intra-arterial injection of a concentrated hyperosmotic solution.

[0115] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously by standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable for the delivery of the pharmaceutical composition of the present disclosure. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be immediately discarded and replaced with a new cartridge containing the pharmaceutical composition. Subsequently, the pen-type delivery device can be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is depleted and the reservoir is empty, the entire device is discarded.

[0116] A number of reusable pen-type and self-injector delivery devices are applicable for subcutaneous delivery of the pharmaceutical compositions of the present disclosure. By way of example only, and not limitation, these include AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices applicable for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, by way of example only, and not limitation, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ self-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park IL).

[0117] In certain situations, a pharmaceutical composition can be delivered by a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed near the target of the composition, and thus only a fraction of the systemic dose is required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0118] Examples of injectable preparations may include dosage forms for intravenous, subcutaneous, intrathecal, intradermal, and intramuscular injections, infusion, etc. These injectable preparations can be prepared by known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying any of the antibodies, antigen-binding fragments, or salts thereof disclosed herein in a sterile aqueous medium or an oily medium conventionally used for injections. Examples of the aqueous medium for injections include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and these may be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. Examples of the oily medium include, for example, sesame oil, soybean oil, etc., and these may be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus prepared is preferably filled into appropriate ampoules.

[0119] The above pharmaceutical composition for oral or parenteral use is advantageously prepared in a dosage form of unit dose suitable for adapting to the dosage of the active ingredient. Examples of such dosage forms of unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the above-described antibody contained is generally about 5 to about 500 mg per dosage form of unit dose; in particular, the above-described antibody or antigen-binding fragment is preferably contained in an amount of about 5 to about 100 mg in the form of an injection and about 10 to about 250 mg in other dosage forms.

[0120] E. Therapeutic Use of Antibodies For any of the methods described herein, the present disclosure contemplates the use of any of the antibodies or antigen-binding fragments of the present disclosure.

[0121] In some embodiments, the present disclosure provides a method of treating a disorder in a subject in which unwanted and / or abnormal PAR2 activity is involved, the method comprising administering any of the antibodies or antigen-binding fragments described herein. As used herein, "disorder," "condition," and "disease" are used interchangeably and refer to any of the disorders, conditions, or diseases disclosed herein. In some embodiments, the disorder / condition / disease in which unwanted and / or abnormal PAR2 activity is involved is a disorder / condition / disease associated with abnormal or unwanted inflammation. Examples of disorders / conditions / diseases in which abnormal or unwanted PAR2 activity is involved include acute or chronic pain, acute or chronic itching, acute or chronic inflammation (e.g., acute or chronic inflammation of joints, lungs, brain, gastrointestinal tract, periodontal tissues, skin, and vascular system), autoimmune disorders, periodontitis, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, arthritis, psoriasis, obesity, diabetes, cardiovascular disease, pancreatitis, cancer (e.g., breast cancer, lung cancer, colorectal cancer, gastric cancer, or prostate cancer), asthma, fibrosis, gastric ulcer, fibrosis or fibrotic disorders, Alzheimer's disease, Parkinson's disease, scleroderma, Crohn's disease, ulcerative colitis, adult respiratory distress syndrome (ARDS), glomerulonephritis, and meningitis. In some embodiments, the present disclosure provides a method of treating a subject having a condition associated with one or more metabolic syndromes such as metabolic syndrome, or visceral fat deposition, hypertension, abnormalities in glucose and insulin homeostasis, insulin resistance, endothelial damage, cardiovascular hypertrophy, inflammation, vascular inflammation, atherosclerosis, ventricular systolic dysfunction, fibrosis, and fatty liver disease. In certain embodiments, the present disclosure provides a method of treating pain, such as pain associated with any of the disorders / conditions / diseases disclosed herein (e.g., osteoarthritis pain). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0122] In some embodiments, the present disclosure provides a method of interfering with the interaction between a protease (e.g., trypsin) and PAR2, the method comprising administering to a cell any of the antibodies or antigen-binding fragments described herein. In some embodiments, the present disclosure provides a method of inhibiting the exposure of the tethered ligand of PAR2 on the cell surface, the method comprising administering to a cell any of the antibodies or antigen-binding fragments described herein. In some embodiments, the present disclosure provides a method of inhibiting the interaction between the tethered ligand of PAR2 and the second transmembrane loop of the PAR2 protein, the method comprising administering to a cell any of the antibodies or antigen-binding fragments described herein. In some embodiments, the present disclosure provides a method of inhibiting the activation of the PAR2 receptor on the cell surface, the method comprising administering to a cell any of the antibodies or antigen-binding fragments described herein. In some embodiments, the cell is a nerve (e.g., a sensory nerve). In some embodiments, the cell is in vitro. In other embodiments, the cell is in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject suffers from any of the disorders disclosed herein.

[0123] For any of the methods described herein, the present disclosure contemplates combinations of any one or more steps of one method with any one or more steps from another method. These methods include administering to an individual in need thereof an effective amount of a compound of the present disclosure appropriate for a particular disease or condition. In certain embodiments, these methods include delivering to the cells of a subject in need thereof any of the antibodies or antigen-binding fragments disclosed herein.

[0124] The terms "treatment", "treating", "alleviate", etc. are used herein generally to mean obtaining a desired pharmacological and / or physiological effect, which can also be used to refer to improving, alleviating, and / or reducing the severity of one or more symptoms of the medical condition being treated. The effect can be prophylactic in terms of a complete or partial delay in the onset or recurrence of a disease, medical condition, or its symptoms, and / or therapeutic in terms of a partial or complete cure of the disease or medical condition and / or the adverse effects contributing to the disease or medical condition. "Treatment", as used herein, encompasses any treatment of a disease or medical condition in a mammal, particularly a human, and includes (a) preventing the onset of a disease or medical condition in a subject who may be susceptible to the disease or medical condition but has not yet been diagnosed as having it; (b) suppressing a disease or medical condition (e.g., arresting its development); or (c) alleviating a disease or medical condition (e.g., causing regression of the disease or medical condition, bringing about improvement of one or more symptoms). For example, "treatment" of pain (e.g., osteoarthritis pain) includes reducing, arresting, alleviating, or eliminating the pain symptoms in the subject being treated. The population of subjects to be treated by the methods described herein includes subjects suffering from an undesirable medical condition or disease and subjects at risk of developing a medical condition or disease.

[0125] For any of the methods described herein, the disclosure contemplates the use of any of the antibodies or antigen-binding fragments described throughout this application. Additionally, for any of the methods described herein, the disclosure contemplates combinations of any one or more steps of one method with any one or more steps from another method.

[0126] In certain embodiments, the present invention provides a method of treating a condition associated with any of the diseases / conditions / disorders disclosed herein, such as acute or chronic pain (e.g., osteoarthritis pain). These methods include administering to an individual a therapeutically effective amount of any of the above antibodies or antigen-binding fragments. These methods are particularly aimed at therapeutic and prophylactic treatment of animals, more specifically humans. The present disclosure contemplates any and all combinations of any of the foregoing aspects and embodiments, as well as combinations with any of the embodiments described in the modes and examples for carrying out the invention.

[0127] The term "therapeutically effective dose" means a dose that produces the desired effect for which administration is intended. The exact dose will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0128] In certain embodiments, either the antibody or antigen-binding fragment of the invention can be administered alone or in combination with one or more additional compounds or therapies for treating any of the diseases / conditions / disorders disclosed herein, such as acute or chronic pain (e.g., osteoarthritis pain). For example, either the antibody or antigen-binding fragment disclosed herein can be co-administered with one or more therapeutic compounds. When co-administration is indicated, the combination therapy can include co- or alternating administration. In addition, the combination can include acute or chronic administration. Optionally, the antibody / antigen-binding fragment and the additional compound act in an additive or synergistic manner for treating any of the diseases / conditions / disorders disclosed herein, such as acute or chronic pain (e.g., osteoarthritis pain). Additional compounds used in combination therapy include, but are not limited to, small molecules, polypeptides, antibodies, antisense oligonucleotides, and siRNA molecules. In some embodiments, the additional compound is any one or more of an anti-inflammatory agent, an analgesic, a non-steroidal anti-inflammatory agent (NSAID), a corticosteroid, hyaluronic acid, acetaminophen, codeine, lorcet, loratab, vicodin, hydrocodone, morphine, oxycontin, oxycodone, percocet, aspirin, celecoxib, pregabalin, arthrodesis, joint replacement, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab, tocilizumab, and tofacitinib. Depending on the nature of the combination therapy, administration of the antibody or antigen-binding disclosure of the present disclosure can be continued during and / or after other therapies are administered. Administration of the antibody or antigen-binding fragment can be by single or multiple doses. In some examples, administration of the antibody or antigen-binding fragment is initiated at least several days prior to the other therapy, while in other examples, administration is initiated either immediately prior to or at the time of administration of the other therapy. In some embodiments, any of the additional compounds disclosed herein are conjugated to any of the antibodies or antigen-binding fragments disclosed herein.

[0129] In another example of combination therapy, either the antibodies or antigen-binding fragments of the present disclosure can be used as part of a treatment regimen in combination with one or more additional therapeutic modalities. As an example, such other therapeutic modalities include, but are not limited to, diet therapy, occupational therapy, physical therapy, psychotherapy, massage, acupuncture, acupressure, mobility aids, assistance animals, and the like.

[0130] The antibodies or antigen-binding fragments disclosed herein can be used in combination with other therapies, but it should be noted that in certain embodiments, the antibodies or antigen-binding fragments are provided as the sole form of therapy. Regardless of whether they are administered alone or in combination with other drugs or treatment regimens, the dosage, frequency, route of administration, and timing of administration of the antibodies or antigen-binding fragments are determined by a physician based on the patient's condition and needs.

[0131] According to certain embodiments of the present disclosure, multiple doses of an anti-PAR2 antibody or an antigen-binding fragment thereof (or a pharmaceutical composition comprising a combination of an anti-PAR2 antibody and any of the additional therapies described herein) can be administered to a subject over a predetermined period of time. A method according to this aspect of the present disclosure includes sequentially administering multiple doses of the anti-PAR2 antibody or antigen-binding fragment of the present disclosure to a subject. As used herein, "administering sequentially" means that each dose of the anti-PAR2 antibody or antigen-binding fragment is administered to the subject on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months), at different times. The present disclosure includes methods that include sequentially administering to a patient one or more second doses of the anti-PAR2 antibody or antigen-binding fragment, optionally followed by one or more third doses of the anti-PAR2 antibody or antigen-binding fragment, following a single initial dose of the anti-PAR2 antibody or antigen-binding fragment.

[0132] The terms "first dose", "second dose", and "third dose" refer to the chronological order of administration of the anti-PAR2 antibody or antigen-binding fragment of the present disclosure. Thus, the "first dose" is the dose administered at the start of the treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose administered after the first dose; and the "third dose" is the dose administered after the second dose. The first, second, and third doses may all contain the same amount of anti-PAR2 antibody or antigen-binding fragment, but generally may differ from each other with respect to the frequency of administration. However, in certain embodiments, the amounts of anti-PAR2 antibody or antigen-binding fragment contained in the first, second, and / or third doses may change relative to each other during the course of treatment (e.g., are upregulated or downregulated as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency.

[0133] F. Diagnostic / Other Uses of the Antibody or Antigen-Binding Fragment The anti-PAR2 antibodies of the present disclosure can also be used to detect and / or measure PAR2 or PAR2-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-PAR2 antibody, or an antigen-binding fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression of PAR2 (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for PAR2 may include, for example, contacting a sample obtained from a patient with an anti-PAR2 antibody of the present disclosure, where the anti-PAR2 antibody is labeled with a detectable label or reporter molecule.

[0134] Alternatively, an unlabeled anti-PAR2 antibody can be used in combination with a secondary antibody that is itself detectably labeled in a diagnostic application. Detectable labels or reporter molecules are 3 H, 14 C, 32 P, 35 S, or 125Radioisotopes such as I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate, or rhodamine; or enzymes such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase may also be used. Specific exemplary assays that can be used to detect or measure PAR2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0135] The compositions of the present disclosure have numerous uses. For example, the antibodies and antigen-binding fragments of the present disclosure are useful for studying the preferential cell and tissue distribution in cells and tissues in vitro and / or in vivo. Similarly, antibodies and antigen-binding fragments conjugated to single or heterologous agents are useful as imaging agents for diagnostic applications, etc. ex vivo or in vivo. For example, antibodies or antigen-binding fragments conjugated to a radioactive moiety are useful for ex vivo or in vivo imaging studies. Similarly, any of the antibodies or antigen-binding fragments of the present disclosure are similarly useful.

[0136] When used in vitro, the antibodies and antigen-binding fragments of the present disclosure are suitable for the identification of the binding partner of the delivered antibody or antigen-binding fragment (e.g., the identification of the protein or peptide that binds to the antibody or antigen-binding fragment), as well as the evaluation of localization and transport. Similarly, when used in vivo, the antibody or antigen-binding fragment is useful for the identification of the binding partner of the delivered antibody or antigen-binding fragment (e.g., the identification of the protein or peptide that binds to the antibody or antigen-binding fragment), the evaluation of localization and transport, the evaluation of biodistribution and half-life, and the evaluation of immunogenicity.

[0137] G. Animal / Cell Model A number of animal models that would be useful for testing either an antibody or a fragment thereof are known to those of skill in the art. See, for example, Kuyinu et al., 2016, J Orthop Surg Res, 11(19):10.1186 / s13018-016-0346-5. In some embodiments, the animal model is a pain model produced by treating an animal with a chemical such as sodium monoiodoacetate (MIA) or carrageenan. In some embodiments, the chemical is injected into a site in the animal where pain will be induced. In some embodiments, the animal model is an animal in which an injury such as anterior cruciate ligament resection, meniscectomy, or medial meniscectomy is induced post-surgically (e.g., incisionally). In some embodiments, the animal model is associated with an inflammatory condition such as lower esophageal hypersensitivity, colonic inflammation, gastric ulceration, bladder inflammation, pancreatic inflammation, and uterine inflammation. See, for example, the animal models referred to in National Research Council Committee on Recognition and Alleviation of Pain in Laboratory Animals, “Models of Pain”, 2009.

[0138] H. Kit In certain embodiments, the invention also provides a pharmaceutical package or kit comprising one or more containers filled with at least one antibody or antigen-binding fragment of the present disclosure. Such containers may optionally be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, reflecting (a) approval by the agency of manufacture, use, or sale for human administration, (b) instructions for use, or both.

Example

[0139] The following examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, but are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0140] Example 1: Generation of Antibodies with pH-Sensitive Binding to PAR2 Generation of Recombinant Human, Rat, and Cynomolgus PAR2 and PAR1 Proteins Human, rat, and cynomolgus (Macaca fascicularis) PAR2 (protease-activated receptor 2) constructs containing extracellular residues 1 - 75 were designed with an N-terminal AviTag™ (Avidity LLC) and C-terminal Flag and polyhistidine tags and cloned into the vector pDEST12.2 OriP FH (Life Technologies). A human PAR1 (protease-activated receptor 1) construct containing extracellular residues 1 - 102 was designed with C-terminal Flag and polyhistidine tags and cloned into the vector pDEST12.2 OriP FH (Life Technologies). This construct was expressed in HEK293 cells and purified from the medium using standard affinity and size exclusion chromatography. To generate biotinylated protein, the AviTag™ was enzymatically biotinylated according to the manufacturer's instructions.

[0141] Construction of a Combinatorial Histidine Scanning Library Split pool oligonucleotides were designed to introduce histidine or wild-type amino acids at each position of either VHCDR2, VHCDR3 or VLCDR3 of Par0067. Subsequently, three Par0067 scFv phage display libraries were constructed in which 0% to 100% histidine residues were present in either VHCDR2, VHCDR3 or VLCDR3.

[0142] Selection of pH-sensitive Par0067 mutant scFv To isolate Par0067 mutants that bind to PAR2 at pH 7.4 but have reduced binding at pH 6.0, a combinatorial histidine scanning library was subjected to affinity-based phage display selection. To achieve this, four rounds of selection were performed on each library using decreasing concentrations of biotinylated recombinant human PAR2 (Hawkins, RE et al., 1992 Aug 5;226(3):889-96). In each round, phage were pre-incubated for 1 hour with paramagnetic beads (dynabeads®) coated with streptavidin to remove streptavidin conjugates. Subsequently, the streptavidin beads were removed and discarded using a DYNAL® magnet, and the remaining phage were added to biotinylated recombinant human PAR2 at pH 7.4. After 2 hours of selection, paramagnetic beads coated with streptavidin were added to capture phage bound to biotinylated recombinant human PAR2. After washing the beads five times with PBS Tween (PBST), specific scFv was eluted in low pH buffer (pH 5.5 - pH 6.0). Next, the selected scFv-phage particles were rescued as previously described (Osbourn JK.et al.Immunotechnology,2(3):181-96,1996) and the selection process was repeated in the presence of biotinylated PAR2 with decreasing concentration (1 nM to 0.05 nM between 4 rounds).

[0143] Reformatting of scFv into IgG1-TM The antibody was basically converted from the scFv into the full-length immunoglobulin G1 triple mutant (IgG1-TM, an IgG1 Fc sequence incorporating the mutations L234F, L235E, and P331S) antibody format as described by Persic et al. (1997, Gene, 187, 9-18), with the following modifications. To facilitate use with CHO transient cells and to enable episomal replication, the expression vector was included with the OriP fragment. The variable heavy chain (VH) domain was cloned into a vector containing the human heavy chain constant domain and regulatory elements to express the full-length IgG1 heavy chain in mammalian cells. Similarly, the variable light chain (VL) domain was cloned into a vector for expression of the human light chain (lambda) constant domain and regulatory elements to express the full-length IgG light chain in mammalian cells. To obtain IgG, these heavy chain and light chain IgG expression vectors were transfected into CHO transient mammalian cells (Daramola et al. Biotechnol Prog 30(1):132-41(2014)). IgG was expressed and secreted into the medium. The recovered product was filtered and then purified, and subsequently IgG was purified using protein A chromatography. The culture supernatant was loaded onto a column of Ceramic Protein A (BioSepra) of appropriate size and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl. The bound IgG was eluted using 0.1 M sodium citrate (pH 3.0) and neutralized by the addition of Tris-HCl (pH 9.0). The eluted material was buffer-exchanged into PBS using a Nap10 column (Amersham, #17-0854-02), and the IgG concentration was determined spectrophotometrically using the extinction coefficient based on the amino acid sequence of IgG (Mach et al., Anal. Biochem. 200(1):74-80(1992)). The purified IgG was analyzed for aggregation and degradation purity using SEC-HPLC and by SDS-PAGE.

[0144] Screening of pH-sensitive Par0067 mutant scFv and IgG To screen for and characterize antibodies with potential pH-dependent binding, a biochemical epitope competition assay format was used. An assay using the technology of homogeneous time-resolved fluorescence (HTRF™) was designed to evaluate the ability of test antibodies (scFv or IgG) to inhibit the binding interaction of the parental Par0067 IgG antibody to the extracellular domain (ECD) of human PAR2. Importantly, the assay was performed at two different pH values (pH 7.4 and pH 6.0).

[0145] First, crude unpurified scFv (bacterial extract) was screened in a single-point 384-well high-throughput screening (HTS) using parallel assays at pH 7.4 and pH 6.0 (as described above). This single-point parallel HTS format enabled the screening of many (thousands) of test scFvs and advanced antibodies that reduced the inhibition of the binding interaction of the parental Par0067 IgG to human PAR2 at pH 6.0 compared to pH 7.4 for further characterization. Subsequently, the same epitope competition assay approach was performed in a multi-point dose-response IC 50 format to test both purified scFv and purified IgG (the latter required minor modifications to the assay design as described in Section C). Additionally, the assay was performed at pH 7.4 and pH 6.0, but in these experiments, the inventors were most interested in the test antibodies, where the dose-response inhibition curve showed a significant rightward shift at pH 6.0 relative to the corresponding dose-response inhibition curve observed at pH 7.4 (i.e., the IC 50 values were significantly increased).

[0146] The following protocol includes methods for both single-point testing of crude unpurified scFv and subsequent testing of purified scFv and IgG.

[0147] Section A: General assay conditions: Assay buffer: It was freshly prepared on the day of using the assay buffer. For experiments at pH 7.4, KF (0.4 M) (VWR, 103444T) and BSA (0.1% w / v) (PAA, K05 - 013) were added to DPBS (Gibco 14190 - 086), and then the pH was re - checked and finely adjusted to pH 7.4 if necessary. For experiments at pH 6.0, while keeping all other buffer components the same as the pH 7.4 assay buffer outlined above, the pH 6.0 assay buffer was prepared using 200 mM MES (Sigma, M - 5287) (in contrast to DPBS) as the base buffer. After the addition of KF (0.4 M) and BSA (0.1% w / v), the 200 mM MES - based buffer was adjusted to pH 6.0 with HCl.

[0148] Assay plate: The assay was performed using a black, low - volume 384 - well plate (round bottom, non - binding) (Corning, 4514).

[0149] Assay volume: 20 μL

[0150] Incubation and plate reading: The assay plate was incubated at room temperature for 2 hours and then read using the standard HTRF (trademark) reading protocol of an Envision plate reader.

[0151] Section B: Testing of scFv: (Unpurified bacterial lysate and purified scFv)

[0152]

Table 1

[0153] Preparation / Addition of Par0067 IgG: Unlabeled purified Par0067 IgG (in-house) was made to a concentration of 4.44 nM (1.11 nM final [assay]) in each of the two assay buffers (pH 7.4 and pH 6.0) described above in Section A. A 4.44 nM Par0067 IgG solution of the appropriate pH at 5 μl / well was added to all wells of the corresponding assay plates (pH 7.4 and pH 6.0).

[0154] Preparation / Addition of Test scFv: a) For parallel single-point HTS of crude unpurified bacterial lysate scFv samples at both pH 7.4 and pH 6.0, the samples were first pre-diluted to 40% of their appropriate concentrations using either assay buffer at pH 7.4 or pH 6.0 as needed. Then, 5 μl of the 40% pre-diluted sample was transferred to the appropriate assay (pH 7.4 or pH 6.0) to obtain a final assay sample concentration of 10.0% (in a 20 μl final assay volume). The parental Par0067 scFv was included in all HTS experiments as a control, and specific pH-dependent antibodies were likewise included if they were available (for reference). b) Multiple-point dose-response IC of purified scFv antibody 50 For the test, the samples were tested starting from 1 / 4 of the maximum final assay of the appropriate undiluted sample (i.e., no prior dilution step was performed). Next, replicate 11-point 1:3 serial dilutions were prepared on 384-well polypropylene Greiner plates in each of the two assay buffers (pH 7.4 and 6.0). 5 μl per well of each serial dilution was transferred from the corresponding scFv dilution plate of the appropriate pH to the corresponding assay plates (pH 7.4 and pH 6.0). 5 μl of assay buffer of the appropriate pH was added to the total and non-specific wells. The parental Par0067 purified scFv was included in all multiple-point dose-response IC 50 experiments as a control, and specific pH-dependent purified scFv were likewise included if they were available (for reference). The data are shown in Table 1.

[0155] Preparation / Addition of Biotinylated Human PAR2 ECD: The in-house biotinylated human PAR2 ECD was diluted in each of two assay buffers (pH 7.4 and pH 6.0) to obtain a standard solution of 4.0 nM (1 nM final assay concentration). Next, 5 μl / well of the appropriate 4.0 nM biotinylated human PAR2 ECD standard solution was added to all wells of the corresponding assay plates (pH 7.4 and pH 6.0) except for the negative binding control wells. 5 μl / well of the assay buffer of the appropriate pH was added to the negative binding wells.

[0156] Preparation / Addition of HTRF Detection Reagent: Europium cryptate-labeled streptavidin (CisBio, 610SAKLB) and XL 665 labeled anti-human Fc (CisBio, 61HFCXLB) were diluted in the assay buffer of each pH (pH 7.4 and pH 6.0) to obtain a combined standard solution with concentrations of 6.0 nM (europium cryptate-labeled streptavidin) and 40 nM (XL 665 labeled anti-human Fc). When diluted 4-fold and added to the assay, this resulted in final assay concentrations of 1.5 nM (europium cryptate-labeled streptavidin) and 10 nM (XL 665 labeled anti-human Fc). Next, 5 μl / well of the corresponding pH combined HTRF™ detection reagent standard solution was added to all wells of the corresponding assay plates (pH 7.4 and pH 6.0).

[0157] Section C: Testing of Purified IgG:

[0158]

Table 2

[0159] Par0067 IgG Dylight650 Labeling: The Dylight 650 label of Par0067 IgG was labeled with Dylight 650Performed using a labeling kit (Thermo Scientific catalog number 84536). Labeling of in-house purified Par0067 IgG was performed according to the labeling procedure recommended by the manufacturer. Final Dylight 650 The labeled Par0067 IgG concentration was determined to be 0.56 mg / ml by the average Dylight for an IgG dye incorporation ratio of 2.7 moles of dye / mole of IgG 650 .

[0160] Dylight 650 Preparation / Addition of Labeled Par0067 IgG: Dylight 650 Labeled Par0067 IgG (0.56 mg / ml, 3,733 nM) was brought to a concentration of 4.44 nM (1.11 nM final [assay]) in each of the two assay buffers (pH 7.4 and pH 6.0) described previously in the Materials section. 5 μl / well of the appropriately pH-adjusted 4.44 nM Dylight 650 Par0067 IgG solution was added to all wells of the corresponding assay plates (pH 7.4 and pH 6.0).

[0161] Serial Dilution / Addition of Test IgG: Native Par0067 IgG (used as a reference / control in all assays) was pre-diluted to obtain a 2000 nM stock in each of the two assay buffers of different pH (to obtain a maximum final assay IgG concentration of 500 nM). All test, or other reference / control IgGs, were tested from 1 / 4 of the maximum final assay concentration of the appropriate undiluted sample (i.e., no prior dilution step was performed). Next, an 11-point 1:3 serial dilution in duplicate was prepared on a 384-well polypropylene Greiner plate in each of the two assay buffers (pH 7.4 and 6.0). 5 μl per well was transferred from the corresponding IgG dilution plate of the appropriate pH to the corresponding assay plates (pH 7.4 and pH 6.0). 5 μl of the appropriately pH-adjusted assay buffer was added to the total and non-specific wells.

[0162] Preparation / Addition of Biotinylated Human PAR2 ECD: The in-house biotinylated human PAR2 ECD was diluted in each of two assay buffers (pH 7.4 and pH 6.0) to obtain a standard solution of 4.0 nM (1 nM final assay concentration). Next, 5 μl / well of the appropriate 4.0 nM biotinylated human PAR2 ECD standard solution was added to all wells of the corresponding assay plates (pH 7.4 and pH 6.0) except for the negative binding control wells. 5 μl / well of the assay buffer of the appropriate pH was added to the negative binding wells.

[0163] Preparation / Addition of HTRF Detection Reagent: The europium cryptate-labeled streptavidin (CisBio, 610SAKLB) was diluted in the assay buffer of each pH (pH 7.4 and pH 6.0) to obtain a standard solution at a concentration of 6.0 nM. When diluted 4-fold and added to the assay, this resulted in a final assay europium cryptate-labeled streptavidin concentration of 1.5 nM. Next, 5 μl / well of the europium cryptate-labeled streptavidin standard solution of the corresponding pH was added to all wells of the corresponding assay plates (pH 7.4 and pH 6.0).

[0164] Section D: Data Analysis First, the 665 nm and 620 nm counts were converted to 665 nm / 620 nm ratio values. Next, Delta F (%) was calculated according to the following formula. Delta F (%) = ((Sample Ratio - Negative Ratio) / Negative Ratio) × 100

[0165] The negative ratio value was calculated from the corresponding negative binding control wells in the absence of Par0067 IgG. Next, the % Specific Binding was calculated according to the following formula. % Specific Binding = {(Sample % Delta F - Negative Binding % Delta F) / (Total Binding % Delta F - Negative Binding % Delta F)} × 100

[0166] For a single point HTS, the % specific binding at pH 6.0 relative to pH 7.4 (higher % specific binding) was plotted on the x and y axes respectively to visualize the distribution of scFvs with reduced inhibition at pH 6.0 relative to pH 7.4.

[0167] For the dose - response curves at multiple points, the values of % specific binding were plotted against the test - purified antibody concentration (scFv or IgG). IC 50 values were determined by sigmoid dose - response inhibition variable slope curve fitting (4 - parameter logistic equation) using Graphpad Prism software.

[0168]

Table 3

[0169]

Table 4

[0170] Recombinant incorporation of multiple histidines into a single scFv Histidine residues derived from scFvs that demonstrated pH - dependent binding in the Par0067 epitope competition binding assay were recombined using site - directed mutagenesis (Reikofski J and Tao BY, 1992, Biotechnol Adv, 10(4):535 - 547). The resulting scFvs containing histidines in two different CDRs were retested in a competition binding assay as full - length immunoglobulin G1 triple mutants (IgG1 - TM) (Table 2) to identify mutants with further improved pH - dependent binding compared to the parental antibody, Par0067.

[0171] Sequence alignments for each of the histidine - modified clones compared to the CDR sequences of the reference Par0067 antibody are provided in Figures 1A - 1B and 2A - 2B.

[0172]

Table 5

[0173] Affinity of anti-PAR2 Fab for human, rat, and cynomolgus PAR2 determined by BIACORE (trademark) Antigen-binding fragments (Fabs) of anti-PAR2 antibodies were expressed (Spooner J. et al. (2015) Biotechnol Bioeng. 112:1472-7), and their affinities for recombinant PAR2 of various species (human, rat, and cynomolgus) were determined by Biacore.

[0174] Biacore affinity analysis The affinity of anti-PAR2 Fab was measured at 25°C using a Biacore T100 at various pH values (pH 7.4, pH 6.0, and pH 5.6). Experiments were performed using recombinant human, rat, and cynomolgus PAR2 with an N-terminal Avi tag and a C-terminal Flag-His tag.

[0175] Using standard amine coupling techniques, streptavidin was covalently immobilized on the C1 chip surface at a concentration of 4 μg / ml in 10 mM sodium acetate pH 4.5. A final streptavidin surface of approximately 30 - 100 RU was reached. Recombinant biotinylated PAR2 species (in-house) were titrated onto the streptavidin chip surface at 4 μg / ml in HBS-EP+ buffer to a saturation state (R max ) such that Fab binding was possible. Binding of this low level of analyte ensured minimal mass transfer effects.

[0176] The anti-PAR2 Fab was serially diluted (0.39 nM to 25 nM) in HBS-EP+ buffer at pH 7.4 or MES-BS-EP+ buffer at pH 6.0 or MES-BS-EP+ buffer at pH 5.6 and flowed over the chip at 50 μl / min with 3 minutes of association and up to 30 minutes of dissociation. Multiple injections of buffer only were performed under the same conditions to allow subtraction of the double reference of the final sensorgram set and analyzed using BiaEval software (version 2.0.1). The chip surface was completely regenerated by application of 4 M MgCl2.

[0177] The BiaCore affinity results for the selected clones are provided below in Tables 3 to 13.

[0178] [Table 6]

[0179] [Table 7]

[0180] [Table 8]

[0181] [Table 9]

[0182] [Table 10]

[0183] [Table 11]

[0184] [Table 12]

[0185]

Table 13

[0186]

Table 14

[0187]

Table 15

[0188]

Table 16

[0189] Example 2: Cell-based PAR2 and PAR1 activity assays Human A549 cells expressing endogenous PAR2, rat KNRK, mouse LL / 2 or cynomolgus monkey CYNOM-K1 cells, or human 1321N1-hPAR2-cl8 cells overexpressing human PAR2 were seeded at 5,000 cells per well (human, cynomolgus monkey) or 7,000 cells per well (mouse, rat) on tissue culture plates (Greiner Bio-One) coated with PDL. Cells were loaded with Fluo-Screen Quest™ Fluo-8 no-wash calcium dye (AAT Bioquest, Inc). Cells were pretreated with IgG or Fab diluted in assay buffer (HBSS, 0.1% BSA, 20 mM HEPES) at room temperature for 1 hour. For mouse and cynomolgus monkey assays, cells were pretreated with 0.5 nM or 10 nM thrombin, respectively, to desensitize PAR1 activity. The calcium response of PAR2 to 11 nM (human), 400 nM (mouse) or 80 nM (rat, cynomolgus monkey) trypsin (Polymun) was measured on a fluorescence imaging plate reader (FLIPR) Tetra (Molecular Devices). To determine the functional activity against human PAR1, the thrombin-driven calcium response in the A549 human cell line was similarly determined on the FLIPR tetra, and in these assays, neutralizing anti-PAR1 IgG WEDE15 (Beckman Coulter) and ATAP2 (Life Technologies) were used as positive controls. To determine the functional activity against various proteases, 1321N1-hPAR2-cl8 cells overexpressing human PAR2 were pretreated with PaB670129 and then calcium release was stimulated with 0.5 nM trypsin, 500 nM tryptase or 1 nM matriptase. Fluorescence was measured before, during, and after the addition of protease, and the peak RFU per well was calculated. The % response (versus protease alone) was calculated against antibody concentration, and the IC50 was determined using GraphPad Prism software.

[0190] The results from human, cynomolgus monkey, rat, and mouse cells in these assays are provided in Figure 3 (A, B, C, and D respectively), the calculated PAR2 IC50 in Figure 3E, and Figure 6 (specificity data for PAR1). The calculated IC50 values demonstrate that PaB670129 potently inhibits the trypsin-induced PAR2 calcium response in human, mouse, rat, and cynomolgus monkey cells expressing endogenous PAR2 (Figure 3E). Further, in human A549 cells, thrombin-induced PAR1 activation is not inhibited by PaB670129 but can be efficiently blocked by vorapaxar and anti-PAR1 monoclonal antibodies (Figure 6). These data demonstrate that PaB670129 is a potent and specific antagonist of PAR2. Application of PaB670129 alone to PAR2-expressing cells does not affect the basal calcium levels of the cells, demonstrating that PaB670129 lacks any agonist activity at PAR2 (Figure 4A). Further, PaB670129 potently antagonizes the PAR2-induced responses to various proteases, including trypsin, tryptase, and matriptase (Figure 4B).

[0191] Primary DRG glia-neuron PAR2 calcium assay Dissociated cultures of spinal dorsal root ganglia (DRGs) from offspring of Sprague-Dawley rats were prepared and grown on tissue culture plates (Greiner Bio-One) coated with laminin and PDL. Prior to use in the assay, the plates were incubated at 37 °C for 24 - 72 hours. Cells were loaded with 2 μM Fura-2 calcium dye (Life Technologies). Cells were incubated in imaging buffer (HBSS, 20 mM HEPES, 0.1 mM sulfinpyrazone, 10 μM PAR1 antagonist vorapaxar) containing 20 nM PAR2 antibody. Next, intracellular calcium in response to application of the agonists thrombin (Sigma) and matriptase (R&D Systems) PAR2 activating peptide LIGRLO (SEQ ID NO: 832), (Peptides International) and high extracellular potassium (50 mM) was quantified by ratiometric imaging of Fura-2 on an Olympus IX81 microscope equipped with a xenon arc lamp that excited at 340 and 380 nm. The number of neurons relative to glia per field of view was calculated (neurons defined as those showing a response to high potassium). Next, the total number of matriptase-sensitive neurons and glia per field of view was calculated. Results from the calcium assay are provided in FIGS. 5A - F, showing that the PaB670129 antibody efficiently reduced sensitivity to matriptase in DRG neurons (FIGS. 5A - C) and non-neuronal cells (FIGS. 5D - F).

[0192] Example 3: Effect of anti-PAR2 antibody in a rat model of inflammatory arthralgia Intra-articular administration of monosodium iodoacetate (MIA) into the ipsilateral knee of Sprague-Dawley rats initially causes a severe and long-lasting hyperalgesia and allodynia accompanied by an inflammatory response. The development of these signs in this animal model is considered clinically relevant; it reflects the symptoms shown by patients presenting with chronic inflammatory pain associated with potential pathologies such as osteoarthritis (OA) or rheumatoid arthritis (Bove et al., 2003; Fernihough et al., 2004; Kalbhen 1987). MIA-induced hyperalgesia (using weight bearing as an evaluation item) has been previously demonstrated to follow a biphasic pattern over time, with an initial predominantly inflammatory component that is Cox-2 sensitive and significantly reduced by the standard drug celecoxib. This initial inflammatory phase is Pregabalin (PGB) sensitive and transitions to a more chronic pain phenotype that is celecoxib insensitive, suggesting a potential more neuropathic component.

[0193] Weight bearing: Untreated rats distribute their body weight evenly between their two hind paws. However, if there is inflammation and / or pain in the injected (left) hind knee, the body weight is redistributed so that less weight is borne by the affected limb (weight bearing on the damaged limb is reduced). The weight bearing through each hind limb is measured using an incapacitance tester for small animals (Linton Instruments, UK).

[0194] Sprague-Dawley rats were placed in an incapacitance tester for small animals with their hind paws on separate sensors, and the average force exerted by both hind limbs was recorded over 4 seconds.

[0195] Procedure: After parturition, rats underwent a minimum acclimation period of 7 days before the start of the test. Untreated rats were acclimated to the treatment room in their home cages with free access to food and water. Acclimation to the weight-bearing chamber was carried out over several days. Baseline weight-bearing recordings were made on the final day.

[0196] After recording the final baseline on day 0, the animals were anesthetized using isoflurane and oxygen mixed at 3:1 under sterile conditions. The left knee area was shaved and purified with Hibiclens dilution solution.

[0197] Osteoarthritis (OA) was induced by injecting a solution of MIA (Sigma, I2512), 25 μl of 80 mg / ml, (2 mg), into the knee joint of the left hindlimb. Sham animals were injected with saline. After the animals were allowed to recover in a warm environment, they were returned to their home cages.

[0198] Animals develop an inflammatory response after MIA, guard the affected area, and may lick it. Therefore, the rats were carefully monitored for signs of pain or unexpected severe pain, and as a result, animals showing such signs could be immediately sorted.

[0199] The animals were weighed daily for the first week and then every few days thereafter. Weight bearing was evaluated for the development of chronic pain on days 3, 7, 10, and 14 after MIA injection. On day 18, weight bearing measurements were taken, the animals were classified, and the treatment groups were randomized according to the MIA window in a Latin square design.

[0200] Antibody administration regimen: On day 18, the animals were treated with Par0067 (PAR2+ve) 10 mg / kg or isotype control (PAR2-ve) 10 mg / kg by intravenous injection, and weight bearing measurements were taken again 4 hours after antibody administration and on days 1, 2, 6, 8, 10, and 14.

[0201] Pregabalin and celecoxib administration regimen: The animals were administered PGB (30 mg / kg orally; 2 ml / kg) or celecoxib (50 mg / kg orally; 2 ml / kg) daily on days 24, 25, 26, 27, and 28 after MIA injection. Weight bearing evaluations were performed 1 hour after administration on days 24, 26, and 28, and further recordings were taken on day 32 after the drug treatment was discontinued.

[0202] On days 1, 2, 6, 10, and 14 after administration, after weight-bearing evaluation, 400 μl of blood was collected from the antibody and isotype control treatment groups (n = 5 / group) via the tail vein for PK analysis.

[0203] Test evaluation: Weight-bearing (g) records were taken on both the left and right hind paws, and the difference was calculated. The data were expressed as the % ratio ipsilateral / contralateral ((weight-bearing left / weight-bearing right) × 100) (mean ± s.e.m.).

[0204] Calculation: Measured value ipsilateral / measured value contralateral × 100. The difference in weight-bearing of the untreated - the difference in weight-bearing before administration was defined as the MIA window.

[0205] Statistical analysis: Planned comparison tests following repeated measures ANOVA using InVivoStat (invivostat.co.uk), (p < 0.05 was considered significant). Data were analyzed by comparing the treatment groups with the vehicle control group at each time point.

[0206] As detected by the change in weight-bearing between the injured and uninjured hind paws, injection of 2 mg of MIA into the knee joint clearly caused significant inflammation and hypersensitivity responses from day 3. This MIA-induced hypersensitivity response remained evident in all groups until day 18 (and thereafter for the vehicle-treated control animals), at which point the first test agent was administered. Injection of saline had no effect on weight-bearing.

[0207] As demonstrated in Figure 7A, after daily administration of pregabalin (30 mg / kg) from day 24 to day 28, a significant and notable recovery of hypersensitivity was seen, and the weakly residual effect was still evident on day 32 after the prior cessation of treatment. In contrast, daily administration of celecoxib (50 mg / kg) from day 24 to day 28 showed only a weak recovery of hypersensitivity at best. This pharmacological profile suggests that the hypersensitivity observed during this period of the MIA response is primarily neurogenic rather than inflammatory in nature.

[0208] As further demonstrated in Figure 7A, a significant recovery of hypersensitivity was seen with Par0067 from 4 hours to 28 days (10 days after dosing). No effect was seen with the isotype control during the same period. Figure 7B shows the effect of treatment with different concentrations of Par0067.

[0209] Example 4: Effect of the PAR2 antibody, PaB670129, on the recovery of mechanically induced allodynia in female C57BL / 6 mice Introduction Partial ligation of the sciatic nerve (PNL), as described by Seltzer (1990), is one of several nerve ligation models reported to function as a preclinical model of neuropathic pain. This results in severe mechanical allodynia that can be measured using an analgesy meter described by Randall and Selitto (1957). This example describes the effect of administration of the anti-PAR2 antibody PaB670129 on allodynia in this nerve injury / neuropathic pain model.

[0210] Procedure Sixty female C57BL / 6 mice received transponder implantation for identification purposes at least 5 days before the start of the study. Mechanical hyperalgesia was determined using an analgesia measurement device (Randall and Selitto 1957) (Ugo Basile). Force was applied sequentially to the dorsum of each hind paw, increasing the force until a withdrawal response was observed. At this point, force application was stopped and the body weight was recorded in grams. Data were expressed as withdrawal thresholds in grams for the ipsilateral and contralateral paws. After baseline recordings were established, the mice were divided into two groups with approximately equal ipsilateral / contralateral ratios that either underwent surgery to partially ligate the sciatic nerve or served as sham-operated controls. Mice undergoing surgery were anesthetized with isoflurane. Subsequently, approximately 1 cm of the left sciatic nerve was exposed by blunt dissection through an incision at the mid-thigh position. Next, a suture (8 / 0 Virgin Silk: Ethicon) was passed through the third of the dorsal nerves and tightly tied. Next, the incision was closed using an adhesive, and the mice were allowed to recover for at least 6 days before the start of the study. Sham-operated mice received the same protocol, but after nerve exposure, the mice were sutured and allowed to recover.

[0211] Mice were tested for the development of hyperalgesia on days 7 and 10 after surgery. Any mice showing an ipsilateral / contralateral ratio greater than 80% were classified as non-responders and excluded from the study. After the day 10 test, the mice were divided into the final treatment groups: A. Group 1: Sham surgery + isotype control 10 mg / kg subcutaneous injection (N = 10) B. Group 2: Nerve ligation + isotype control 10 mg / kg subcutaneous injection (N = 9) C. Group 3: Nerve ligation + etanercept 0.3 mg / kg subcutaneous injection (N = 9) D. Group 4: Nerve ligation + PaB670129 3 mg / kg subcutaneous injection (N = 9) E. Group 5: Nerve ligation + PaB670129 10 mg / kg subcutaneous injection (N = 9) F. Group 6: Nerve ligation + PaB670129 50 mg / kg subcutaneous injection (N = 9) and further subdivided into the indicated groups.

[0212] Mice were administered a control or test molecule diluted in phosphate buffered saline (PBS) on day 13, and re-tested for changes in mechanical hyperalgesia 4 hours after administration and on days 1, 2, 4, and 7 after administration.

[0213] Data analysis Ipsilateral and contralateral recordings were obtained for each animal at each test time point. Weight bearing by the ipsilateral and contralateral hind limbs was expressed as a ratio, and group data were analyzed using two-way ANOVA (PRISM), and pairwise comparisons were made using the Tukey method as needed.

[0214] Results Partial ligation of the sciatic nerve resulted in mechanical hyperalgesia, which manifested as a significant decrease in the ipsilateral / contralateral ratio on days 7 and 10 when compared to sham-operated controls. After treatment with an isotype control, the operated mice showed no change from pre-treatment levels in the level of mechanical hyperalgesia, indicating a lack of effect. Administration of the internal standard drug etanercept (0.3 mg / kg subcutaneous injection) resulted in a significant recovery of hyperalgesia from 4 hours after administration to day 7, consistent with results seen in previous studies. PaB670129 dose-dependently resulted in recovery of the ipsilateral / contralateral ratio, with peak effects seen at both 10 mg / kg and 50 mg / kg. The lowest dose of 3 mg / kg was significant on days 1, 2, and 7 after administration but showed a lower effect (see Figure 8).

[0215] Partial ligation of the sciatic nerve induced long-term mechanical hyperalgesia, consistent with previously reported results. Without being bound by theory, this is thought to serve as a preclinical correlate of pain observed in neuropathic pain. Administration of PaB670129 demonstrated a significant and dose-dependent recovery of this hyperalgesia, indicating a promising use of PAR2 antibodies in the treatment of neuropathic pain.

[0216] References: · Persic, L., et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 9 - 18 (1997). · Reikofski J and Tao BY (1992) Polymerase chain reaction (PCR) techniques for site - directed mutagenesis. Biotechnol Adv, 10(4):535 - 547. · Bove SE, Calcaterra SL, Brooker RM, Huber CM, Guzman RE, Juneau PL, et al. Weight bearing as a measure of disease progression and efficacy of anti - inflammatory compounds in a model of monosodium iodoacetate - induced osteoarthritis. Osteoarthritis Cartilage 2003;11(11):821 - 30. eng. · Fernihough J, Gentry C, Malcangio M, Fox A, Rediske J, Pellas T, et al. Pain related behaviour in two models of osteoarthritis in the rat knee. Pain 2004;112(1 - 2):83 - 93. eng. · Kalbhen DA. Chemical model of osteoarthritis--a pharmacological evaluation. J Rheumatol 1987;14 Spec No:130 - 1. eng. · Clark RA,Shoaib M,Hewitt KN,Stanford SC,Bate ST(2012). A comparison of InVivoStat with other statistical software packages for analysis of data generated from animal experiments,J Psychopharmacology,26(8),1136-1142. · Myska Improving Biosensor Analysis.Journal of Molecular Recognition.1999 · D.G.Myska Improving Biosensor Analysis.Journal of Molecular Recognition.1999;12:279-284. · Myska DG,Improving Biosensor Analysis.Journal of Molecular Recognition.1999;12:279-284. · A.W.Drake,M.L.Tang,G.A.Papalia,G.Landes,M.Haak-Frendscho,S.L.Klakamp,Biacore surface matrix effects on the binding kinetics and affinity of an antigen / antibody complex,Anal.Biochem.429(2012)58-69 · Pace CN,Vajdos F,Fee L,Grisley G and Grey T,How to measure and predict the molar absorption coefficient of a protein,Protein Sci.1995;4:2411-2423. · Spooner J, Keen J, Nayyar K, Birkett N, Bond N, Bannister D, Tigue N, Higazi D, Kemp B, Vaughan T, Kippen A, Buchanan A. (2015) Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab. Biotechnol Bioeng. 112:1472-7. · Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W, Field R (2014) A High yielding CHO transient system: co-expression of genes encoding EBNA-1 and GS enhances transient protein expression. Biotechnol Prog. 30(1):132-41. · Mach H, Middaugh CR, Lewis RV (1992) Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal. Biochem. 200(1):74-80. · Seltzer Z, Dubner R, Shir Y (1990). A novel behavioural model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 43:205-218 · Randall LO, Selitto JJ (1957). A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther. 111(4):409-419

[0217] Sequence Listing:

[0218]

Table 17

[0219]

Table 18

[0220]

Table 19

[0221]

Table 20

[0222]

Table 21

[0223]

Table 22

[0224]

Table 23

[0225]

Table 24

[0226]

Table 25

[0227]

Table 26

[0228]

Table 27

[0229]

Table 28

[0230]

Table 29

[0231]

Table 30

[0232]

Table 31

[0233]

Table 32

[0234]

Table 33

[0235]

Table 34

[0236] Array No. 801 - Human PAR2 Preproprotein (Genbank Accession No. NP_005233.3)

Chem.

Chem.

Chem.

Chem.

[0237] Incorporation by reference All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference.

[0238] Although specific embodiments of the present disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will be apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the present disclosure should be determined by reference to the claims in their full scope, along with their equivalents, and to this specification in light of such variations.

Claims

1. An antibody or antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH is: i) VH-CDR1 having the amino acid sequence of SEQ ID NO: 3, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 3; ii) VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 4; and iii) VH-CDR3 having the amino acid sequence of SEQ ID NO: 5, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 5; and the VL is: i) VL-CDR1 having the amino acid sequence of SEQ ID NO: 8, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 8; ii) VL-CDR2 having the amino acid sequence of SEQ ID NO: 9, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 9; and iii) VL-CDR3 having the amino acid sequence of SEQ ID NO: 10, but optionally having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions in the sequence of SEQ ID NO: 10; wherein the amino acid substitutions, deletions, or insertions, when tested at a pH of 7.4 in a PAR2 binding assay, reduce the binding affinity of the antibody or antigen-binding fragment thereof for human PAR2 by 1000, 800, 700, 500, 400, 300, 200, 100, 50, or 10-fold or less compared to an antibody or antigen-binding fragment having a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO:

7. An antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid substitutions, deletions, or insertions include conservative substitutions.

3. An antibody or antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH is: i) VH-CDR1 having the amino acid sequence of SEQ ID NO: 3; ii) VH-CDR2 having the amino acid sequence of SEQ ID NO: 4, but optionally having histidine present at any one or more of the amino acid positions corresponding to positions 1 to 17 of SEQ ID NO: 4; and iii) It includes a VH - CDR3 having the amino acid sequence of SEQ ID NO: 5, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 8 of SEQ ID NO: 5; and said VL is: i) a VL - CDR1 having the amino acid sequence of SEQ ID NO: 8; ii) a VL - CDR2 having the amino acid sequence of SEQ ID NO: 9; and iii) an antibody or an antigen - binding fragment thereof that includes a VL - CDR3 having the amino acid sequence of SEQ ID NO: 10, wherein histidine is optionally present at any one or more of the amino acid positions corresponding to positions 1 to 14 of SEQ ID NO:

10. **Claim 4** Said VH is: i) a VH - CDR1 having the amino acid sequence of SEQ ID NO: 3, ii) a VH - CDR2 having the amino acid sequence of SEQ ID NO: 4, iii) includes a VH - CDR3 having the amino acid sequence of SEQ ID NO: 5, and said VL is: i) a VL - CDR1 having the amino acid sequence of SEQ ID NO: 8, ii) a VL - CDR2 having the amino acid sequence of SEQ ID NO: 9, iii) includes a VL - CDR3 having the amino acid sequence of SEQ ID NO: 10, The antibody or an antigen - binding fragment thereof according to claim 3. **Claim 5** Said VH is: i) a VH - CDR1 having the amino acid sequence of SEQ ID NO: 13, ii) a VH - CDR2 having the amino acid sequence of SEQ ID NO: 14, iii) includes a VH - CDR3 having the amino acid sequence of SEQ ID NO: 15, and said VL is: i) a VL - CDR1 having the amino acid sequence of SEQ ID NO: 18, ii) a VL - CDR2 having the amino acid sequence of SEQ ID NO: 19, iii) includes a VL - CDR3 having the amino acid sequence of SEQ ID NO: 20, The antibody or an antigen - binding fragment thereof according to claim 3. **Claim 6** Histidine is present at the amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO: 4; and histidine is present at the amino acid positions corresponding to positions 2 and 3 of SEQ ID NO:

5. The antibody or an antigen - binding fragment thereof according to claim 3. **Claim 7** Histidine is present at the amino acid position corresponding to position 5 of SEQ ID NO:

4. The antibody or an antigen - binding fragment thereof according to any one of claims 3 or 5 - 6. **Claim 8** Histidine is present at the amino acid position corresponding to position 7 of SEQ ID NO:

4. The antibody or an antigen - binding fragment thereof according to any one of claims 3 or 5 - 7. **Claim 9** Histidine is present at the amino acid position corresponding to position 8 of SEQ ID NO:

4. The antibody or an antigen - binding fragment thereof according to any one of claims 3 or 5 - 7. **Claim 10** The antibody or antigen-binding fragment thereof according to any one of claims 1 or 5 to 7, wherein histidine is present at the amino acid position corresponding to position 12 of SEQ ID NO:

4.

11. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 10, wherein histidine is present at the amino acid position corresponding to position 15 of SEQ ID NO:

4.

12. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 11, wherein histidine is present at the amino acid position corresponding to position 16 of SEQ ID NO:

4.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 5 to 11, wherein histidine is present at the amino acid position corresponding to position 17 of SEQ ID NO:

4.

14. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 11, wherein histidine is present at the amino acid positions corresponding to positions 5 and 8 of SEQ ID NO:

4.

15. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 11, wherein histidine is present at the amino acid positions corresponding to positions 5, 8, 12, and 16 of SEQ ID NO:

4.

16. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 11, wherein histidine is present at the amino acid positions corresponding to positions 5, 8, 12, 16, and 17 of SEQ ID NO:

4.

17. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 16, wherein histidine is present at the amino acid position corresponding to position 2 of SEQ ID NO:

5.

18. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 17, wherein histidine is present at the amino acid position corresponding to position 3 of SEQ ID NO:

5.

19. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 18, wherein histidine is present at the amino acid positions corresponding to positions 2 and 3 of SEQ ID NO:

5.

20. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 19, wherein histidine is present at the amino acid position corresponding to position 1 of SEQ ID NO:

10.

21. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 20, wherein histidine is present at the amino acid position corresponding to position 5 of SEQ ID NO:

10.

22. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 21, wherein histidine is present at the amino acid position corresponding to position 6 of SEQ ID NO:

10.

23. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 22, wherein histidine is present at the amino acid position corresponding to position 14 of SEQ ID NO:

10.

24. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, 544, 554, 564, 574, 584, 594, 604, 614, 624, 634, 644, 654, 664, 674, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774, 784, 794, and 811 to 818.

25. The antibody or antigen-binding fragment thereof according to claim 3 or 24, wherein the VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, and 819 to 820.

26. The antibody or antigen-binding fragment thereof according to any one of claims 3, 24 or 25, wherein the VL-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 and 800.

27. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 14; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO:

20.

28. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 811; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 819; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20.

29. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 814; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 820; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20.

30. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 816; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20.

31. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH-CDR2 comprises an amino acid sequence corresponding to SEQ ID NO: 818; the VH-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 15; and the VL-CDR3 comprises an amino acid sequence corresponding to SEQ ID NO: 10 or 20.

32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the VH comprises a framework region that is at least 90% identical to each of SEQ ID NOs: 803 to 806.

33. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the VH comprises a framework region that is at least 95% identical to each of SEQ ID NOs: 803 to 806.

34. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the VH comprises a framework region corresponding to SEQ ID NOs: 803 to 806.

35. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the VL comprises a framework region that is at least 90% identical to each of SEQ ID NOs: 807 to 810.

36. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the VL comprises a framework region that is at least 95% identical to each of SEQ ID NOs: 807 to 810.

37. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the VL comprises a framework region corresponding to SEQ ID NOs: 807 to 810.

38. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 562, 572, 582, 592, 602, 612, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 762, 772, 782, 792 and 821-831.

39. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, 397, 407, 417, 427, 437, 447, 457, 467, 477, 487, 497, 507, 517, 527, 537, 547, 557, 567, 577, 587, 597, 607, 617, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727, 737, 747, 757, 767, 777, 787, and 797.

40. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence corresponding to SEQ ID NO: 821 and the VL comprises an amino acid sequence corresponding to SEQ ID NO: 7 or 17.

41. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence corresponding to SEQ ID NO: 824 and the VL comprises an amino acid sequence corresponding to SEQ ID NO: 7 or 17.

42. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence corresponding to SEQ ID NO: 827 and the VL comprises an amino acid sequence corresponding to SEQ ID NO: 7 or 17.

43. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence corresponding to SEQ ID NO: 831 and the VL comprises an amino acid sequence corresponding to SEQ ID NO: 7 or 17.

44. The antibody or antigen-binding fragment thereof according to any one of claims 3 to 39, wherein the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 12, and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO:

17.

45. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 44, wherein the antibody or antigen-binding fragment is an antigen-binding fragment.

46. The antibody or antigen-binding fragment thereof according to claim 45, wherein the antigen-binding fragment is scFv.

47. The antibody or antigen-binding fragment thereof according to claim 45, wherein the antigen-binding fragment is Fab'.

48. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 44, wherein the antibody or antigen-binding fragment is an antibody.

49. The antibody or antigen-binding fragment thereof according to claim 48, wherein the antibody is a monoclonal antibody.

50. The antibody or antigen-binding fragment thereof according to claim 48 or 49, wherein the antibody is an IgG antibody.

51. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 50, wherein the antibody or antigen-binding fragment is humanized.

52. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 50, wherein the antibody or antigen-binding fragment is human.

53. The antibody or antigen-binding fragment according to any one of claims 1 to 52, wherein the VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841.

54. The antibody or antigen-binding fragment according to claim 53, wherein the VH is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841.

55. The antibody or antigen-binding fragment according to claim 53, wherein the VH is encoded by a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841.

56. The antibody or antigen-binding fragment according to any one of claims 1 to 55, wherein the VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.

57. The antibody or antigen-binding fragment according to claim 56, which is encoded by a nucleic acid comprising a nucleotide sequence in which the VL is at least 95% identical to any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.

58. The antibody or antigen-binding fragment according to claim 56, which is encoded by a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.

59. The antibody or antigen-binding fragment according to any one of claims 1 to 58, which is encoded by a nucleic acid comprising a nucleotide sequence in which the VH is at least 90% identical to SEQ ID NO:

11.

60. The antibody or antigen-binding fragment according to claim 59, which is encoded by a nucleic acid comprising a nucleotide sequence in which the VH is at least 95% identical to SEQ ID NO:

11.

61. The antibody or antigen-binding fragment according to claim 59, which is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:

11.

62. The antibody or antigen-binding fragment according to any one of claims 1 to 61, wherein the VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO:

16.

63. The antibody or antigen-binding fragment according to claim 62, wherein the VL is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO:

16.

64. The antibody or antigen-binding fragment according to claim 62, wherein the VL is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:

16.

65. The antibody or antigen-binding fragment according to any one of claims 1 to 64, wherein the antibody or antigen-binding fragment binds to PAR2.

66. The antibody or antigen-binding fragment according to any one of claims 1 to 65, wherein the antibody or antigen-binding fragment inhibits the interaction of trypsin, tryptase, and / or matriptase with PAR2.

67. The antibody or antigen-binding fragment according to any one of claims 1 to 66, wherein the antibody or antigen-binding fragment inhibits the cleavage of PAR2 by trypsin, tryptase, and / or matriptase.

68. The antibody or antigen-binding fragment according to any one of claims 1 to 67, wherein the antibody or antigen-binding fragment inhibits the cleavage of the extracellular domain of PAR2.

69. The antibody or antigen-binding fragment according to any one of claims 1 to 68, wherein the antibody or antigen-binding fragment inhibits the exposure of the tethered ligand.

70. The antibody or antigen-binding fragment according to any one of claims 1 to 69, wherein the antibody or antigen-binding fragment inhibits the interaction of the tethered ligand with the second transmembrane loop of PAR2.

71. The antibody or antigen-binding fragment according to any one of claims 1 to 70, wherein the antibody or antigen-binding fragment binds to PAR2 with a higher affinity at pH 7.4 than at pH 6.

0.

72. The antibody or antigen-binding fragment has a K of less than about 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 650 pM, 600 pM, 500 pM, 200 pM, 100 pM or 50 pM Ｄ and binds to PAR2 at pH 7.4, the antibody or antigen-binding fragment according to any one of claims 1 to 70.

73. The antibody or antigen-binding fragment has a K of less than about 700 pM Ｄ and binds to PAR2 at pH 7.4, the antibody or antigen-binding fragment according to claim 72.

74. The antibody or antigen-binding fragment has a K greater than about 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM, or 100 nM and binds to PAR2 at pH 6.

0. The antibody or antigen-binding fragment according to any one of claims 1 to 70. Ｄ ​

75. The antibody or antigen-binding fragment has a K greater than about 30 nM Ｄ and binds to PAR2 at pH 6.0, the antibody or antigen-binding fragment according to any one of claims 1 to 70.

76. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 7.4 in a PAR2 binding assay, an IC of less than 100 nM ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 75, having.

77. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 7.4 in a PAR2 binding assay, an IC of less than 50 nM ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 76, having.

78. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 7.4 in a PAR2 binding assay, an IC ５０ is provided, the antibody or antigen-binding fragment according to any one of claims 1 to 77.

79. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 6.0 in a PAR2 binding assay, an IC greater than 500 nM ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 78, having

80. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 6.0 in a PAR2 binding assay, it has an IC greater than 1000 nM. ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 79, having the above.

81. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 at pH 6.0 in a PAR2 binding assay, an IC greater than 1100 nM ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 80, having.

82. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay, an IC that is more than 20-fold lower at pH 7.4 than at pH 6.0 ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 81, having.

83. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay, the IC is more than 25-fold lower at pH 7.4 than at pH 6.0 ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 82, having the same.

84. When the antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment having the CDRs of SEQ ID NOs: 3-5 and 8-10 in a PAR2 binding assay, having an IC that is more than 30-fold lower at pH 7.4 than at pH 6.0 ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 83.

85. The antibody or antigen-binding fragment has an IC －１０ 50 less than 3.0×10 ５０ M in the calcium influx assay in human A549 cells, and the antibody or antigen-binding fragment according to any one of claims 1 to 84.

86. The antibody or antigen-binding fragment has an IC －１０ of less than 1.5 × 10 ５０ M in a calcium influx assay in human A549 cells. The antibody or antigen-binding fragment according to any one of claims 1 to 85.

87. The antibody or antigen-binding fragment has an IC of less than 7.0×10 －１０ M in a calcium influx assay in rat KNKR cells ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 86.

88. The antibody or antigen-binding fragment has an IC －１０ of less than 5.5×10 －１０ M in a calcium influx assay in rat KN-RK cells ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 87.

89. The antibody or antigen-binding fragment has an IC －１１ 50 less than 7.0×10 ５０ M in a calcium influx assay in cynomolgus CYNO-K1 cells. The antibody or antigen-binding fragment according to any one of claims 1 to 88.

90. The antibody or antigen-binding fragment has an IC －１１ of less than 5.0×10 －１１ M in a calcium influx assay in cynomolgus CYNO-K1 cells ５０ ５０ and is the antibody or antigen-binding fragment according to any one of claims 1 to 89.

91. The antibody or antigen-binding fragment has an IC －１１ in a calcium influx assay in mouse LL / 2 cells of less than 6.0×10 －１１ M, and is the antibody or antigen-binding fragment according to any one of claims 1 to 90. ５０ ​

92. The antibody or antigen-binding fragment has an IC of less than 4.0×10 －１１ M in a calcium influx assay in mouse LL / 2 cells ５０ The antibody or antigen-binding fragment according to any one of claims 1 to 91.

93. The antibody or antigen-binding fragment according to any one of claims 92, wherein the antibody or antigen-binding fragment lacking histidine modification disappears from the serum of the treated patient more slowly than an antibody or antigen-binding fragment lacking histidine modification.

94. When the optionally present histidine or histidines are tested at a pH of 7.4 in a PAR2 binding assay, the binding affinity of the antibody or antigen-binding fragment for human PAR2 is 1000, 800, 700, 500, 400, 300, 200, 100, 50 or 10 times lower than that of an antibody or antigen-binding fragment having a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO:

7. The antibody or antigen-binding fragment thereof according to any one of claims 3 or 5 to 93.

95. A nucleic acid capable of expressing the antibody or antigen-binding fragment according to any one of claims 1 to 94.

96. A nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833-841.

97. The nucleic acid according to claim 96, comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841.

98. The nucleic acid according to claim 96, comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 481, 491, 501, 511, 521, 531, 541, 551, 561, 571, 581, 591, 601, 611, 621, 631, 641, 651, 661, 671, 681, 691, 701, 711, 721, 731, 741, 751, 761, 771, 781, 791, and 833 - 841.

99. A nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.

100. The nucleic acid according to claim 99, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.

101. The nucleic acid according to claim 99, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 486, 496, 506, 516, 526, 536, 546, 556, 566, 576, 586, 596, 606, 616, 626, 636, 646, 656, 666, 676, 686, 696, 706, 716, 726, 736, 746, 756, 766, 776, 786, and 796.

102. A nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO:

11.

103. The nucleic acid according to claim 102, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

11.

104. The nucleic acid according to claim 102, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

11.

105. A nucleic acid comprising a nucleotide sequence that is at least 90% identical to SEQ ID NO:

16.

106. The nucleic acid according to claim 105, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

16.

107. The nucleic acid according to claim 105, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

16.

108. A vector comprising the nucleic acid according to any one of claims 95 to 107.

109. A set of vectors comprising: a) the nucleic acid according to any one of claims 96 to 98 or 102 to 104; and b) the nucleic acid according to any one of claims 99 to 101 and 105 to 107.

110. A host cell comprising one or more of the vectors according to claim 108 or 109.

111. A composition comprising a pharmaceutically acceptable carrier and the antibody or antigen-binding fragment according to any one of claims 1 to 94.

112. A lyophilized composition comprising the antibody or its antigen-binding fragment according to any one of claims 1 to 94.

113. A reconstituted lyophilized composition comprising the antibody or its antigen-binding fragment according to any one of claims 1 to 94.

114. The composition according to any one of claims 111 to 113, formulated for administration by buccal, spray, oral administration, delayed release or sustained release, transmucosal administration, syrup, mucoadhesion, buccal formulation, mucoadhesive tablets, topical administration, parenteral administration, injection, subcutaneous administration, oral solution, rectal administration, buccal administration or transdermal administration.

115. A kit comprising an antibody or antigen-binding fragment according to any one of claims 1 to 94 or a composition according to any one of claims 111 to 114.

116. A method for treating pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 94.

117. A method for treating pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 111 to 114.

118. The method according to claim 116 or 117, wherein the pain is selected from the group consisting of nociceptive pain, neuropathic pain, and mixed pain.

119. The method according to any one of claims 116 or 117, wherein the pain is associated with headache, chronic headache, migraine, cancer, viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease, liver disease, multiple sclerosis, spinal cord injury, postherpetic neuralgia, diabetic neuropathy, low back pain, inflammatory heart disease, kidney disease, gastritis, gingivitis, periodontal disease, asthma, chronic obstructive pulmonary disease, autoimmune disease, irritable bowel syndrome, fibromyalgia, lower limb pain, restless legs syndrome, diabetic neuropathy, allergic conditions, surgical procedures, acute or chronic physical injuries, fractures or contusions, spinal cord injury, inflammatory diseases, non-inflammatory neuropathic pain conditions or functional nociceptive pain conditions, or combinations thereof.

120. The method according to claim 119, wherein the pain is osteoarthritis pain.

121. The method according to any one of claims 116 to 120, wherein the subject is human.

122. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 94, comprising the steps of expressing a nucleic acid according to any one of claims 95 to 106 in cultured cells and purifying the antibody or antigen-binding fragment.

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