Anti-par2 antibodies

Abstract

The present disclosure provides antibodies and antigen-binding fragments capable of binding to human PAR2, including antibodies capable of acting as dual inhibitors of PAR2. The dual inhibitor antibodies are capable of inhibiting both protease cleavage mediated PAR2 activation (e.g., by trypsin) and peptide mediated PAR2 activation (e.g., by PAR2-AP or PAR1-AP). The present disclosure also provides methods for making and using the antibodies and antigen binding fragments.

Description

Technical Field

[0001] The present invention relates to an antibody or antigen binding fragment capable of binding to human PAR2. The present invention also relates to an antibody that specifically binds to an epitope of a human PAR2 receptor and blocks, antagonizes, inhibits or prevents activation of human PAR2. The present invention relates to a pharmaceutical composition comprising the antibody, and a preparation method and a use method thereof. Background Art

[0002] Chronic pain and chronic inflammation are two of the largest global health burdens. Chronic pain alone affects approximately 50 million American adults, or 20% of the population. Chronic pain is a debilitating condition defined as pain that persists and is experienced on most days or every day for 6 months or longer (https: / / uspainfoundation.org / wp-content / uploads / 2018 / 09 / Chronic-pain-facts-infographic.pdf). Chronic inflammation plays a central role in diseases that cause a large number of deaths, including cancer, cardiovascular disease, and diabetes. It is predicted that by 2020, chronic diseases will account for approximately three-quarters of all deaths worldwide (Helamo, Delil and Dileba, 2017). Despite the global burden of chronic pain, patients only receive 30% pain relief from currently available treatments (Rice, Smith and Blyth, 2016).

[0003] Protease activated receptor 2 (PAR2) is a G protein coupled receptor belonging to the protease activated receptor (PAR) family. PAR2 is considered to have a key role in inflammation, pain and other pathophysiological responses (wherein finding protease levels increase). PAR2 is widely expressed with a particularly high level in pancreas, liver, kidney, small intestine and colon. Moderate expression is detected in many epithelial and endothelial cells and organs, and the evidence of expression in brain or skeletal muscle is limited. In addition, PAR2 is also expressed in immune and inflammatory cells (such as T cells, monocytes, macrophages, neutrophils, mast cells and eosinophils).

[0004] The literature suggests that blockade of PAR2 may have clinical benefits in atopic dermatitis, asthma, cancer (multiple cancers including breast cancer, melanoma, head and neck cancer), pain (inflammatory, postoperative, neuropathic, fracture, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, lymphangioleiomyomatosis, allergic rhinoconjunctivitis (AR), eosinophilic esophagitis (EoE), and diseases related to epithelial barrier function (in Yau et al., 2013; Heuberger and (reviewed in Schuepbach, 2019).

[0005] Therefore, PAR2 antagonists are thought to potentially provide benefit to a wide variety of patients and may have the potential to alleviate pain and / or inflammation-related conditions. PAR2 is therefore viewed as a valuable therapeutic target for the treatment of several disease indications.

[0006] There is a need to identify therapeutic moieties that can specifically inhibit PAR2. It would be particularly desirable if such an agent could inhibit all PAR2 activation mechanisms. Summary of the invention

[0007] Antibodies and antigen-binding fragments thereof that bind to PAR2 are provided herein. The antibodies and antigen-binding fragments of the present disclosure are particularly useful for inhibiting PAR2-mediated signaling, and for treating diseases and disorders caused by or associated with PAR2 activity and / or signaling.

[0008] The antibodies or antigen-binding fragments thereof provided herein specifically bind to PAR2 and inhibit the activity of PAR2, wherein the antibodies or fragments thereof bind to epitopes comprising the extracellular loop (ECL) and N-terminal segment (comprising spirals 0 and 1) of PAR2. It is believed that binding to both of these regions can result in comprehensive functional inhibition of PAR2 activity. Therefore, the antibodies provided herein have dual activity, i.e., they can inhibit both: protease-mediated PAR2 activation (e.g., by trypsin) and peptide-mediated PAR2 activation (e.g., by PAR2-AP or PAR1-AP).

[0009] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a discontinuous epitope of PAR2, wherein the epitope comprises one or more regions of helix 0 / 1, the helix 0 / 1 region, and the non-helical segment 1 preceding ECL3, optionally wherein when numbered according to the human PAR2 sequence of SEQ ID NO: 1, the regions of segment 1, helix 0 / 1, and ECL3 are selected from V55-F77, L306-Y311, and F312-Y326 of PAR2.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the activity of PAR2 and comprises a VH domain, wherein the VH domain contains a HCDR3, wherein: (a) a HCDR3 comprising or consisting of: SEQ ID NO: 5, 22 or 30, or an amino acid sequence of SEQ ID NO: 5, 22 or 30 having 3, 2 or 1 amino acid substitutions therein; (b) a HCDR3 comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, 22 or 30; or d) a HCDR3 amino acid sequence that is defined according to Kabat or Chothia and is derived from a VH domain selected from SEQ ID NO: 2, 10, 13, 16, 19 or 27.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain, wherein the VH domain comprises: i. a HCDR1 amino acid sequence selected from SEQ ID NO: 3, 11, 14, 17, 20 or 28, optionally with 3, 2 or 1 amino acid substitutions therein; and / or ii. a HCDR2 amino acid sequence selected from SEQ ID NO: 4, 12, 15, 18, 21 or 29, optionally with 3, 2 or 1 amino acid substitutions therein.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL domain, optionally a VL domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6, 23 or 31.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a LCDR3, wherein a) the LCDR3 amino acid sequence is selected from SEQ ID NO: 9, 26 or 33, optionally wherein there are 3, 2 or 1 amino acid substitutions; or b) the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a VL domain according to SEQ ID NO: 6, 23 or 31, optionally wherein LCDR3 comprises 3, 2 or 1 amino acid substitutions.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL domain, wherein the VL domain comprises: a) i) a LCDR1 amino acid sequence of SEQ ID NO: 7, 24 or 32, optionally wherein there are 3, 2 or 1 amino acid substitutions; or ii) a LCDR1 amino acid sequence, which is derived from a VL domain according to SEQ ID NO: 6, 23 or 31 as defined by Chothia or Kabat, optionally wherein the LCDR1 sequence comprises 3, 2 or 1 amino acid substitutions; and / or b) i) a LCDR2 amino acid sequence of SEQ ID NO: 8 or 25, optionally wherein there are 3, 2 or 1 amino acid substitutions; or ii) a LCDR2 amino acid sequence, which is derived from a VL domain according to SEQ ID NO: 6, 23 or 31 as defined by Chothia or Kabat, optionally wherein the LCDR2 sequence comprises 3, 2 or 1 amino acid substitutions.

[0015] Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to PAR2, comprising a VH region selected from SEQ ID NO: 2, 10, 13 and 16, 19 or 27, or an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical thereto; and a VL region according to SEQ ID NO: 6, 23 or 31, or an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical thereto.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. HIn some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID 16. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID 19. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. L Area, where V L The region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, the antibody or antigen-binding fragment thereof comprises V L Area, where V L The region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Area, where V H The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 2, and V L The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Area, where V H The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 10, and VL The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Area, where V H The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 13, and V L The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Area, where V H The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 16, and V L The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Area, where V H The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 19, and V L The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 23. In some embodiments, the antibody or antigen-binding fragment thereof comprises V H Area, where V H The region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 27, and V L The region comprises an amino acid sequence that is identical to, or at least 90% identical to, SEQ ID NO:31.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V H The invention comprises: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; and wherein V LThe invention comprises: (d) LCDR1 having an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:9 containing 3, 2 or 1 amino acid substitutions.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V H comprising: (a) HCDR1 having an amino acid sequence of SEQ ID NO:11 or SEQ ID NO:11 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO:12 or SEQ ID NO:12 containing 3, 2 or 1 amino acid substitutions; and (c) HCDR3 having an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and wherein VL comprises: (d) LCDR1 having an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:9 containing 3, 2 or 1 amino acid substitutions.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V Hcomprising: (a) HCDR1 having an amino acid sequence of SEQ ID NO:14 or SEQ ID NO:14 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO:15 or SEQ ID NO:15 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and wherein VL comprises: (d) LCDR1 having an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:9 containing 3, 2 or 1 amino acid substitutions.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V H comprising: (a) HCDR1 having an amino acid sequence of SEQ ID NO:17 or SEQ ID NO:17 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO:18 or SEQ ID NO:18 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and wherein VL comprises: (d) LCDR1 having an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:9 containing 3, 2 or 1 amino acid substitutions.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V Hcomprising: (a) HCDR1 having an amino acid sequence of SEQ ID NO:20 or SEQ ID NO:20 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO:21 or SEQ ID NO:21 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO:22 or SEQ ID NO:22 containing 3, 2 or 1 amino acid substitutions; and wherein VL comprises: (d) LCDR1 having an amino acid sequence of SEQ ID NO:24 or SEQ ID NO:24 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO:25 or SEQ ID NO:25 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having an amino acid sequence of SEQ ID NO:26 or SEQ ID NO:26 containing 3, 2 or 1 amino acid substitutions.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V H comprising: (a) HCDR1 having an amino acid sequence of SEQ ID NO:28 or SEQ ID NO:28 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO:29 or SEQ ID NO:29 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO:30 or SEQ ID NO:30 containing 3, 2 or 1 amino acid substitutions; and wherein VL comprises: (d) LCDR1 having an amino acid sequence of SEQ ID NO:32 or SEQ ID NO:32 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO:25 or SEQ ID NO:25 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having an amino acid sequence of SEQ ID NO:33 or SEQ ID NO:33 containing 3, 2 or 1 amino acid substitutions.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 77. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 78. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 79. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 80. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 83. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 84. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 84.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises V H domain, where V HThe domain comprises: a) a HCDR3 amino acid sequence having SEQ ID NO:5 or SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO:3 or SEQ ID NO:3 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO:4 or SEQ ID NO:4 containing 3, 2 or 1 amino acid substitutions; b) a HCDR3 amino acid sequence having SEQ ID NO:5 or SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO:11 or SEQ ID NO:11 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO:12 or SEQ ID NO:12 containing 3, 2 or 1 amino acid substitutions; c) a HCDR3 amino acid sequence having SEQ ID NO:5 or SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR2 amino acid sequence having SEQ ID NO:13 or SEQ ID NO:14 containing 3, 2 or 1 amino acid substitutions; NO:14 or a HCDR1 amino acid sequence of SEQ ID NO:14 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence of SEQ ID NO:15 or a HCDR2 amino acid sequence of SEQ ID NO:15 containing 3, 2 or 1 amino acid substitutions; d) a HCDR3 amino acid sequence of SEQ ID NO:5 or a HCDR3 amino acid sequence of SEQ ID NO:5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence of SEQ ID NO:17 or a HCDR1 amino acid sequence of SEQ ID NO:17 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence of SEQ ID NO:18 or a HCDR2 amino acid sequence of SEQ ID NO:18 containing 3, 2 or 1 amino acid substitutions; e) a HCDR3 amino acid sequence of SEQ ID NO:22 or a HCDR3 amino acid sequence of SEQ ID NO:22 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence of SEQ ID NO:20 or a HCDR1 amino acid sequence of SEQ ID NO:20 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR NO:21 or a HCDR2 amino acid sequence of SEQ ID NO:21 containing 3, 2 or 1 amino acid substitutions; f) a HCDR3 amino acid sequence of SEQ ID NO:30 or a HCDR3 amino acid sequence of SEQ ID NO:30 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence of SEQ ID NO:28 or a HCDR1 amino acid sequence of SEQ ID NO:28 containing 3, 2 or 1 amino acid substitutions; and / or ii.A HCDR2 amino acid sequence having SEQ ID NO: 29 or SEQ ID NO: 29 containing 3, 2 or 1 amino acid substitutions; g) a HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V. H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 2 H domain and comprising 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO: 2. H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 2 H domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 2 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 2 H h) a HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 10 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 10 H domain and comprising 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 10. H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 10 H domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 10. H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 10 Hdomain and comprising 3, 2 or 1 amino acid substitutions; i) a HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 13 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 13 H domain and comprising 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 13. H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 13 H domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 13. H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 13 H j) a HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 16 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 16 H domain and comprising 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 16. H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 16 H domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 16. H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 16 Hk) a HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 19 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 19 H domain and comprising 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 19. H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 19 H domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 19. H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 19 H domain and comprising 3, 2 or 1 amino acid substitutions; 1) a HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 27 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 27 H domain and comprising 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 27. H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 27 H domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 27. H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 27 H domain and contain 3, 2 or 1 amino acid substitutions.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises V L domain, where V L The domain comprises a) a LCDR3 amino acid sequence having SEQ ID NO:9 or SEQ ID NO:9 containing 3, 2 or 1 amino acid substitutions; and i. a LCDR1 amino acid sequence having SEQ ID NO:7 or SEQ ID NO:7 containing 3, 2 or 1 amino acid substitutions; and / or ii. a LCDR2 amino acid sequence having SEQ ID NO:8 or SEQ ID NO:8 containing 3, 2 or 1 amino acid substitutions; b) a LCDR3 amino acid sequence having SEQ ID NO:26 or SEQ ID NO:26 containing 3, 2 or 1 amino acid substitutions; and i. a LCDR1 amino acid sequence having SEQ ID NO:24 or SEQ ID NO:24 containing 3, 2 or 1 amino acid substitutions; and / or ii. a LCDR2 amino acid sequence having SEQ ID NO:25 or SEQ ID NO:25 containing 3, 2 or 1 amino acid substitutions; c) a LCDR3 amino acid sequence having SEQ ID NO:33 or SEQ ID NO:33 containing 3, 2 or 1 amino acid substitutions; and i. a LCDR2 amino acid sequence having SEQ ID NO:34 or SEQ ID NO:34 containing 3, 2 or 1 amino acid substitutions. NO:32 or a LCDR1 amino acid sequence of SEQ ID NO:32 containing 3, 2 or 1 amino acid substitutions; and / or ii. a LCDR2 amino acid sequence of SEQ ID NO:25 or a LCDR2 amino acid sequence of SEQ ID NO:25 containing 3, 2 or 1 amino acid substitutions; d) a LCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO:6 L domain, or wherein the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 6 L domain and comprising 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO: 6 L domain, or wherein the LCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 6 L domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a LCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO: 6 Ldomain, or wherein the LCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 6 L domain and comprising 3, 2 or 1 amino acid substitutions; e) a LCDR3 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO: 23 L domain, or wherein the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 23 L domain and comprising 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence defined by Chothia or Kabat and derived from a V selected from SEQ ID NO: 23. L domain, or wherein the LCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 23 L domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a LCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO: 23. L domain, or wherein the LCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 23 L f) a LCDR3 amino acid sequence defined by Chothia or Kabat and derived from a V domain selected from SEQ ID NO: 31 L domain, or wherein the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 31 L domain and comprising 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence defined by Chothia or Kabat and derived from a V selected from SEQ ID NO: 31 L domain, or wherein the LCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 31 L domain and comprising 3, 2 or 1 amino acid substitutions; and / or ii. a LCDR2 amino acid sequence defined according to Chothia or Kabat and derived from a V selected from SEQ ID NO: 31. L domain, or wherein the LCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 31L domain and contain 3, 2 or 1 amino acid substitutions.

[0027] In a preferred embodiment, the antibodies of the invention are isolated or purified.

[0028] In some embodiments, the antibody or its antigen binding fragment inhibits the activation of PAR2 by PAR2 peptide. In some embodiments, the antibody or its antigen binding fragment specifically binds to PAR2 and inhibits the activity of PAR2, wherein the inhibition of PAR2 activity includes binding to segment 1, helix 0 / 1, and ECL3 of the PAR2 receptor. In some embodiments, the antibody or its antigen binding fragment specifically binds to PAR2 and inhibits PAR2 activation, wherein the inhibition of PAR2 activation includes inhibiting the binding of PAR2 bound ligands. In some embodiments, the antibody or its antigen binding fragment specifically binds to PAR2 and inhibits PAR2 activation, wherein the inhibition of PAR2 activation includes inhibiting the cross-activation of PAR1 bound ligands in PAR1-PAR2 heterodimers. In some embodiments, the antibody or its antigen binding fragment specifically binds to PAR2 and inhibits the binding of PAR2 activation peptides to PAR2. In some embodiments, the antibodies provided herein can inhibit the following two: protease cleavage-mediated PAR2 activation (e.g., by trypsin) and peptide-mediated PAR2 activation (e.g., by PAR2-AP or PAR1-AP).

[0029] In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope as the epitope specifically bound by an antibody or fragment selected from clones Y022065, Y022870, Y022877, Y022883, Y022054, and / or Y021171.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope, wherein the epitope to which the antibody or fragment binds is identified by hydrogen deuterium exchange (HDX) and / or by site-directed mutagenesis and flow cytometry.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of the PAR2 activating peptide to PAR2, wherein the antibody or fragment has an IC of 1 to 100 nM. 50 Inhibiting PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation, optionally wherein PAR2 peptide-mediated IP accumulation is determined using a PAR2 peptide-stimulated IP signaling assay.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated PAR2 activation. In some embodiments, the antibody or antigen-binding fragment thereof has an IC of 1 to 300 nM. 50 Inhibiting trypsin-mediated IP accumulation, optionally wherein trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof has an IC of 1 to 100 nM. 50 Inhibiting PAR2 activating peptide mediated inositol monophosphate (IP) accumulation, optionally wherein PAR2 peptide inhibition is determined using a HTRF assay.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits calcium mobilization mediated by the PAR2 activating peptide, optionally with an IC of 1 to 100 nM. 50 Inhibition, and optionally wherein calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization, optionally with an IC of 1 to 200 nM. 50 Inhibition, and optionally wherein calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof is not internalized into the cell upon binding to PAR2 on the cell surface, optionally wherein internalization is determined by quantifying antibody or fragment binding using FACs.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof does not inhibit ligand SFLLR-mediated PAR1 activation, wherein PAR1 activation is determined by an IP signaling assay stimulated with the ligand SFLLR.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof has an EC of 600 pM to 5 nM or less. 50 Binding to cynomolgus monkey PAR2, optionally wherein cynomolgus monkey PAR2 binding is determined using flow cytometry.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof has a K of 100 pM to 10 nM. D Binding to human PAR2, optionally wherein the binding affinity is determined using surface plasmon resonance (SPR) or KinExA.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of a PAR2 peptide to PAR2, wherein binding of the antibody or fragment to PAR2 is pH independent between pH 7.5 and 6.0.

[0041] In some embodiments, the antibody or its antigen binding fragment is not combined with PAR1, and optionally wherein PAR1 is combined with flow cytometry or ELISA to determine. In some embodiments, the antibody or its antigen binding fragment is not combined with PAR3, and optionally wherein PAR3 is combined with flow cytometry or ELISA to determine. In some embodiments, the antibody or its antigen binding fragment is not combined with PAR4, and optionally wherein PAR4 is combined with flow cytometry or ELISA to determine.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of a PAR2 peptide to PAR2, wherein 3 mg / kg of the antibody or fragment inhibits a leukocyte response induced by a PAR2 stimulator by >95% over a period of 30 days, wherein the inhibition is measured by determining a gene signature induced by the stimulator. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of a PAR2 peptide to PAR2, wherein 1 mg / kg of the antibody or fragment inhibits a leukocyte response induced by a PAR2 peptide by >90% over a period of 30 days, wherein the inhibition is measured by determining a gene signature induced by the stimulator.

[0043] In some embodiments, the antibody or antigen binding fragment thereof competes with the functional ligand AZ8838 for binding to PAR2. In some embodiments, the antibody or antigen binding fragment thereof competes directly with AZ8838 for binding to PAR2.

[0044] In some embodiments, the antibody or antigen binding fragment thereof binds to a PAR2 homodimer.In some embodiments, the antibody or antigen binding fragment thereof binds to a PAR2-PAR1 heterodimer, optionally wherein the binding inhibits cross-activation of PAR2 by a PAR1 bound ligand.

[0045] Provided herein are antibodies and antigen-binding fragments thereof that bind to PAR2 for use in therapy.

[0046] In some embodiments, the antibodies or antigen-binding fragments thereof are used to treat a PAR2-mediated disease or condition, such as atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fractures, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

[0047] In some embodiments, the antibodies or antigen-binding fragments thereof are used in the preparation of a medicament for treating a PAR2-mediated disease or condition, such as atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fractures, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

[0048] Provided herein are methods for treating a PAR2-mediated disease or condition (e.g., pain, optionally wherein the pain is independently selected from chronic pain, inflammatory pain, postoperative pain, neuropathic pain, fracture-related pain, gout-related pain, cancer-related pain, gastrointestinal pain associated with inflammatory bowel disease, etc.) in a patient, comprising administering to the patient (e.g., a human) a therapeutically effective amount of an antibody or fragment thereof of the present disclosure, wherein the PAR2-mediated disease or condition is thereby treated.

[0049] In some embodiments, the antibodies of the present disclosure may be combined or administered concurrently with an additional therapy, optionally wherein the additional therapy comprises one or more additional therapeutic agents independently selected from analgesics, including anti-inflammatory drugs (e.g., NSAIDS, including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g., codeine, morphine, oxycodone, fentanyl, buprenorphine), amitriptyline, gabapentin; Cancer drugs, including alkylating agents (e.g., nitrogen mustards, nitroureas), antimetabolites (e.g., folic acid analogs, pyrimidine and purine analogs), antibiotics and enzymes (e.g., actinomycin D, daunorubicin, doxorubicin, L-asparaginase), natural agents (e.g., vinca alkaloids, taxens, tecans), hormones and antagonists (e.g., progestins, estrogens, GnRH, antiestrogens), hydroxyurea, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecular targeted therapeutics (e.g., antibody-conjugated drugs), platinum-based therapeutics (e.g., cisplatin, carboplatin, oxaliplatin); and / or optionally wherein the additional treatment is selected from radiation therapy and / or surgical removal of the tumor.

[0050] Provided herein are pharmaceutical compositions comprising an antibody or fragment of the present disclosure and a pharmaceutically acceptable excipient, diluent or carrier, and optionally further comprising one or more additional therapeutic agents independently selected from the group consisting of analgesics, including anti-inflammatory drugs (e.g., NSAIDS, including aspirin, ibuprofen, diclofenac, naproxen), acetaminophen, opioids (e.g., codeine, morphine, oxycodone, fentanyl, buprenorphine), amitriptyline, gabapentin; anticancer drugs, including alkylating agents (e.g., nitrogen mustard, nitroureas) , antimetabolites (e.g., folate analogs, pyrimidine and purine analogs), antibiotics and enzymes (e.g., actinomycin D, daunorubicin, doxorubicin, L-asparaginase), natural agents (e.g., vinca alkaloids, paclitaxel, tecans), hormones and antagonists (e.g., progestins, estrogens, GnRH, antiestrogens), hydroxyurea, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecular targeted therapeutics (e.g., antibody-conjugated drugs), platinum-based therapeutics (e.g., cisplatin, carboplatin, oxaliplatin).

[0051] Provided herein are pharmaceutical compositions of the disclosure or kits comprising the same, wherein the compositions are used to treat a PAR2-mediated disease or condition, e.g., selected from atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fractures, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

[0052] Provided herein are pharmaceutical compositions of the disclosure, or kits of the disclosure, in combination with a label or instructions for treating a disease or condition in a patient; optionally wherein the label or instructions include a marketing authorization number (e.g., an FDA or EMA authorization number); optionally wherein the kit comprises an IV or injection device containing the antibody or fragment.

[0053] In some embodiments, amino acid replacement comprises homologous replacement. These amino acid replacements can be conservative replacements. Conservative amino acid replacement refers to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, the predicted non-essential amino acid residues in the anti-PAR-2 ​​antibody are replaced by other amino acid residues from the same side chain family. Methods for identifying conservative substitutions of amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0054] In some embodiments, the amino acid substitutions reduce the binding affinity of the antibody or antigen-binding fragment thereof for human PAR2 by no more than 1000-fold, 800-fold, 700-fold, 500-fold, 400-fold, 300-fold, 200-fold, 100-fold, 50-fold, or 10-fold compared to an antibody or antigen-binding fragment having a VH with an amino acid sequence of SEQ ID NO: 16 and a VL with an amino acid sequence of SEQ ID NO: 6 when tested in a PAR2 binding assay (e.g., an SPR or Kinexa assay) at a pH of 7.4 to 7.6.

[0055] The antibodies provided herein can be human. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody or its antigen binding fragment is an antigen binding fragment. In one embodiment, the antigen binding fragment is a scFv. In some embodiments, the antigen binding fragment is a Fab. In one embodiment, the antibody or its antigen binding fragment is humanized.

[0056] The antibodies provided herein bind to human PAR2, and may also bind to Cynomolgus PAR2 and Rhesus PAR2, but not to mouse and / or rat PAR2.

[0057] In some embodiments, the antibody or its antigen binding fragment prevents trypsin, tryptase and / or protease from interacting with PAR2. In some embodiments, the antibody or its antigen binding fragment inhibits PAR2 activation of trypsin. In some embodiments, the antibody or its antigen binding fragment inhibits exposure of bound ligands. In some embodiments, the antibody or its antigen binding fragment prevents bound ligands from interacting with PAR2.

[0058] In some embodiments, provided herein are nucleic acids capable of expressing antibodies or antigen-binding fragments thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 34, 70, and 72. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 34, 70, and 72. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 35, 36, 37, 38, 71, and 73. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 35, 36, 37, 38, 71, and 73. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 35, 36, 37, 38, 71, and 73. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 35, 36, 37, 38, 71, and 73. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 34. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 34. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 34. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 38. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 38. In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 38.

[0059] In a preferred embodiment, the nucleic acids of the invention are isolated or purified.

[0060] In some embodiments, the disclosure provides vectors comprising any of the nucleic acids disclosed herein. In some embodiments, the disclosure provides a collection of vectors comprising any one or more of the nucleic acids disclosed herein.

[0061] In some embodiments, the disclosure provides host cells comprising any one or more of the vectors disclosed herein.

[0062] In some embodiments, the present disclosure provides such a composition comprising a pharmaceutically acceptable carrier and any antibody or antigen-binding fragment disclosed herein. In some embodiments, the present disclosure provides such a lyophilized composition comprising any antibody or antigen-binding fragment thereof disclosed herein.

[0063] In some embodiments, the present disclosure provides such a reconstituted lyophilized composition comprising any antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the composition is formulated for administration by: tablets, sprays, oral administration, delayed release or sustained release, transmucosal administration, syrups, mucoadhesives, buccal preparations, mucoadhesive tablets, topical administration, parenteral administration, injection, subcutaneous administration, oral solution, rectal administration, buccal administration, or transdermal administration.

[0064] In some embodiments, the present disclosure provides a kit comprising any antibody or antigen-binding fragment disclosed herein, or any composition disclosed herein.

[0065] In some embodiments, the present disclosure provides a method for treating pain in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of any composition disclosed herein. In some embodiments, the pain is selected from: nociceptive, neuropathic, 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, post-herpetic 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, leg pain, restless legs syndrome, diabetic neuropathy, allergic conditions, surgical procedures, acute or chronic physical injuries, fractures or crush injuries, spinal cord injuries, inflammatory diseases, non-inflammatory neuropathic or dysfunctional pain conditions, or combinations thereof. In some embodiments, the pain is osteoarthritis pain. In some embodiments, the subject is a human.

[0066] In some embodiments, the present disclosure provides methods of producing any antibody or antigen-binding fragment disclosed herein, comprising the steps of expressing any nucleic acid disclosed herein in cultured cells, and purifying the antibody or antigen-binding fragment. Specific implementation plan

[0067] The present invention provides an antibody or antigen-binding fragment that binds to human PAR2 receptor. The antibody of the present invention can be used to inhibit PAR2 and its downstream signal transduction cascade.

[0068] Before describing the present disclosure, it is to be understood that the present disclosure is not limited to the particular methods and experimental conditions described, as 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.

[0069] PAR2 or GPR11 (PAR2; human PAR2 Uniprot protein ID: P55085) is a 44 kDa G protein-coupled receptor belonging to the protease-activated receptor (PAR) family. It is encoded by the gene F2RL1 (coagulation factor II receptor-like 1). PAR2 belongs to the protease-activated receptor (PAR) family, which is activated by proteolytic cleavage within the extracellular N-terminus. The family contains four members, PAR1 to PAR4, which are activated by different proteases. PAR2 is mainly activated by the serine proteases trypsin and trypsin, while other PAR family members are mainly activated by thrombin, although protease 3, factor VIIa and factor Xa have also been described to participate in PAR activation. For reviews, see Yau et al., 2016; Mrozkova et al., 2016; Hamilton and Trejo, 2017; Kagota et al., 2016.

[0070] PAR2 is activated by three main mechanisms. One of the mechanisms involves protease cleavage of the extracellular N-terminal domain. This results in the exposure of the bound ligand, which binds to a conserved region on the extracellular loop 2 of the receptor and triggers intracellular signaling. Alternatively, PAR2 can be activated by a short synthetic peptide called the activation peptide (PAR2-AP) that mimics the first six amino acids of the bound N-terminal ligand. Additionally, PAR2 can be activated by cross-activation of PAR1 bound ligands in PAR1-PAR2 heterodimerization. Following PAR2 activation, G αq and G i The protein is activated, which in turn leads to calcium influx, induction of MAPK signaling and downstream inflammatory signaling. This leads to subsequent biological responses, such as the proliferation or secretion of proinflammatory cytokines (e.g., IL-6, IL-8 (also known as CXCL8) and GM-CSF).

[0071] PAR2 expression has been shown to increase in synovial lining, chondrocytes and tissues in human rheumatoid arthritis and in animal models of arthritis (Amiable et al 2009). PAR2 also enhances signal transduction through channels such as TRPV1 (Dai et al 2007), a ligand-gated ion channel involved in inflammatory pain. PAR2 signal transduction is also known to sensitize TRPV1 in vivo, leading to thermal hyperalgesia (Amadesi et al., 2006).

[0072] It has been shown that PAR2 activation is the cause of multiple inflammatory signaling pathways. In mice lacking PAR2 receptors, there is a delayed onset of inflammation in response to inflammatory mediators (Lindner et al, 2000). Other rodent PAR2 knockout studies have shown that PAR2 plays an important role in many disease conditions, such as pain, musculoskeletal inflammation (including osteoarthritis), neuroinflammatory conditions, airway inflammation, itching, dermatitis, colitis and the pathophysiology of related conditions (Yau et al2013). It has also been shown that PAR2 receptor antagonists such as GB88 block inflammatory responses in vivo (including collagen-induced arthritis rat model) (Lohman et al 2012).

[0073] Known small molecule PAR2 antagonists are not ideal therapeutic agents because of low efficacy and wide specificity. In view of the scope of such diseases in which PAR2 is considered to be critical, it is therefore necessary to determine effective, specific PAR2 antagonists. Antibody of the present invention is an effective and specific PAR2 antagonist that inhibits the PAR2 activation mediated by the cutting of the N-terminal domain.

[0074] Unless defined otherwise, 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 belongs.

[0075] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0076] As used in this specification and the appended claims, an unqualified noun means "one, more than one, " unless the context clearly dictates otherwise.

[0077] It is convenient to point out here that "and / or" used in this article should be regarded as specifically disclosing each of the two specific features or components with or without the other. For example, "A and / or B" is regarded as specifically disclosing each of the following: (i) A, (ii) B, and (iii) A and B, just as if each was listed separately in this article.

[0078] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0079] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate. It should be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about". It should also be understood that, although not always explicitly stated, the reagents described herein are only examples and that such equivalents are known in the art.

[0080] As used herein, the terms "human protease activated receptor 2" or "human PAR2" or "PAR2" and the like refer to human PAR2 (wild type or wt), which has UniProt ID No.: P55085, re-represented herein as SEQ ID NO: 1. Human PAR2 includes any sequence having at least 99% or 100% identity to the amino acid sequence of any SEQ ID NO: 1 or a biologically active fragment thereof.

[0081] The term "binding ligand" as used herein refers to a region of the N-terminal portion of PAR2 that is exposed after proteolytic cleavage by a protease (e.g., trypsin) and once cleaved binds to the proximal binding site on the PAR2 receptor to activate it. The binding ligand at the N-terminus of the human PAR2 receptor comprises SEQ ID NO: 39.

[0082] By "PAR2-AP", "PAR2 activated peptide or PAR2 activating peptide" is meant a synthetic short peptide that mimics the cleaved tethered N-terminal ligand. The human peptide is SEQ ID NO:39, and the murine peptide comprises SEQ ID NO:40.

[0083] The terms "antibody", "antibodies against PAR2" or "anti-PAR2" used herein refer to complete antibodies or antigen binding fragments. The antibody used herein interacts with PAR2 (e.g., by binding, steric hindrance, stabilization / instability, spatial distribution) and interferes with the activation of PAR2. Naturally occurring "antibodies" refer to glycoproteins comprising at least two heavy (heavy, H) chains and two light (light, L) chains interconnected by disulfide bonds. The heavy chain comprises a variable region (VH) and a constant region, and the light chain comprises a variable region (VL) and a constant region. The VH and VL regions can be further divided into hypervariable (HV) and framework (FR) regions. Each VH and VL is composed of three complementarity determining regions (CDRs) and four FRs. The term "antibody" can refer to, for example, a monoclonal antibody, a human antibody, a humanized antibody, a shark antibody, a camelid antibody or a chimeric antibody.

[0084] Both the light chain and the heavy chain are divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the heavy chain and the light chain determine antigen recognition and specificity. In contrast, the light chain constant domain (CL) and the heavy chain constant domain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0085] According to the amino acid sequence of its heavy chain constant domain, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes (isotypes) of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses, for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and generally described in, for example, Abbas et al., (2000). Antibodies can be part of larger fusion molecules, which are formed by covalent or non-covalent association of antibodies with one or more other proteins or peptides.

[0086] A "complementarity determining region" ("CDR") is an amino acid sequence whose boundaries are determined using any of a number of well-known schemes, such as the "Kabat" and "Chothia" numbering schemes shown in Table 1. For example, under the Kabat scheme, the CDR amino acid residues in the heavy chain, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); whereas under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3).

[0087] Table 1: CDR definitions (numbering according to Kabat nomenclature).

[0088]

[0089] As used herein, the term "antigen binding portion" or "epitope binding fragment" of an antibody, etc., includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen binding fragments of an antibody can be derived from, for example, a complete antibody molecule using any suitable standard techniques involving manipulation and expression of DNA encoding antibody variable domains and optional constant domains, such as proteolytic digestion or recombinant genetic engineering techniques. Such DNA is known and / or can be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, so that one or more variable and / or constant domains are arranged in a suitable configuration, or so that codons are introduced, cysteine ​​residues are generated, amino acids are modified, added or deleted, etc.

[0090] Although the two fragment variable (Fv) domains VL and VH are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods to enable them to become a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (called single chain Fv (scFv); see, e.g., Bird et al., 1988; and Huston et al., 1988). Such single chain antibodies are also intended to be encompassed within the terms "fragment", "epitope binding fragment" or "antibody fragment". These fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0091] Other 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) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single domain antibodies, camelid antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, double antibodies, three antibodies, four antibodies, mini antibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), adnectins, small modular immune-pharmaceuticals (SMIPs) and shark variable IgNAR domains are also encompassed in the expression "antigen-binding fragment" used herein.

[0092] The antigen-binding fragment of an antibody will generally comprise at least one variable domain (e.g., at least one of VH or VL). The variable domain may have any size or amino acid composition, and will generally comprise at least one CDR, which is adjacent to or in the same frame as one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and comprise a VH-VH, VH-VL, or VL-VL dimer. Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric VH or VL domain.

[0093] In certain embodiments, the antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Some non-limiting exemplary configurations of variable and constant domains that may be present in the antigen-binding fragment of an antibody of the present disclosure include:

[0094] (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; (vi (ii) 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.

[0095] In any configuration of variable domains and constant domains (including any exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other, or may be connected by all or part of a hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that cause a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule. In some embodiments, the hinge region comprises a glycine-serine linker.

[0096] Furthermore, antigen-binding fragments of the antibodies of the present disclosure may comprise homodimers or heterodimers (or other multimers) of any of the above-listed variable and constant domain configurations non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0097] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, each of which is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format may be suitable for use in the context of an antigen-binding fragment of an antibody of the present disclosure.

[0098] As used herein, the term "biparatopic antibody" refers to a bispecific antibody that binds to two different epitopes on a single PAR2 target.

[0099] The term "monovalent antibody" as used herein refers to an antibody comprising one epitope binding moiety. The term "bivalent antibody" as used herein refers to an antibody comprising two epitope binding moieties.

[0100] The term "multivalent antibody" refers to a single binding molecule with more than one valence, wherein "valence" is described as the number of antigen binding moieties present in each molecule of the antibody construct. Therefore, a single binding molecule can bind to more than one binding site on a target molecule. Some examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, etc., as well as multispecific antibodies and biparatopic antibodies. For example, for PAR2, a multivalent antibody (e.g., a PAR2 biparatopic antibody) has two different binding moieties for two different epitopes of PAR2, respectively.

[0101] The term "monoclonal antibody" or "mAb" as used herein refers to polypeptides, including antibodies, bispecific antibodies, etc., having substantially the same amino acid sequence or derived from the same genetic source. The term also includes preparations of antibody molecules having a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0102] The term "fragment crystallizable region" or "Fc region" as used herein refers to a polypeptide comprising at least a portion of the hinge region of the constant domain of an antibody and CH3 and CH2. Optionally, the Fc region may include a CH4 domain, which is present in certain antibody classes. The Fc region may include the entire hinge region of the constant domain of an antibody. In one embodiment, the constant region is modified compared to the wild-type constant region. That is, the polypeptide of the present invention disclosed herein may include changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and / or to the light chain constant region domain (CL). Some exemplary modifications include the addition, deletion or replacement of one or more amino acids in one or more domains. Such changes may include optimizing effector function, half-life, etc.

[0103] The term "dual-active" or "dual-activity" or "dual-activity antibody" as used herein refers to an Ab provided herein that is capable of inhibiting both protease cleavage-mediated PAR2 activation (e.g., by trypsin) and peptide-mediated PAR2 activation (e.g., by PAR2-AP or PAR1-AP).

[0104] As used herein, the term "reference antibody," "reference mAb," or "reference Ab," or "benchmark," or "benchmark antibody" refers to any antibody used during experiments for reference to the anti-PAR2 antibodies of the present invention in the present disclosure (e.g., for positive or negative controls and to establish assay conditions). The benchmark antibodies used in the experiments herein include: Benchmark 1, which binds to the N-terminal epitope of PAR2 (Giblin et al 2011); Benchmark 2 (WO2018167322A1), also known as R001053 or PaB670129; Benchmark 3, Regeneron Ab, also known as R001044 or H4H581P; Benchmark 4, Amgen Ab, also known as R001048 or 1A1; MAB3949, a murine R&D systems mAb; and Benchmark 6 (Giblin et al 2011).

[0105] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (called a paratope) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on the antigen and may have different biological effects. Epitopes may be conformational (e.g., discontinuous) or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a portion of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0106] As used herein, the term "binding site" encompasses the region on the PAR2 target molecule to which an antibody or antigen-binding fragment selectively binds.

[0107] Generally speaking, an antibody specific for a particular target antigen will bind to an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0108] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity. The term "high affinity" as used herein for an IgG antibody or a fragment thereof (e.g., a Fab fragment) refers to an antibody having a KD of 10 for a target antigen. -8 M or smaller, 10 -9 M or smaller, or 10 -10 M, or 10 -11 M or smaller, or 10 -12 M or smaller, or 10 -13 M or less. However, high affinity binding can vary for other antibody isotypes. For example, for an IgM isotype, high affinity binding means an antibody has a KD of 10 -7 M or smaller, or 10 -8 M or smaller.

[0109] The term "avidity" as used herein refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It is controlled by three main factors: antibody epitope affinity; the valency of both the antigen and the antibody; and the structural arrangement of the interacting parts. Ultimately, these factors determine the specificity of the antibody, i.e., the likelihood that a particular antibody will bind to a precise antigen epitope.

[0110] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function. The following are some non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (messenger RNA, mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, shRNA, RNAi agents and primers. Polynucleotides may be modified or replaced at one or more bases, sugars and / or phosphates with any of a variety of modifications or replacements described herein or known in the art. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If there is a modification, the modification of the nucleotide structure may be performed before or after polymer assembly. The sequence of nucleotides may be interspersed with non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present invention with respect to polynucleotides encompasses both situations, namely encompassing a double-stranded form and encompassing each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0111] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a specific polypeptide or protein after transcription and translation. The polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene associated therewith. Methods for isolating larger fragment sequences are known to those skilled in the art.

[0112] The term "amino acid" as used herein refers to natural and / or non-natural or synthetic amino acids, both D and L optical isomers of any amino acid and amino acid analogs. If the peptide chain is short, the peptide of three or more amino acids is generally referred to as an oligopeptide. If the peptide chain is long, the peptide is generally referred to as a polypeptide or protein. The terms "biomarker" or "marker" are used interchangeably herein. A biomarker is a nucleic acid, polypeptide or other organic or inorganic molecule expressed in a human, and the presence or absence of a mutation or differential expression of a biomarker is used to determine sensitivity to any treatment comprising an anti-PAR2 antibody according to the present invention. For example, when a protein is defective, mutated, missing or reduced in post-translational modification, production, expression, level, stability and / or activity compared to the same protein in normal (non-cancerous) cells or control cells, the protein is a biomarker for cancer cells.

[0113] The terms "polypeptide", "peptide", "peptide mimetic" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The residues may be linked by peptide bonds or other bonds, such as esters, ethers, etc.

[0114] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence, meaning that when comparing two sequences, the bases (or amino acids) of this percentage are identical. This comparison and homology percentage or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al., (1987). Preferably, comparison is performed using default parameters. Preferred comparison tools are provided on the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) webpage, using default parameters. Other programs include BLAST, BLASTN, and BLASTP using the following default parameters: genetic code=standard; filter=none; strands=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; order by=HIGH SCORE; database=nonredundant.

[0115] As used herein, the term "expression product" or "gene product" refers to a nucleic acid or amino acid (eg, a peptide or polypeptide) produced when a gene is transcribed and translated.

[0116] As used herein, "expression" refers to the process by which DNA is transcribed into mRNA, and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0117] " differential expression " applied to gene refers to the difference generation of mRNA transcribed from gene or protein product encoded by gene.Compared with the expression level of normal or control cell, the gene of differential expression may be overexpressed or underexpressed.However, overexpression used in this article is the raising of gene expression, and is usually at least 1.25 times, or alternatively at least 1.5 times, or alternatively at least 2 times, or alternatively at least 3 times, or alternatively at least 4 times of expression detected in normal or control counterpart (counterpart) cell or tissue.Low expression used in this article is the reduction of gene expression, and is usually at most 1.25th, or alternatively at most 1.5th, or alternatively at most 1.2th, or alternatively at most 1.3rd, or alternatively at most 1.4th, or alternatively at most 1.5 ...

[0118] The high expression level of a gene can occur due to the overexpression of the gene or the increase of the gene copy number. Due to the imbalance or disappearance of a negative regulator, the gene can also be transcribed and translated into the protein level raised. Finally, the high expression of a gene can occur due to the improvement of protein stability or the reduction of degradation, thereby causing the accumulation of protein.

[0119] The term "inhibition (inhibit or inhibiting)" used herein etc. refers to interference, antagonism or blocking PAR2 receptor activation. Such term represents the quantitative difference between two states, for example, refers to a statistically significant difference between two states. For example, "effectively inhibiting the amount of PAR2 activation" means that the activation of the PAR2 caused by agonist and PAR2 blocking antibodies will have at least a statistically significant difference compared with the cell processed with agonist alone.

[0120] The terms "treatment", "treating", "alleviation", etc. are generally used herein to mean obtaining a desired pharmacological and / or physiological effect, and may also be used to refer to improving, alleviating, and / or reducing the severity of one or more symptoms of the treated condition. The effect may be preventive in terms of completely or partially delaying the onset or recurrence of a disease, condition, or its symptoms, and / or may be therapeutic in terms of partial or complete cure of a disease or condition and / or adverse effects attributable to the disease or condition. "Treatment" as used herein covers any treatment of a disease or condition in mammals, particularly humans, and includes any one or more of the following: (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed with the disease or condition; (b) inhibiting a disease or condition (e.g., preventing its development); or (c) alleviating a disease or condition (e.g., causing the disease or condition to subside, providing an improvement in one or more symptoms). For example, "treatment" of pain (e.g., chronic or neuropathic pain) involves reducing, preventing, alleviating, or eliminating the pain symptoms of the treated subject. The subject population treated by the disease methods includes subjects suffering from the undesirable condition or disease, as well as subjects at risk of developing the condition or disease.

[0121] The term "therapeutically effective dose" means a dose that produces the desired effect when administered. The exact dose will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., The Art, Science, and Technology of Pharmaceutical Compounding, 3rd edition, 2008).

[0122] Once the nucleotide sequence encoding such an antibody is determined, chimeric antibodies can be produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using materials and procedures generally known in the art and disclosed herein.

[0123] An "isolated" or "purified" antibody or protein is an antibody or protein that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). For example, an antibody or protein is substantially free of cellular material or other contaminating proteins from a cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when synthesized chemically. The phrase "substantially free of cellular material" includes antibody preparations in which the antibody is separated from the cellular components of the cell from which it is separated or recombinantly produced. Thus, antibodies substantially free of cellular material include antibody preparations having less than about 30%, 20%, 10% or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the antibody is recombinantly produced, it is preferably also substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10% or 5% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in protein synthesis.

[0124] Thus, such antibody preparations have less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a preferred embodiment, the antibodies of the invention are isolated or purified.

[0125] Provided herein are PAR2 directed antibodies and antigen binding fragments thereof that bind to PAR2. In some embodiments, the antibody is an antagonistic, neutralizing and / or blocking anti-PAR2 antibody or antigen binding fragment. "Antagonistic", "neutralizing" or "blocking" antibody or antigen binding fragment used herein is intended to refer to such an antibody or antigen binding fragment that binds to PAR2 and performs the following: (i) inhibiting the binding of PAR2 activating peptides to PAR2, or the activation of PAR2 by PAR2 activating peptides; and / or (ii) interfering with the interaction between the bound ligands exposed by PAR2 and PAR2; and / or (iii) interfering with the interaction between PAR2 and proteases (e.g., trypsin, tryptase, protein lyase, legumain); and / or (iv) inhibiting PAR2 signaling (e.g., inhibiting PAR2-mediated IP accumulation or intracellular Ca 2+ and / or (vi) inhibiting PAR2 activation; and / or (vii) leading to inhibition of at least one biological function of PAR2 (e.g., PAR2-mediated inflammatory response); and / or (viii) inhibiting the activation of PAR2 through the main G aqPAR2 secondary messenger signaling driven by activation of a signaling partner. In some embodiments, the antibodies or antigen binding fragments of the present disclosure inhibit activation of PAR2. In some embodiments, the antibodies or antigen binding fragments inhibit exposure of bound ligands. In some embodiments, the antibodies or antigen binding fragments inhibit activation of PAR2 receptors by their exposed bound ligands. In some embodiments, the antibodies or antigen binding fragments inhibit activation of PAR2 by exposed bound ligands. In some embodiments, the antibodies or antigen binding fragments inhibit binding of exposed bound ligands to the second extracellular loop (ECL2) of PAR2. In some embodiments, the antibodies or antigen binding fragments thereof bind to discontinuous epitopes, wherein binding to the epitopes blocks binding of exposed bound ligands to ECL2 by steric hindrance. The inhibition caused by anti-PAR2 neutralizing, blocking or antagonistic antibodies need not be complete, as long as it is detectable using appropriate assays. In some embodiments, compared with uninhibited active PAR2, antibody or its Fab suppresses PAR2 activity at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.Some examples of the active mensuration for detecting representative anti-PAR2 antibody or Fab are described in the example part.Technician knows other anti-PAR2 antibody activity mensuration.

[0126] In some embodiments, antibodies or antigen binding fragments that interfere with the interaction between PAR2 and a protease are provided herein. In some embodiments, the protease is trypsin. In some embodiments, the protease is neutrophil elastase. In some embodiments, the protease is neutrophil protease 3. In some embodiments, the protease is mast cell tryptase. In some embodiments, the protease is tissue factor / factor Vila / factor Xa. In some embodiments, the protease is a kallikrein-related peptidase. In some embodiments, the protease is membrane-bound serine protease-1 / protein lyase 1. In some embodiments, the protease is a parasite cysteine ​​protease.

[0127] In some embodiments, the antibody or antigen-binding fragment inhibits / reduces inflammation-induced pain.

[0128] Provided herein are PAR2-directed antibodies and antigen-binding fragments thereof that bind to PAR2 molecules with high affinity at physiological extracellular pH (ie, pH 7.5).

[0129] The antibodies or antigen-binding fragments of the present disclosure may have one or more of the above biological characteristics or any combination thereof. Other biological characteristics of the antibodies of the present disclosure will be apparent to those of ordinary skill in the art by reading the present disclosure, including the examples provided herein.

[0130] In some embodiments of the present disclosure, the anti-PAR2 antibodies of the present disclosure are human antibodies. The term "human antibody" used herein is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, and in some embodiments in CDR3. However, the term "human antibody" used herein is not intended to include antibodies in which CDR sequences derived from another mammalian species (e.g., mouse) germline have been grafted to human framework sequences.

[0131] In some embodiments, the antibody of the present disclosure can be a recombinant human antibody. The term "recombinant human antibody" used herein is intended to include all human antibodies prepared, expressed, produced or separated by recombinant means, such as antibodies expressed by recombinant expression vectors transfected into host cells, antibodies separated from recombinant combinatorial human antibody libraries, antibodies separated from transgenic animals (such as mice) for human immunoglobulin genes (see, for example, Taylor et al. 1992), or antibodies prepared, expressed, produced or separated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using transgenic animals for human Ig sequences, in vivo somatic cell mutagenesis is performed), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies are such sequences, although they are derived from and related to human germline VH and VL sequences, they may not naturally exist in the human antibody germline library in vivo.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2 activating peptide to PAR2, optionally wherein the antibody or fragment inhibits the accumulation of inositol monophosphate (IP) mediated by the PAR2 activating peptide, and its IC 50 The value is less than about 50pM, 100pM, 200pM, 400pM, 800pM, 1nM, 5nM, 10nM, 20nM, 40nM, 80nM, 100nM, 200nM or 300nM, optionally wherein the PAR2 peptide-mediated IP accumulation is determined using a PAR2 peptide-stimulated IP signaling assay. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2 activating peptide to PAR2, wherein the antibody or fragment inhibits the accumulation of inositol monophosphate (IP) mediated by a PAR2 activating peptide, and its IC 50The value is less than about 40nM, 50nM, 60nM, 70nM or 80nM, optionally wherein the PAR2 peptide-mediated IP accumulation is determined using a PAR2 peptide-stimulated IP signaling assay. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2 activating peptide to PAR2, wherein the antibody or fragment has an IC of 40nM or less. 50 Inhibiting PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation, optionally wherein PAR2 peptide-mediated IP accumulation is determined using a PAR2 peptide-stimulated IP signaling assay. In some embodiments, the IP signaling assay is a Cisbio IP-One HTRF assay.

[0133] In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated IP accumulation, and its IC 50 The value is less than about 50pM, 100pM, 200pM, 400pM, 800pM, 1nM, 5nM, 10nM, 20nM, 40nM, 80nM, 100nM, 200nM or 300nM; optionally wherein trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay. In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated IP accumulation, and its IC 50 The value is less than about

[0134] 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, 200nM, 210nM, 220nM, 230nM, 240nM, 250nM, 260nM, 270nM, 280nM, 290nM or 300nM;

[0135] Optionally, trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay. In some embodiments, the antibody or antigen-binding fragment thereof has an IC of 70 nM or less. 50 Inhibiting trypsin-mediated IP accumulation; optionally wherein trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof inhibits PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation, and its IC 50The value is less than about 50 pM, 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 100 nM, 200 nM or 300 nM; optionally wherein PAR2 peptide inhibition is determined using an HTRF assay.

[0137] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits calcium mobilization mediated by the PAR2 activating peptide, optionally wherein its IC 50 The value is less than about 50pM, 100pM, 200pM, 400pM, 800pM, 1nM, 5nM, 10nM, 20nM, 40nM, 80nM, 100nM, 200nM or 300nM, and optionally wherein calcium mobilization is determined using a PAR2 peptide-stimulated calcium mobilization assay. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits PAR2-activated peptide-mediated calcium mobilization, and optionally its IC 50 The value is less than about 50pM, 100pM, 200pM, 400pM, 800pM, 1nM, 5nM, 10nM, 20nM, 40nM, 80nM or 160nM, and optionally wherein calcium mobilization is determined using a calcium mobilization assay stimulated by a PAR2 activating peptide. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits calcium mobilization mediated by a PAR2 activating peptide, and optionally its IC 50 is 140 nM or less, and optionally wherein calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization, optionally with an IC 50 is less than about 50 pM, 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 100 nM, 200 nM or 300 nM, and optionally wherein calcium mobilization is determined using a calcium mobilization assay stimulated by a PAR2 activating peptide. In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization, optionally wherein its IC 50 is less than about 50 pM, 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 100 nM, 200 nM or 250 nM, and optionally wherein calcium mobilization is determined using a calcium mobilization assay stimulated by a PAR2 activating peptide. In some embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization, optionally wherein its IC50 is 200 nM or less, and optionally wherein calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey PAR2 and its EC 50 is less than about 50 pM, 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 100 nM, 200 nM or 300 nM, optionally wherein cynomolgus monkey PAR2 binding is determined using flow cytometry. In some embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey PAR2 and its EC 50 is less than about 400 pM, 800 pM, 1 nM, 5 nM or 10 nM, optionally wherein cynomolgus monkey PAR2 binding is determined using flow cytometry.

[0140] In some embodiments, the antibody or antigen-binding fragment thereof binds to human PAR2 and its K D is less than about 50 pM, 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 100 nM, 200 nM or 300 nM, optionally wherein the binding affinity is determined using surface plasmon resonance (SPR) or KinExA. In some embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM or 20 nM. D Binds to human PAR2, optionally wherein the binding affinity is determined using surface plasmon resonance (SPR) or KinExA. In some embodiments, the antibody or antigen-binding fragment thereof has a K of 10 nM or less. D Binding to human PAR2, optionally wherein the binding affinity is determined using surface plasmon resonance (SPR) or KinExA. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1: Some examples of specific PAR2 nanodisc (FL-StaR ND) binding relative to empty nanodisc (empty ND) by positive control mAb 'Benchmark 1' and commercially available PAR2 mAb 'R&D anti-huPAR2 mAb (MAB3949)' and the dose-dependent binding observed for these and the three antibody clones of PAR2 phage origin shown. Antibodies were coated directly onto the plates at 5 μg / mL. No significant background binding was observed in the case of the negative control, MOR03207 (anti-lysozyme antibody). Data are shown as signal divided by background (S / BG).

[0142] Figure 2 : Example of degree of competition (full, partial and no competition) with commercially available positive control mAb 'MAB3949' (R&D anti-huPAR2) using full-length PAR2 StaR Nanodiscs in a panel of PAR2 mAbs (derived from a Ylanthia phage antibody library) and thereby demonstrating diversity. 50 nM candidate mAbs were titrated against a dose response of R&D anti-huPAR2 (MAB3949) in competition with full-length StaR Nanodiscs. Both full and partial competition showed dose-dependent binding. No dose-dependent binding or competition with MAB3949 was observed for reference Ab 'Benchmark 1' (N-terminal binder) or 'MOR03207' (anti-lysozyme antibody used as negative control). Data are shown as signal divided by background (S / BG).

[0143] Figure 3 : Dose-dependent binding of the following measured by FACS: A) Dose-dependent binding of benchmark 1 mAb and MAB3949 to cell surface expressed PAR2 on CHO_V5HIS-huPAR2 cells vs CHO parental cells to demonstrate the validity of the assay setup; BG = background; B) Dose-dependent binding of clone Y022065 to recombinantly expressed PAR2 on CHO_V5HIS-huPAR2 cells vs CHO parental cells; C) Dose-dependent binding of clone Y022065 to A549 cells endogenously expressing human PAR2 and CHO cells expressing mouse PAR2. BG = background. Data are shown as relative luminescence units (RLU).

[0144] Figure 4: Flow cytometry was used to understand the ability of antibody clones to bind to full-length huPAR2 (FL-N, second row) and huPAR2 with an N-terminal truncation (truncated, first row). The data obtained showed that it was possible to identify not only antibody clones that preferentially bind to the N-terminus (full-length receptor, e.g. Y022066), but also other antibody clones that bind to both truncated and full-length receptors (e.g. Y022065, Y022071) (where the epitope also includes a portion of the extracellular domain) as well as clones that bind to truncated receptors but not to the full-length receptor (e.g. YY22075).

[0145] Figure 5 : Multiplex profiling using IntelliCyt. The figure shows the binding of purified IgG1f_AEASS on cells expressing human PAR2. The data are exemplary data for clone 'Y022065' (functional candidate). Top) HEKFlp-In TRex 293_huPAR2 expression induced, HEK Flp-In TRex 293_huPAR2 uninduced, Flp-InCHO_V5His_huPAR2, parental Flp-In CHO cells and Flp-In CHO_huPAR1. Bottom) HEK293F infected with BacMam WT FL-huPAR2 and uninfected HEK293F. Data are shown as signal divided by background (S / BG).

[0146] Figure 6 : Used in peptide inhibition assays IP-One Gαq assays were performed in duplicate at two concentrations for functional characterization of Y021171, Y022063, and Y022054 & Y022065 hIgG1f_AEASS. At the highest tested concentration, statistically significant inhibition of activating peptide-induced human PAR2 agonism was observed for the four clones in response to 6.28 nM agonist alone, ranging from 17% to 43%. Y022065 was determined to be the most active candidate. Data are shown as the average response with standard deviation of 2 independent experiments run in duplicate.

[0147] Figure 7 : Human PAR2 embedded in nanodiscs and purified soluble Koff-ranking ELISA performed on proteins where the parental candidates represent the lineage of 22 mature IgG candidates identified as functionally dual active. Data are shown as signal divided by background (S / BG).

[0148] Figure 8: Binding specificity of affinity matured clones shown by FlpIn CHO-V5His-huPar2 relative to the parental lineage on FlpIn CHO parental cells.

[0149] Fig. 9 : Affinity matured clones formatted as Fab fragments bind to CHO cells expressing human PAR2 as assessed by flow cytometry. Data are shown as signal divided by background (S / BG).

[0150] Fig.10 : Affinity matured clones formatted as IgG or fAb bind to cynomolgus monkey (IgG) and human-PAR2 (fAb) BacMam infected HEK293 cells as assessed by flow cytometry. Data are shown as signal divided by background (S / BG).

[0151] Fig.11 : Determination of affinity-matured lead mAb panel binding to cells expressing human and cynomolgus monkey PAR2 as Fab fragments and IgG compared to benchmark mAbs. Antibodies were incubated with human PAR-2 ​​and cynomolgus monkey PAR-2 ​​overexpressing cells at about 5°C with sodium azide overnight. All antibodies showed significant binding to cynomolgus monkey PAR-2 ​​and human PAR-2 ​​in both cases as Fab fragments and as complete IgG. Data are shown as signal divided by background (S / BG).

[0152] Fig.12 : Functional characterization of lead optimized clones in inhibition assays against PAR2 activating peptides. The IP-One assay was used to measure IP accumulation as a function of in vitro human PAR2 activity and Gαq activation. Each value represents the inhibition % normalized to 10 μM MAB3949 at the highest concentration tested in duplicate for each lead clone. Lead clones have been compared with parental IgG. Inhibition of activating peptides was observed for lead clones matured from all parental IgGs. Relative to R&D System anti-human PAR2 MAB3949, the percentage of inhibition in the clones tested was 0 to about 250%, indicating that at comparable concentrations, a large number of lead clones showed greater inhibition of activating peptide excitement of human PAR2 when compared to MAB3949. Lead clones derived from parental IgG Y021171 and Y022065 obtained by affinity maturation showed the largest proportion of actives.

[0153] Fig.13: Functional characterization of optimized lead clones in an inhibition assay against bovine trypsin. The IP-One assay was used to measure IP accumulation as a function of in vitro human PAR2 activity and Gαq activation. Each value represents the inhibition % normalized to 1 μM benchmark 1 at the highest concentration tested in duplicate for each lead clone. The lead clones have been compared to the parental IgG. Inhibition of bovine trypsin was observed for lead clones matured from all parental IgGs. The percentage inhibition ranged from 0 to about 130% in the clones tested relative to benchmark 1. The lead clones identified from the maturation of IgG Y022065 showed the largest proportion of active substances against the bovine trypsin challenge. The two representatives derived from Y021171 showed the highest inhibition %.

[0154] Fig.14 : Graphical representation of dose response inhibitor curves for parental clone (Y022065) and affinity matured lead representatives (Y022870, Y022877, Y022883) compared to Benchmark 1 and Benchmark 2 in a calcium mobilization assay measured in response to HT-29 challenge with bovine trypsin to activate endogenous PAR2. Data are shown as the average response with standard deviation of 3 independent experiments run in duplicate wells.

[0155] Fig.15 : SPR evaluation by Biacore to determine the effect of pH on Y022883 binding to PAR2.

[0156] Fig.16 : Affinity determination of lead candidates Y022870 and Y022883 to HEK-293F-human PAR2-expressing cells by KinExA.

[0157] Fig.17 : Epitope binning matrix of affinity matured clones derived from parental family derivation. Anti-huPAR2 is MAB3949.

[0158] Fig.18 : Back view of PAR2 (ECL3, segment 1 and helix 0 / 1). The region of PAR2 that interacts with Y022883 derived from HDX is shown as a shaded area.

[0159] Fig.19 : Top view of PAR2 (ECL3, segment 1 and helix 0 / 1). The region of PAR2 that interacts with Y022883 derived from HDX is shown as a shaded area.

[0160] Fig. 20: Side view of PAR2 (ECL3, segment 1 and helix 0 / 1). The region of PAR2 that interacts with Y022883 derived from HDX is shown as a shaded area.

[0161] Fig.21 : Expression profiles of WT and mutant (S60W, D62F, G318F, D62G & E63G) human PAR2 detected by flow cytometry on BacMam infected HEK293F cells stained with Benchmark 1 (N-terminal binder) or Y022883. nd : secondary FACS antibody only; ni: uninfected.

[0162] Fig. 22 : Rat serum mAb concentrations relative to time after a single intravenous dose of 10 mg / kg Y022883. Data are shown as means (open circles) with 95% confidence intervals (solid bars) from 3 male adult animals; individual animal data (filled circles) are also shown; detectable mAb concentrations at or below BLOQ (5 ng / mL) are reported as BLOQ / 2 (dashed line).

[0163] Fig.23 : Cynomolgus monkey serum mAb concentrations relative to time after a single intravenous dose of 10, 3 or 1 mg / kg Y022883. Data are shown as means (10 mg / kg open circles, 3 mg / kg closed squares, 1 mg / kg closed triangles) with 95% confidence intervals (solid bars) from 3 male adult animals; individual animal data are also shown (10 mg / kg closed circles, 3 mg / kg open squares, 1 mg / kg open triangles); detectable mAb concentrations at or below BLOQ (5 ng / mL) are reported as BLOQ / 2 (dashed line).

[0164] Fig.24 : In vitro pharmacodynamics in cynomolgus monkeys following a single 10 mg / kg dose of Y022883. Data are shown as the percentage of the gene signature induced by the stimulant before dosing (PAR2-AP as circles, trypsin as squares, LPS as triangles).

[0165] Fig.25 : In vitro pharmacodynamics in cynomolgus monkeys following a single 3 mg / kg dose of Y022883. Data are shown as the percentage of the gene signature induced by the stimulant before dosing (PAR2-AP as circles, trypsin as squares, LPS as triangles).

[0166] Fig.26: In vitro pharmacodynamics in cynomolgus monkeys following a single 1 mg / kg dose of Y022883. Data are shown as the percentage of the gene signature induced by the stimulant before dosing (PAR2-AP as circles, trypsin as squares, LPS as filled triangles).

[0167] Fig. 27 : Inhibition of p38-MAPK and pERK phosphorylation in trypsin-induced T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data show levels relative to treatment with vehicle and are normalized to total ERK or p38-MAPK.

[0168] Fig.28 : Inhibition of p38-MAPK and pERK phosphorylation in PAR2-AP-induced T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data show levels relative to treatment with vehicle and are normalized to total ERK or p38-MAPK.

[0169] Fig.29 : Inhibition of pERK (left) and p38-MAPK (right) phosphorylation in T84 cells induced by trypsin and PAR2-AP by Y022883 (SH-C) and Benchmark II (SH-D). Data show levels as percentage of difference between vehicle and positive control.

[0170] Example

[0171] Certain aspects and embodiments of the invention will now be illustrated by way of examples and with reference to the above-described figures.

[0172] Example 1 - Generation of full-length and N-terminally truncated human PAR2 constructs

[0173] Design of PAR2 constructs

[0174] Based on Hutchison et al., 1978, the gene of human protease activated receptor 2 (PAR2) was edited using a standard site-directed mutagenesis strategy to stabilize the receptor into an antagonist conformation by introducing nine point mutations (Cheng et al 2017). The stabilized receptors were also genetically truncated at the N-terminus by 54 amino acids (the first residue in the encoded construct was V55), or kept full length (FL-N) using polymerase chain reaction (PCR, based on Saiki et al., 1985), which is widely known in the art. All four PAR2 proteins were also genetically truncated at the C-terminus by 20 amino acids (the last residue K377) as part of the stabilized receptor process.

[0175] The following constructs were generated:

[0176] PAR2-1:P151:55-377, glycosylated, pFastBacHisStrepII, truncated

[0177] PAR2-2: P166: 1-377, deglycosylated: N30Q, N222Q, pFastBacHis StrepII, full length

[0178] PAR2-3:P157:1-377, glycosylated, pBacMamHisStrepII, full length

[0179] PAR2-4:P155:55-377, glycosylated, pBacMamHisStrepII, truncated

[0180] The rationale for generating an N-terminally truncated PAR2 receptor that is sufficiently stable for use as an antigen is to enable the discovery and identification of antibodies that bind to regions of the receptor other than just the N-terminus, thereby generating new antibodies.

[0181] By site-directed mutagenesis of the glutamine residues in FL-N protein PAR2-2, the potential glycosylation sites N30 and N222 were replaced.

[0182] StrepII and ten-histidine tags are added to the C-terminus by PCR and endonuclease cloning strategies known to the technician to allow affinity purification and specific selection in ELISA assays. PAR2-1 and PAR2-2 genes are cloned into pFastBac1 vectors (Thermofisher, #10360014). The multiple cloning site of the vector is modified to allow the use of NheI as a restriction endonuclease to insert the gene. The pFastBac1 vector is a part of the commercial Bac-to-Bac expression system (Smith et al., 1983, Thermofisher, #10359016) for insect cells, which is known in the art and widely used. We followed the manufacturer's instructions. The Bac-to-Bac expression system is used for PAR2-1 protein and PAR2-2 protein. The pFastBac1 vector was modified to generate the pBacMam vector by introducing the human cytomegalovirus promoter 3' behind the polyhedrin promoter, allowing protein expression of proteins PAR2-3 and PAR2-4 in mammalian cells. The description of the Bac-to-Bac expression system also applies to pBacMam virus production.

[0183] Express

[0184] Proteins PAR2-3 and PAR2-4 were expressed in human embryonic kidney 293F cells (Gibco 293F cells, Thermofisher Scientific, #11625019) by viral infection with 2.7% virus at a cell density of 2.5×10^6 / ml for 48 hours in Lonza Pro293s CDM medium (#BE02-025Q) with 10% fetal bovine serum albumin (FBS, Sigma-Aldrich, #F9665) and supplemented with 5mM sodium butyrate (Sigma-Aldrich, #303410). Cells were harvested and processed as described below for Sf9-cell expression. Mammalian cell lines were used for construct expression to provide the option of using antigens containing mammalian-like glycosylation patterns.

[0185] Protein PAR2-1 and protein PAR2-2 were expressed in Spodoptera frugiperda Sf9 cells (Thermofisher, #89070101) in expression system ES921 medium (#96-001-01) with 10% FBS (Sigma-Aldrich, #F9665). Cells were infected at a multiplicity of infection (MOI) of 2 at a cell density of 3.5×10^6 / ml. Cells were harvested by centrifugation after 48 hours, washed with 50mM HEPES pH 7.5, 250mM NaCl (PAR2 buffer) supplemented with Roche EDTA-free complete protease inhibitor (protease inhibitor, PI, Sigma-Aldrich, #5056489001), and stored at -80°C. To prepare cell membranes, cells were resuspended in PAR2 buffer supplemented with PI tablets (PAR2 / PI) and then homogenized via a single pass through microfluidic microfluidizer processor. Membranes were collected by ultracentrifugation at 135,000 g for 1 hour in a Beckman 45Ti rotor, homogenized into PAR2 / PI buffer and stored at -80°C. All subsequent purification steps were performed at 4°C.

[0186] purification

[0187] Thawed membranes were dissolved by adding 1% (w / v) LMNG (Anatrace, #NG310) / 0.1% (w / v) cholesterol hemisuccinate (CHS, Anatrace, #210) mixture for 1 hour. Insoluble material was removed by ultracentrifugation at 205,000 g for 30 minutes in a Beckman 45Ti rotor. Proteins were bound to 8 ml NiNTA Superflow resin (Qiagen, #30430) in batches for 2 hours in the presence of 8 mM imidazole. The resin was loaded into a XK 16 / 20 column (GE Healthcare, #GE28-9889-37) and washed with 15 column volumes (CV) of high salt buffer A1 (50 mM HEPES pH 7.5, 500 mM NaCl, 0.02% LMNG, 0.002% CHS, 75 mM imidazole) and 3 CV of buffer A2 (50 mM HEPES pH 7.5, 250 mM NaCl, 0.02% LMNG, 0.002% CHS, 75 mM imidazole). The protein was eluted with buffer A2 supplemented with 300 mM imidazole and concentrated to 0.5 ml using an Amicon Ultra-15 regenerated cellulose concentrator (Millipore, #UFC900308) with a 100 kDa molecular weight cutoff. The material of aggregation is removed by ultracentrifugation at 220,000g for 10 minutes in Beckman Coulter desktop centrifuge using TLA-100.2 rotor.Then the sample is made on Superdex 200 10 / 300GL post (GE Healthcare, #17-5175-01), in 50mM HEPES pH 7.5, 150mM NaCl, 0.02%LMNG, 0.002%CHS, size exclusion chromatography is performed.The fraction corresponding to the monomeric substance is merged, and the Vivaspin 500 concentrator (Vivaproducts, #VS0242) with 100kDa molecular weight cutoff is used to be concentrated to 6 to 8mg / ml.Sample purity and monodispersity are analyzed by SDS-PAGE and analytical gel filtration.The protein absorbance at molecular weight, extinction coefficient and 280nm is used to determine protein concentration using Nanodrop spectrophotometer (Thermofisher Scientific). Proteins were examined by mass spectrometry to identify the protein of interest.

[0188] Preparation of human PAR2 StaR protein embedded in nanodiscs

[0189] Preparation of nanodiscs is known in the art and is based on Banerjee et al., 2008, using zebrafish apolipoprotein-1 (ZAP1) as a scaffold protein with an N-terminal hexa-histidine tag. Protein PAR2-1 and protein PAR2-2 were reconstituted into nanodiscs at 100 to 200 μM and used as antigens for Fab selection. To reconstitute nanodiscs, the following lipids were first resuspended in an aqueous 200 mM sodium deoxycholate solution (Sigma-Aldrich, #6750): POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Anatrace, #P616), POPG (1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoglycerol, Anatrace, #P516) and cholesterol (Anatrace, #CH200) in a ratio of 3:2:0.5. The final molar ratio of PAR2:POPC:POPG:cholesterol:sodium deoxycholate was 1:72:48:12:264 respectively.The final sodium deoxycholate concentration was kept above 15 mM and adjusted with 10 mM HEPES 7.4, 150 mM NaCl buffer.

[0190] After 30 minutes of incubation on ice, zebrafish apolipoprotein 1 (ZAP1) was added at a molar ratio of 1:2PAR2:ZAP1. The mixture was incubated on ice for 1 hour. The detergent was then removed by stirring the sample overnight with a 1:1 ratio of protein solution to dry bio-bead weight (e.g., 900 μl protein to 900 μg bio-bead) using Bio-bead SM-2 (Bio-Rad, #1528920). The nanodisc was recovered as the supernatant and the beads were washed with 50 mM HEPES pH 7.5, 150 mM NaCl, two volumes for one weight of beads (e.g., 1.8 mL for 900 μg beads). The protein solution was concentrated to less than 1 mL, centrifuged at 13,500 g for 15 minutes in a tabletop centrifuge, and the supernatant was subjected to size exclusion chromatography on a Superdex200 10 / 300 GL column (GE Healthcare) as used above in 50 mM HEPES pH 7.5, 150 mM NaCl. Fractions representing the nanodisc peak were pooled and concentrated to 1 to 2 mg / mL in an Amicon Ultra-4 regenerated cellulose concentrator (100 kDa molecular weight cutoff, Millipore, #UFC810096) and frozen in aliquots at -80°C.

[0191] Example 2 - Screening

[0192] The antibodies or antigen-binding fragments of the present disclosure are identified from phage display libraries. Various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. The phage display libraries used to identify the antibodies provided herein are based on the concept of Ylanthia (Tiller et al. 2013). Phagemid and CysDisplay TM The Fabs were displayed on the surface of bacteriophage using the technology (Lohning et al. 2000).

[0193] Ten panning strategies (including solid phase panning, capture panning, and whole cell panning using techniques known in the art) were designed, with a focus on solid phase panning using full-length or N-terminally truncated nanodisc-embedded human PAR2 StaR proteins. Various antigen presentation techniques were implemented to ensure the integrity and stability of the nanodisc-embedded PAR2 StaR and to reduce the risk of enrichment of candidates with specificity for other undesired epitopes during the panning process. Human PAR2 StaR variants embedded in nanodiscs were used as panning antigens (i.e., detergent-free state). For most panning strategies, the nanodisc-embedded human PAR2 StaR protein was presented by a tag-specific capture antibody. Alternation of capture modes was implemented to reduce the risk of enrichment of candidates with specificity for the capture antibody Benchmark 1 (Giblin et al 2011), anti-histidine-Ab IgG1 (StrepMAB-Immo), or reagent (NiNTA). For some strategies, differential whole-cell panning was performed on Flp-In CHO and BacMam-infected HEK293F cells expressing human PAR2. Alternating panning for StaR followed by cell panning was used to reduce the risk of enrichment of candidates with specificity for other cellular targets (off-target binding). To amplify phage-antibodies that compete with functionally active ligands of human PAR2, nanodisc-embedded human PAR2 StaR variants PAR2-2 P166:1-377, deglycosylated: N30Q, N222Q, pFastBacHis StrepII or hPAR2 (55-377_10xH_Sx2 (truncated) PAR2-1: P151:55-377, glycosylated, pFastBacHisStrepII) were used, which were saturated with commercially available small molecule ligands AZ8838 and AZ3451 (Cheng et al 2017) for panning. These pannings led to the identification of a large number of Candidates, which have mostly good specificity for human PAR2 receptor expressed in many different cell backgrounds. In order to more strictly not select non-specifically bound phages, unrelated antigen components were pre-adsorbed before each round of solid phase panning. Unrelated antigen components included Ni-NTA, empty nanodiscs, and capture antibodies anti-StrepII mAb (StrepMAB-Immo) and anti-poly 6x histidine-Ab IgG1 (R&D MAB050), or alternative GPCRs embedded in nanodiscs (only for mature panning).

[0194] Phage preparation

[0195] Production of phage particles presenting Fab: Each round of selection produces new phage particles presenting Fab fragments on their surface. Therefore, E. coli TG1 cultures were infected with phages from the previous round of selection. After centrifugation, the bacterial pellet was resuspended in fresh medium and plated on agar plates. After outgrowth, the colonies were scraped off the plates and used for phage rescue, polyclonal amplification of selected clones, and phage production. The next panning round was started with purified phage. After the last round of panning, single clones were picked from the agar plates and placed in the wells of sterile microtiter plates pre-filled with culture medium. After outgrowth, culture medium containing glycerol was added to each well of the master plate; the plates were sealed with aluminum foil and stored at -80°C.

[0196] Example 3 - Identification of hits by ELISA and whole cell binding by FACS and IntelliCyt screening

[0197] ELISA

[0198] ELISA technology has been used for both screening of single Fab clones identified from panning output for target antigens and characterization of purified antibodies. Optimal antigen and antibody concentrations and blocking conditions were determined according to prior art methods.

[0199] Direct coating of antigen

[0200] The antigen is fixed on a microtiter plate. The plate is blocked and incubated with antibodies such as Fab containing crude E. coli lysate or purified Fab or IgG samples. The bound antibody is detected using alkaline-phosphatase (AP) coupled secondary antibodies and in combination with 'AttoPhos' fluorescent substrate. Multiple washing steps are performed between each assay step.

[0201] Antigen capture

[0202] In other ELISA settings, antigens are captured to the plate by tag-specific antibodies (e.g., anti-StrepII, anti-His, or Benchmark 1 reference mAbs) coated on microtiter plates. Bound antibodies are detected using corresponding alkaline phosphatase (AP)-conjugated secondary antibodies in combination with 'AttoPhos' fluorescent substrate. Multiple washing steps are performed between the various assay steps.

[0203] Fab expression examination by anti-Fd ELISA

[0204] To confirm Fab expression in crude bacterial lysates, plates were coated with Fd fragment specific antibodies. Bound Fabs were detected using the corresponding alkaline phosphatase (AP) coupled anti-Fab specific antibodies in combination with 'AttoPhos' fluorescent substrate. Multiple washing steps were performed between the individual assay steps.

[0205] K off Sorting ELISA

[0206] For K off It is estimated that the ELISA plate was subjected to additional frequent and stringent washing after the initial readout and a second test was performed to identify candidates with slow kd. Before detection, washing was performed using the following conditions: 5 washes with 10 minute incubation, followed by overnight incubation, and then 5 washes with 10 minute incubation, followed by 1 hour incubation, followed by 5 washes with 5 minute incubation. Bound antibodies were detected using the corresponding alkaline phosphatase (AP)-conjugated secondary antibodies and in combination with 'AttoPhos' fluorescent substrate.

[0207] Implementation of ELISA method

[0208] ELISA technology is used for both: screening of single Fab clones identified from panning output for target antigens and characterization of purified antibodies. Evaluate optimal antigen and antibody concentrations and blocking conditions, and adjust settings.

[0209] After the panning selection, 368 clones from every third panning round output were processed as bacterial lysates in the preliminary screening. The panning output of solid phase panning was screened by FACS on magnetic beads coated with StaR or in ELISA using labeled StaR variants or StaR embedded in nanodiscs. The screening was mainly performed on PAR2-1 P151 variant (55-377, glycosylated, pFastBacHisStrepII). Counter screening mainly included empty nanodiscs and anti-Fd mAb and mouse gamma globulin.

[0210] The secondary screening was mainly performed in ELISA. The antigen panel contained PAR2-1: P151: 55-377, glycosylated, pFastBacHisStrepII and PAR2-2 embedded in nanodiscs: P166: 1-377, deglycosylated: N30Q, N222Q, pFastBacHis StrepII, which was saturated with ligands AZ8838 and AZ3451. Similarly, the counter-screening was also extended to the capture antibodies StrepMAB-Immo and bRIL-His-StrepII. Despite the low binding signal on cells, all target clones showed good correlation between cell binding and the binding spectrum obtained for the recombinant nanodisc-embedded StaR material.

[0211] These were selected for sequencing and IgG was converted to the silent h_IgG1f_AEASS format (IgG1 L234AL235E G237A A330S P331S; this format has been clinically validated in Novo Nordisk's second-generation anti-C5aR antibody NNC0215-0384 (Wagner et al., 2014)), and expressed and purified for characterization at advanced micro scale and / or exploratory scale. IgG was rescreened by ELISA before functional characterization. Figure 1 and 2 ) and rescreened the cells for binding by IntelliCyt to re-determine binding specificity.

[0212] FACS and IntelliCyt

[0213] Cells screened by flow cytometry and IntelliCyt for positive identification of PAR2 binders Combination

[0214] Using crude E. coli lysate or purified antibodies from panning output, binding events to cell surface expressed antigens were identified by flow cytometry. High-throughput preliminary screening of panning outputs of differential whole cell panning (DWCP) was mainly performed for the following: Flp-In CHO cells (Flp-In CHO_huPAR2) expressing human wild-type PAR2, GNTI BacMam wild-type PAR2 cells and GNTI BacMam truncated PAR2 StaR relative to parental Flp-In CHO cells (PAR2 negative) or uninfected HEK293GNTI- cells. Candidates that showed increased background binding to parental Flp-In CHO cells were classified as nonspecific or off-target binding agents. All steps were performed in FACS buffer (including FCS and sodium azide) to prevent potential receptor internalization. The cell suspension was transferred to a microtiter plate and the antibody sample was added, and the plate was subsequently incubated at 4°C. The sample volume and cell number were adjusted for the type of plate used. After incubation, the cells were centrifuged and washed with FACS buffer. A fluorophore-conjugated secondary reagent was used to detect the bound antibody. The plates were measured using BD FACS arrays or IntelliCyt HTFC / iQue systems, and data were analyzed using FlowJo, ForeCyt or proprietary software tools ( Figure 3 ).

[0215] Evaluation of whole cell binding / specificity properties (FACS, IntelliCyt)

[0216] Binding specificity as assessed by flow cytometry on Sf9 cells expressing human PAR2

[0217] FACS-based analysis of initial PAR2 candidates was performed using Sf9 insect cells expressing PAR2. To understand whether the antibodies preferentially bind to the N-terminus or the rest of the extracellularly exposed portion of the receptor, cells were incubated with expressing either the full-length N-terminus of PAR-2StaR (PAR2-3,1-–377, described herein as FL-N StaR) or an N-terminally truncated receptor (PAR2-4,55–377, described herein as NΔ53StaR, or in Figure 4 and 7 "truncated" in the text) for infection.

[0218] 200 μL Sf9-cells at 4×10^6 cells / mL were incubated with 20 nM of the antibody of interest at room temperature for 1 hour in FACS buffer (PBS (Sigma, #F9665), 1% BSA (Sigma, #A9647) and Roche complete protease inhibitor (#11836145001)). The cells were pelleted and washed three times with 200 μL FACS buffer. Sequentially, the cells were incubated with 200 μL of secondary phycocyanin (APC)-conjugated 20 nM anti-human IgG for 1 hour and washed again three times with 200 μL FACS buffer.

[0219] Readout was monitored using a BD FACS Canto II FACS instrument.

[0220] A series of binders with different binding profiles (as determined by flow cytometry) were generated, with Y022075 preferentially binding to the truncated receptor and Y022065 binding to both the truncated receptor and full-length PAR2. In contrast, antibody clone Y22066 bound almost exclusively to the FL-N construct. These observations reflect the diversity of epitopes ( Figure 4 ).

[0221] Binding specificity as determined by IntelliCyt

[0222] Binding specificity was determined for purified IgG and performed in duplicate as a 16-point titration on cells expressing human PAR2 as n=2 experiments. The following cell lines were included in the analysis:

[0223] Flp-In CHO_V5His_huPAR2 relative to parental Flp-In CHO cells

[0224] Flp-In CHO_huPAR1 relative to parental Flp-In CHO cells

[0225] HEK Flp-In TRex 293_huPAR2 expression was induced, compared with uninduced cells

[0226] HEK293F cells infected with BacMam WT FL-huPAR2 compared to uninfected cells

[0227] HEK293F cells infected with BacMam WT FL-marmoset PAR2 compared to uninfected cells

[0228] All target clones originate from the PAR2 nanodisc panning strategy, which enables directional presentation by means of antibody capture through labeled receptors and / or nanodisc scaffolds, and enables selective binding to cells expressing PAR2 in different cell backgrounds. PAR2 StaR nanodisc preparations present high-quality PAR2 receptors comparable to the natural receptors expressed on cells in a detergent-free environment, and lead to the identification of promising, functionally active candidates. In addition, panning stringency is easily controlled using recombinant materials (e.g., StaR nanodiscs).

[0229] Multiplexing via IntelliCyt

[0230] In order to evaluate species cross-reactivity and / or undesirable binding to counter-targets simultaneously, screening was performed using the HTFC / iQue screening platform of IntelliCyt in a 384-well plate format. The HTFC / iQue screening system is also used to evaluate binding to multiple target cell lines or to evaluate undesirable / non-specific binding simultaneously, that is to say multiplexing. Different cell populations can be distinguished by pre-labeling with different amounts of fluorescent dyes (e.g., calcein or cell-tracker green), establishing unique fluorescence intensity characteristics for each cell population, thereby generating a fluorescence coding system. The color-coded cell lines are then physically combined and mixed with the antibodies to be tested. Each cell line can be identified by the fluorescence of the corresponding cell lines that have been pre-labeled. Crude bacterial cell lysates are combined with cells and incubated for 1 hour at room temperature in the dark under gentle shaking. Fluorescence measurements are performed using the IntelliCyt HTFC / iQue device. Between incubation steps, washing is not required. The raw data is evaluated with the aid of 'ForeCyt' software. After data acquisition, cell lines from each sample can be identified based on their fluorescence signature and individually evaluated for antibody binding. Staining conditions were optimized for each cell line in order to find an assay setup that allows separation of the different cell lines ( Figure 5 ).

[0231] Y022065, the parent of the final lead set, was obtained by a panning strategy using captured deglycosylated full-length hPAR2 (PAR2-2, 1-377, N30Q, N222Q)_10xH_Sx2 embedded in nanodiscs. In the first and third rounds, the antigen was captured with anti-StrepII antibody. In the second round, the antigen was captured with benchmark 1 or coated on Ni-NTA plates. In summary, specificity was assessed by: ELISA-based assessment of binding to FL-StaR nanodiscs and truncated StaR nanodiscs in the presence or absence of ligands AZ8838 and AZ3451; ELISA-based assessment of binding to counter-targets including empty nanodiscs, bRIL-His-StrepII, StrepMAB-Immo (capture antibody), and mouse gamma globulin; and assessment of binding to the following PAR2-expressing cell lines (n>2): CHO V5His_huPAR2, HEK Flp-InTRex293_huPAR2 induced, HEK293F infected with BacMam WT FL-huPAR2, A549, Flp-In CHO_maPAR2 (rhesus, Macaca mulatta), Flp-In CHO_moPAR2 (mouse), and PAR2-negative cell lines (parental Flp-In CHO, Jurkat, Flp-In CHO_huPAR1).

[0232] Example 4 - Expression and purification of Fab fragments and IgG

[0233] Subcloning into Fab expression vector

[0234] If desired and to facilitate rapid expression of soluble Fab in E. coli, the selected The Fab encoding insert of the phage was subcloned from the pYPdis10 display vector into a Fab expression vector (pYBex10_h_Fab-FH) containing the desired features. Subcloning was performed by triple digestion with XbaI | EcoRI-HF | PstI-HF.

[0235] For full-length FabCys expression in HKB11 cells, selected candidates were also cloned into the pYMex10_h_FabCys-AviH expression vector. Subcloning was performed by excising the antibody encoding fragment from the pYMex_h_IgG1f_AEASS source vector with restriction enzymes NdeI│XhoI and ligating into the target vector pYMex10_h_FabCys-Avi-His.

[0236] Subcloning into IgG expression vector

[0237] For full-length IgG expression in HKB11 cells, selected candidates were cloned into pYMex10_h_IgG1f_AEASS expression vector. Subcloning was performed using a method that conveniently and efficiently converts a large number of sequence-unique Fab clones into IgG format. In brief, VH coding fragments (flanked by restriction sites of NheI│XhoI), VL coding fragments (flanked by restriction sites of NdeI│KpnI), and κ or λ-specific eukaryotic pYMin expression cassettes (flanked by restriction sites of KpnI│NheI) were cloned into a receptor vector for expression in mammalian cells (digested with NdeI│XhoI) with one-step or two-step cloning. After transformation of E. coli XL-1blue cells, quality control of single clones was performed by colony PCR and sequencing of the entire insertion region.

[0238] For large-scale expression in HKB11 cells, selected candidates were cloned into the pCMex003 / 004_kan_h_IgG1f_AEASS expression vector containing the Zeocin resistance gene for stable pool generation. Briefly, the VH encoding DNA fragment, the VL encoding DNA fragment and the lambda-specific eukaryotic pYMin expression cassette were cloned into the acceptor vector pCMex003 / 004_kan_h_IgG1f_AEASS.

[0239] Generation of bacterial lysates containing Fab fragments

[0240] Glycerol stocks from the master plate were used to inoculate 96-well / 384-well microtiter plates prefilled with growth medium (2xYT containing chloramphenicol, IPTG and 0.1% glucose). The plates were incubated at 37°C for bacterial growth and shaken overnight at 22°C for Fab expression. The next day, the expression culture was lysed by adding BEL buffer containing borate buffer, EDTA and lysozyme. Depending on the selected plate format and application, the volume was adjusted and the blocking scheme was adjusted accordingly. If the lysate is used for sensitive cell screening, such as functional assays, EDTA is omitted.

[0241] Exploratory scale production of Fab fragments

[0242] The expression of the Fab fragment encoded by the bacterial expression vector in Escherichia coli TG1 F- cells was performed in shake flask cultures using 500 mL of 2xYT medium supplemented with 0.1% glucose and 34 μg / mL chloramphenicol. The culture was shaken until OD600 reached a value of 0.5. Fab expression was induced by adding IPTG (isopropyl-β-D-thiogalactoside) and further culturing for 20 hours. Cells were harvested and destroyed using lysozyme. The His6-tagged Fab fragment was separated by IMAC (Bio-Rad│Germany) and eluted using imidazole. Buffer exchange was performed to 1×Dulbecco's PBS (pH 7.2) using a 'PD10' column (GE Healthcare│Germany). The sample was sterile filtered (0.2 μm). Protein concentration was determined by UV-spectrophotometry. The purity of the sample was analyzed in denaturing, non-reducing 15% SDS-PAGE. The homogeneity of the Fab preparation in the native state was determined by size exclusion chromatography (HP-SEC) using calibration standards.

[0243] Microscale production of IgG in mammalian cells

[0244] Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both IgG heavy and light chains. Cell culture supernatants were harvested 7 days after transfection and protein A affinity chromatography was performed using a liquid handling station (MabSelect SURE│GE Healthcare). Unless otherwise specified, the samples were retained in neutralized elution buffer (NaPS: 137mM Na phosphate, 81mM NaCl, pH 7). The samples were sterile filtered (0.2 μm pore size). Protein concentration was determined by UV-spectrophotometry, and CE-SDS (LabChip GXII│Perkin Elmer│USA) was used to analyze the purity of IgG under denaturing, reducing conditions. HP-SEC was performed to analyze the IgG preparations in the native state.

[0245] Exploratory scale production of IgG

[0246] Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both IgG heavy and light chains. Cell culture supernatants were harvested on the 3rd or 6th day after transfection and subjected to standard protein A affinity chromatography analysis (MabSelect SURE│GE Healthcare). Unless otherwise stated, buffer exchange was performed to 1×Dulbcecco's PBS (pH 7.2│Invitrogen), and the samples were sterile filtered (0.2 μm pore size). Protein concentration was determined by UV-spectrophotometry, and CE-SDS (LabChip GXII│Perkin Elmer│USA) was used to analyze the purity of IgG under denaturing, reducing and non-reducing conditions. HP-SEC was performed to analyze the IgG preparations in the native state.

[0247] Exploratory scale production of AviHis-tagged FabCys

[0248] Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of disulfide-bridged FabCysAviHis. Cell culture supernatants were harvested on day 3 or 7 after transfection and subjected to metal ion affinity chromatography (Protino Ni-NTA | Macherey Nagel). Unless otherwise specified, buffer exchange was performed to 1× Dulbecco's PBS (pH 7.2 | Invitrogen) and the samples were sterile filtered (0.2 μm pore size). Protein concentration was determined by UV-spectrophotometry, and CE-SDS (LabChip GXII | Perkin Elmer | USA) was used to analyze the purity of FabCysAviHis under denaturing, reducing and non-reducing conditions. HP-SEC was performed to analyze FabCysAviHis preparations in the native state.

[0249] IgG production for in vivo characterization

[0250] Material production is carried out by establishing a stable HKB11 cell pool. Eukaryotic HKB11 cells are transfected with mammalian expression vector DNA encoding both IgG heavy and light chains. In order to produce a stably expressed cell pool, the corresponding vector also contains a zeocin resistance gene in addition. Three days after transfection, selection is started by adding 160 μg / mL zeocin to the cell suspension. During selection, cell count and viability are initially reduced. 20 to 30 days after transfection, cells begin to recover. When reaching about 80% viability, the stable library is scaled up to the desired amount. Harvest cell culture supernatant on the 6th day after transfection, and perform protein A affinity chromatography analysis (MabSelect SURE│GE Healthcare). If necessary, a second purification step (preparative SEC│Superdex 200│GE Healthcare) is performed to remove aggregates. Buffer exchange is performed to 1×Dulbecco's PBS (pH 7.2│Invitrogen), and the sample is sterile filtered (0.2μm pore size). Protein concentration was determined by UV-spectrophotometry, and CE-SDS (LabChip GXII│Perkin Elmer│USA) was used to analyze the purity of IgG under denaturing, reducing and non-reducing conditions. HP-SEC was performed to analyze the IgG preparations in the native state. Endotoxin levels were determined by KQCL assay (Lonza). Mass spectrometry (100MDL43) was used to determine protein identity.

[0251] Example 5 - Analysis of clone profiles of lead isolates

[0252] A PCR-based assay for human PAR2 was performed to identify functional primary hits. IP-One Gαq assay of cells

[0253] After full validation of the clones in ELISA and IntelliCyt, functional characterization was performed to determine the PAR2 binding status. IP-One Gαq assay, the exploratory scale purified IgG1f_AEASS clone was tested in duplicate at two concentrations for its ability to inhibit activation of the PAR2 receptor by a synthetic agonist, the activating peptide (2-Furyol-LIGRO).

[0254] HEK293F cells were infected with 2% v / v human PAR2 BacMam virus in growth medium (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin / streptomycin) in the presence of 0.5 mM sodium butyrate and cultured in suspension for 24 hours in a humidified incubator with a 5% CO2 atmosphere at 37°C. On the day of the experiment, cells were harvested and resuspended in an assay buffer containing LiCl and 0.5% bovine serum albumin (BSA, Sigma) at a density of 1×10^6 / ml (1 part stimulation buffer (Cisbio): 5 parts ddH20). Antibodies were tested at stock concentrations or after an initial 1:2 dilution in PBS. 5 μl samples of antibodies at either concentration were added in duplicate to a half-area white 96-well plate (Corning), followed by 25 μl PAR2 cell suspension (PAR2 of cellsuspension). The plate was incubated at 37°C for 30 minutes, and then 5 μl / well of the activation peptide was added to make the final agonist attack concentration of 6.28 nM. The final assay antibody screening was performed at a 1:7 or 1:14 dilution of the Ab stock. The plate was incubated at 37°C for another 30 minutes, and then 10 μl IP-One detection kit (Cisbio) was added in the lysis buffer. The cell plate was placed on a plate oscillator for 1 hour at room temperature (RT), and then the plate was read using a standard HTRF protocol on a PHERAstar FS microplate reader (BMG Labtech), where excitation was at 335 nm and emission was read at 620 nm and 665 nm. The HTRF ratio was calculated by equation 1. The response was normalized to the inhibition % of the activation peptide IP-One response at a final assay concentration of 6.28 nM (EC80) ( Figure 6 ).

[0255] Equation 1: Calculation of HTRF ratio

[0256]

[0257] Table 2: Inhibition of peptide-induced human PAR2 agonism

[0258] Use in peptide inhibition assays Functional characterization of Y021171, Y022063, and Y022054 & Y022065 hIgG1f_AEASS was performed in duplicate at the highest concentration by IP-One Gαq assay. Figure 6 Shown in.

[0259]

[0260] Affinity determination

[0261] K by SPR using an antibody capture setup D , ka and kd determination

[0262] For K D For determination, monomeric fractions (at least 90% monomer content as analyzed by analytical SEC) of the antibody protein (Fab fragment or IgG) were used. Affinity was determined by kinetic characterization using Biacore (Biacore T200) or Octet (QK384 or HTX) instruments as described below.

[0263] Antibodies were captured on an anti-human IgG chip surface followed by injection of 100 nM PAR 2. Data were fit to a 1 :1 interaction model, but clone 'Y021160' was excluded, in which case a heterogeneous ligand model was used, and the slowly dissociating component was used for comparison.

[0264] Table 3: Biacore analysis of lead isolate clones

[0265]

[0266] A suitable high capacity capture surface is prepared, for example by covalently immobilizing a suitable capture ligand to a CM5 chip (Biacore, GE Healthcare) or AR2G sensor (forteBIO, Pall Corp.) using EDC / NHS chemistry, or by loading a SA sensor with a biotinylated capture reagent. Some examples of suitable capture systems are anti-hu-Fc antibodies (Biacore, GE Healthcare), protein A sensors (forteBIO, Pall Corp.), MabSelect Sure ligand (GE Healthcare), and anti-His-tag antibodies (Genscript). During the kinetic experiments, six to eight different analyte concentrations (2 nSerial dilutions) were used for analysis. After each cycle, the sensor surface was regenerated to remove the captured antibody / antigen complex while maintaining the integrity of the capture surface. A blank injection of running buffer was used for reference. In all experiments, the assay buffer was matched with the formulation of PAR2 protein, i.e., basic buffer and detergent. IgG was captured by Fc-fragment, and FL-N PAR2 StaR (PAR2-3) dissolved by detergent (LMNG / CHS) was used as the analyte in solution. 50mM HEPES pH 7.5, 150mM NaCl, 0.02% LMNG were used as assay buffer. CM3 sensor chip was used as the basis for fixing high-density anti-hu-Fc capture antibody (BR-1008-39, GE Healthcare). IgG was captured at a relatively high level of about 500RU to achieve a higher ratio of specific binding to nonspecific binding (while still maintaining conditions suitable for kinetic characterization). Sensorgrams were evaluated using the evaluation software of the corresponding instrument, i.e., Biacore T200 Evaluation Software 3.x (Biacore, GE Healthcare) or forteBIO Octet Data Analysis (forteBIO, Pall Corp.). All sensorgrams were fitted to a 1:1 binding model to determine the ka and kd rate constants, which were used to calculate K D In the case of Y021160, a heterogeneous ligand model was applied and the slowly dissociating component was used to calculate ka, kd, and K D .

[0267] For three purified IgG1f_AEASS clones Y021171, Y022054 and Y022063, the affinity of them to human PAR2-3 variants (P157: 1-377, glycosylated, pBacMamHisStrepII, full length) was determined by SPR fixed IgG and probed with FL-N PAR2 StaR dissolved by detergent (LMNG / CHS). For Y022063 and Y021171, the affinity was 22nM and 20nM respectively. Compared with other candidates, Y022054 showed a 10-fold higher affinity of 2.5nM. Y021171 showed rapid association (ka), but also showed very rapid dissociation (kd). From binding affinity to functional inhibition, it is assumed that the affinity required for antagonistic anti-PAR2 antibodies is within the range of <100pM. During affinity maturation activities, an “off rate selection” strategy (Hawkins et al. 1992) was implemented to improve K D Features, especially for Y021171.

[0268] Biacore T200 instrument (GE Healthcare) is used to carry out the mensuration of PAR2 and anti-PAR2 antibody interaction.By amine coupling, anti-human IgG antibody (human antibody capture kit (Human Antibody Capture Kit), GE Healthcare) is fixed on sensor chip CM3 (GE Healthcare) so that surface density is 3500 to 4500 resonance units (resonance unit, RU).In HBS-EP+ buffer (GE Healthcare), fix at 25 ℃.Then buffer is changed to 50mM HEPES, pH 7.5,150mM NaCl, 0.02%LMNG, 0.002%CHS. Each cycle of the PAR2-antibody interaction assay consisted of antibody capture (injection of 100 nM antibody for 2 min at a flow rate of 5 μL / min), PAR2 injection (blank or 250 nM, contact and dissociation times of 2 min and 3 min, respectively, at a flow rate of 20 μL / min), and chip surface regeneration (20 μL / min for 30 sec) using a regeneration solution from a human antibody capture kit (GE Healthcare). Blank-subtracted data were fitted to a 1:1 interaction model to obtain association and dissociation rate constants ka (kon) and kd (koff) and the affinity constant K D The measured values ​​between each experimental dataset are within the acceptable range of the method.

[0269] Table 4: Affinity determination of functional candidates Y021171, Y022063 and Y022054 by SPR

[0270] Antibodies were captured on an anti-human IgG chip surface followed by injection of 250 nM PAR2. Data were fit to a 1:1 interaction model.

[0271]

[0272] Example 6 - Affinity maturation / lead optimization and phage selection

[0273] In the IP One assay, several IgG clones, including Y021171, Y022054, Y022063, and Y022065, partially inhibited PAR2-AP activation. Several other clones (but not any of the four functional candidates) showed cross-reactivity to non-human primate (NHP) PAR2, and despite the high degree of identity between human and rhesus PAR2 sequences in the extracellular domain, none of the clones showed significant cross-reactivity to FlpIn CHO_maPAR2 (rhesus, Macaca mulatta) or FlpIn CHO_moPAR2 (mouse) prior to affinity maturation.

[0274] Mature Library

[0275] Generation of phage maturation library: Cloning of mature library was performed on CysDisplay encoding parental Fab fragments. TM If the DNA sequence encoding the parent Fab fragment is not already present in the CysDisplay TM vector, they were transferred to the corresponding vector by restriction digestion and ligation before library cloning. In order to improve affinity and biological activity and reduce the non-specificity of the selected antibody candidates, the CDR-L3 and CDRH-1 / CDR-H2 regions were optimized in parallel. Due to its modular structure, the MorphoSys antibody library is designed for affinity optimization by CDR exchange, in which a single CDR of an antibody is excised and replaced by a single member of a large CDR library (Prassler et al. 2009). Mature Modules ( maturation module (YMM) is designed based on the Ylanthia library and uses The maturation library was pre-constructed using the Ylanthia maturation stuffer (YMS) technology (van den Brulle et al. 2008). The maturation library was generated separately for each maturation candidate. To monitor the cloning efficiency, the parental CDR-L3 was replaced with the Ylanthia maturation stuffer (YMS) and then the diversified LCDR-L3 YMM was inserted. The digested vector fragment was ligated with a 2-fold molar excess of the insert fragment carrying the diversified CDR-L3. The same procedure was applied to the diversification of CDRH-1 & CDR-H2. The ligation mixture was electroporated in Escherichia coli MC1061F' cells to produce >108 independent colonies. The library was amplified as described in the literature (Tiller et al. 2013). For quality control, approximately 10 single clones of each library were randomly picked and Sanger sequenced.

[0276] Mature candidates are selected based on the following scientific evidence and criteria:

[0277] 1. Avoid candidates with increased risk of off-target binding due to increased binding to:

[0278] PAR2-negative antigen (ELISA): empty nanodiscs, mouse IgG, anti-StrepII, bRIL

[0279] PAR2-negative cell lines (FACS): parental Flp-In CHO cells, Flp-In CHO_huPAR1 and Jurkat cells

[0280] 2. Not selecting candidates that preferentially bind to the membrane distal part of the N-terminus (assessed by comparative ELISA of FL-StaR Nanodiscs versus truncated StaR Nanodiscs)

[0281] The human PAR2-specific IgG1f_AEASS candidates were ranked as follows:

[0282] 1. Functionally active candidates;

[0283] 2. Binding to human PAR2 expressed on the cell surface, with a focus on the functionally active HEK293F cell line infected with WT FL-huPAR2BacMam as a prerequisite for assessing functionality;

[0284] 3. Compete with the binding of the functionally active candidate Y021171 candidate and / or the functionally active R&D anti-PAR2 reagent mAb MAB3949, as the published PAR2 structure (Cheng et al., 2017) suggests that this reagent antibody interacts with the extracellular loop.

[0285] Table 5: Overview of mature candidates

[0286]

[0287] The main goal of affinity maturation is to improve existing characteristics and does not usually result in displacement of the target epitope. It was observed that in terms of IgG characteristics, functional candidates did not have cross-reactions to rhesus monkey PAR2, whereas non-functional candidates bound to the substance. Due to the lack of recombinant rhesus monkey PAR2 StaR antigenic material, a differential whole-cell panning strategy (DWCP) was implemented.

[0288] The SPR data highlighted the fast dissociation rate of Y021171 (kd (1 / sec) of 0.01). Therefore, during the third round of maturation panning, a so-called "dissociation rate selection" procedure (Hawkins et al., 1992) was implemented to enrich for candidates with improved dissociation rates. Since SPR data were not available for all maturation candidates, this approach was limited to panning strategies that included functional candidates.

[0289] During maturation, wash stringency is increased compared to the panning rounds, e.g., by increasing the number and duration of wash steps (e.g., overnight washes). Long or overnight washes are used in off-rate selection strategies along with the addition of soluble antigen to the wash buffer to prevent any rebinding of antibody-phage to the immobilized antigen.

[0290] Nine parental clones were progressed to affinity maturation activities covering all available binding spectra and potential modes of action. To improve the affinity and bioactivity of previously selected antibody fragments, CDR-L3 or CDR-H1 & CDR-H2 regions were exchanged in parallel via diversification modules (Prassler et al. 2009). The maturation module (YMM) is used to preserve fixed VH / VL human germline framework pairs to avoid cross-cloning or alternative framework combinations. Before affinity maturation library cloning, the parental Fab fragments are transferred from the corresponding expression vectors to the CysDisplay TMvector. CDR-L3 and CDR-H1 & CDR-H2 libraries were cloned separately for each maturation candidate and combined. Affinity maturation was customized for 6 individual candidates, namely all functional candidates (Y021171, Y022054, Y022063, Y022065) as well as Y022059 and Y022069. Weaker candidates were matured in the library (Y021160, Y022075 and Y022079). Maturation libraries were generated for individual diversity of CDR-L3 and CDR-H1 & CDR-H2. Maturation panning was designed to focus on high stringency solid phase panning using full-length or N-terminally truncated nanodisc-embedded PAR2 StaR, as well as on the generation or improvement of rhesus macaque PAR2 cross-reactivity. After affinity maturation and screening, the project team successfully identified a large number of highly specific Antibody candidates. Clones progressed to IgG conversion and in-depth characterization.

[0291] Affinity maturation panning selection

[0292] In order to improve the affinity and bioactivity of previously selected antibody fragments, the CDR-L3 and CDR-H2 regions were exchanged in parallel by means of diversification modules (Prassler et al. 2009). Before library cloning for affinity maturation, the parental Fab fragments were transferred from the corresponding expression vectors to CysDisplay TM The vector was added to the vector. To select candidates with improved affinity, three rounds of mature panning were performed on phages derived from the mature library. The panning stringency was increased by reducing the antigen concentration in each panning round (Low et al. 1996). In addition to antigen reduction, the selected strategies were also subjected to off-rate selection (Hawkins et al. 1992). These strategies were combined with long washing steps and the addition of soluble antigen to the washing buffer to prevent any rebinding of antibody-phage to the fixed antigen.

[0293] After affinity maturation, the DNA fragment encoding the modified variable region was directly substituted into the pYMex10_h_IgG1f_AEASS expression vector encoding the parental IgG. Briefly, the vector components encoding the parental VH or VL were removed with appropriate restriction enzymes (NheI│XhoI for VH, NdeI│KpnI for VL), and the fragment encoding the affinity matured variable region was inserted and expressed and purified (as described above) to produce full-length IgG.

[0294] Example 7 - Lead optimisation and clonal profiling

[0295] Purified The IgG profiling included assessment of binding specificity and cross-reactivity to human, mouse, rhesus and marmoset PAR2-expressing cells, such as good binding of StaR embedded in nanodiscs to PAR2 (reconfirmed by ELISA) and good binding of PAR2-expressing cells to PAR2 (reconfirmed by flow cytometry). After affinity maturation, several IgG clones (mainly derived from the functionally active parental IgG Y022065) were identified as having antagonistic activity, as they were able to completely inhibit human PAR2 receptor activation, thereby emphasizing their specificity for functionally relevant epitopes.

[0296] Preliminary screening of truncated or FL PAR2 StaR nanodiscs in ELISA showed excellent improvement in binding compared to the parental antibody. Kd ranked ELISA was performed for all panning subcodes. Plotting of the data obtained with the classical ELISA setup relative to the Koff ranked ELISA setup showed a clear linear correlation of the signals, indicating that most mature candidates have slow Koff values. For analysis, the loss of binding signal was calculated as the ratio of the signal over background (S / BG) ​​of the classical ELISA setup to the S / BG of the Koff ranked ELISA: [truncated / FL PAR2 StaR nanodisc S / BG] / [Koff ELISA truncated / FL PAR2 StaR nanodisc S / BG]. The poor Koff estimates obtained by ELISA ranking were associated with low signals in the classical ELISA setup. Candidates with a >2-fold loss in signal ratio were classified as candidates with potential fast degradation rates. 94% of mature candidates showed no or small signal changes (<2-fold), of which 67% were derivatives of the parental candidate Y021171.

[0297] In the Koff ranking ELISA, parent Y021171 showed the greatest loss of binding signal, while other parent antibody candidates showed no or small signal changes. The results are consistent with the SPR data previously generated for Y021171, in which the candidates were classified as fast dissociation rates, kd (1 / sec) = 0.01058. 6% of the mature candidates showed an increase in signal change (2 to 4.5 times), indicating a fast degradation rate. 92% of these were mature derivatives of Y021171 (approximately 33% of all Y021171 candidates) ( Figure 7 ).

[0298] Whole-cell binding to PAR2 as determined by IntelliCyt

[0299] In Intellicyt, screening of 2200 lead candidates on FlpIn CHO-V5His-huPAR2 relative to FlpIn CHO parental cells showed that most mature derivatives had improved binding compared to their respective parent antibody candidates. The best improvements were achieved with mature panning of Y021171, Y022065, Y022069 and the mature parents in the library, with derivatives of Y022079 expected to be the main family. All candidates showed low binding to FlpIn CHO parental cells (S / BG <1.5). Over 800 candidates showed good or excellent binding to FlpIn CHO-V5His-huPAR2, with improvements of 2 to 5 fold, 5 to 10 fold, or >10 fold (Signal / Signal Parent) compared to FlpIn CHO parental cells. Figures 8 to 11 ).

[0300] PAR2-specific protein panel profiling (3P)

[0301] For proteomic profiling (Frese et al. 2013), 32 different proteins and controls were coated at a concentration of 1.0 μg / mL on two 384-well MSD standard plates (Meso Scale Discovery, MTP 384-well MA6000, #L21XA) at 4°C overnight. The coating solution was discarded and the plates were blocked with 50 μL of 3% (w / v) BSA or 3% (w / v) skim milk powder in PBS on a microtiter plate shaker (about 500 rpm) at room temperature for one hour, followed by three washing steps with 50 μL of wash buffer (PBS with 0.05% (v / v) Tween 20). Antibody samples (Fab fragments or IgG) were diluted to 100 nM and 10 nM in assay buffer (PBS with 0.5% (w / v) BSA, 0.05% (v / v) Tween 20). MOR reference mAb anti-lysozyme MOR03207 Fab or IgG (depending on the sample form) and assay buffer were used as controls. Samples and controls (30 μL / well) were added and incubated on a microtiter plate oscillator for three hours at room temperature. The plate was washed three times, and 30 μL of detection antibody (ECL-labeled anti-human Fab, 1:2000 dilution) was added to each well, and incubated on a microtiter plate oscillator (about 500 rpm) for one hour. After the MSD plate was washed and 35 μL / well of MSD Read Buffer T with a surfactant was added, the electrochemiluminescent signal was detected using Sector Imager 6000 (Meso Scale Discovery│Gaithersburg│MD│USA). In order to evaluate, the signal of the antibody sample on a certain protein was divided by the corresponding signal of the anti-lysozyme reference mAb MOR03207 to obtain the binding rate. Most clones, including all later mature clones, showed no non-specific binding tendency or a small non-specific binding tendency.

[0302] Example 8 - Functional Characterization of Lead Optimized Clones

[0303] Functional assay: Cell-based IP-One Gαq assay

[0304] The assay method followed that of Example 5. The antibody stock was serially diluted 1:2 according to a 10-point concentration curve to determine the IC 50 In separate experiments, lead optimized antibody clones were tested in challenges against an activation peptide (2-Furyol-LIGRO, 6.28 nM; Table 6), bovine trypsin (2 nM; Table 7), or a PAR1 activation peptide (SFLLR-NH2, 632 nM; Table 8).

[0305] The IP-One HTRF results obtained when screening the parental antibody clones in challenge with the activation peptide were normalized to the effect of 10 μM MAB3949 ( Fig.12 ).

[0306] The IP-One HTRF results obtained when screening the parental antibody clones in a challenge against bovine trypsin were normalized to the effect of a 1 μM benchmark 1 ( Fig.13 ).

[0307] IP-One HTRF results were normalized to the effect of 10 μM MAB3949 when screened against the PAR1 peptide SFFLR-NH2 trypsin challenge (Table 8).

[0308] Normalized data were fitted to a 4-parameter sigmoidal dose-response curve in GraphPad Prism version 7.04 (Equation 2).

[0309] The data in Table 8 demonstrate that the Y022883 antibody is able to block PAR2 activation by the PAR1 activating peptide.

[0310] Equation 2: 4-parameter sigmoid-dose response fit

[0311] Y = lower limit + (upper limit - lower limit) / (1 + ((X^ Hill Slope) / (IC50^ Hill Slope)))

[0312] Among them, IC 50 is the concentration (in nM) that inhibits the AP / trypsin response by 50%, and the %max inhibition is the maximal inhibition of AP / trypsin (taken from the smallest asymptote, ie the 'bottom', of the curve).

[0313] Table 6: Functional characterization of lead optimized clones against activation peptides. The IP-One Gαq kit showed activity in duplicate in the IP-One Human PAR2 Antagonist assay and was also active in the bovine trypsin challenge assay.

[0314]

[0315] Table 7: Functional characterization of the lead optimized clones against bovine trypsin. Activity was shown in the IP-One Human PAR2 Antagonist assay using the IP-One Gαq Kit and was also active in the activating peptide challenge assay. Where activity could be determined in only one replicate, this is indicated in the column called 'Number of Replicates'.

[0316]

[0317] Affinity matured lead clones derived from the parental IgG Y022065 showed the greatest proportion of active lead clones active against both activation peptide and trypsin. Tables 6 and 7 list the functional IgG actives that exhibited IC 50 Values ​​n≥ 1. Table 8: IP-One HTRF results normalized to the effect of 10 μM MAB3949 when screened against trypsin challenge of the PAR1 peptide SFFLR-NH2.

[0318]

[0319] Functional assay: Cell-based FLIPR calcium mobilization Gαq assay using HT-29 cell line

[0320] IC of lead clones in the IP-One recombinant human PAR2 in vitro assay 50 Following this determination, the activity was determined on an immortalized human colon adenocarcinoma cell line (HT-29) that endogenously expresses the PAR2 receptor.

[0321] HT-29 cells (ATCC HTB-38) were maintained in continuous culture using DMEM medium with high glucose (25 mM), without sodium pyruvate, but with GlutaMAX (Gibco, Paisley, UK), 10% heat-inactivated fetal bovine serum, and penicillin / streptomycin (100 units / mL penicillin and 100 μg / mL streptomycin) in a humidified incubator at 37° C. with a 5% CO atmosphere. The medium was changed every 2 days from the second day after seeding, and cells were harvested by 0.05% trypsin / EDTA in the logarithmic growth phase after reaching 80% to 90% confluence.

[0322] Cells were plated at a cell density of 5,000 cells / well in 50 μL / well culture medium in 384-well black wall plates (Corning) and incubated at 37°C in a humidified incubator with a 5% CO2 atmosphere for 24 hours. On the day of the experiment, the cell culture medium was removed and 50 μL of assay buffer (HBSS 20 mM HEPES pH 7.4 buffer containing 0.1% BSA) containing calcium 5 dye diluted 1:20 from stock (Molecular Devices) was added. The plates were incubated at 37°C for another 45 minutes, and the cells were then equilibrated at room temperature for another 15 minutes.

[0323] IgG lead clone is prepared in assay buffer, and serial dilution is to produce 10 point curves.Use FLIPRTetra (Molecular Devices) pipette (10 μ L) to add IgG dose response curve online, and measure calcium response in the time period of 5 minutes.At 37 ℃, plate is incubated again for 60 minutes, then add activation peptide (630nM) or bovine trypsin (63nM) online with 10 μ L / well, and further measure the people PAR2 activated by calcium mobilization carried out in the time period of 5 minutes.

[0324] Data were analyzed by extracting the Max-Min raw data files and analyzed by Equation 2. For the activation peptide and bovine trypsin assays, data were normalized to MAB3949 or benchmark 1, respectively.

[0325] Table 9: Functional characterization of lead clones showing full dose-dependent inhibition n≥3 in the HT-29 FLIPR calcium mobilization assay normalized to the corresponding antibody control against bovine trypsin or activation peptide (AP).

[0326]

[0327] Functional assays: Cell-based IP-One Gαq assays for cynomolgus monkey PAR2 and rhesus monkey PAR2

[0328] use The IP-One Gαq assay was functionally characterized in duplicate at two concentrations to test the lead clone cross-species antagonist activity against cynomolgus and rhesus PAR2 receptors in response to challenge with the synthetic agonist SLIGKV.

[0329] HEK293f cells were infected with 2.5% v / v cynomolgus PAR2 BacMam virus or 2.5% v / v rhesus PAR2 BacMam virus in growth medium (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin / streptomycin) in the presence of 0.5 mM sodium butyrate and cultured in suspension for 24 hours at 37° C. in a humidified incubator with a 5% CO2 atmosphere. On the day of the experiment, cells were harvested and resuspended in an assay buffer containing LiCl and 0.5% bovine serum albumin (BSA, Sigma) at a density of 1×10^6 / ml (1 part stimulation buffer (Cisbio): 5 parts ddH20).

[0330] Antibody stocks were serially diluted 1:2 using a 10-point concentration curve to determine the IC 50Value. Antibody lead clones were tested against SLIGKV (1 μM) attack. 5 μl aliquots of either concentration of antibody were added in duplicate to a half-area white 96-well plate (Corning), followed by 25 μl PAR2 cell suspension (PAR2 of cellsuspension). The plate was incubated at 37°C for 30 minutes, and then 5 μl / well of SLIGKV was added to make the final agonist attack concentration of 1 μM. The plate was incubated at 37°C for another 30 minutes, and then 10 μl IP-Onedetection kit (Cisbio) was added in lysis buffer. The cell plate was placed on a plate oscillator for 1 hour at room temperature (RT), and then the plate was read using a standard HTRF protocol on a PHERAstar FS microplate reader (BMG Labtech), with excitation at 335nm and emission readout at 620nm and 665nm. The HTRF ratio was calculated by equation 1. Responses were normalized to the % fold inhibition at 1 μM SLIGKV alone final assay concentration, and results were fit to a 4-parameter sigmoidal dose-response curve in GraphPad Prism version 7.04 (Equation 2).

[0331] Table 10: Functional characterization of lead clones against SLIGKV that reproducibly showed antagonist activity in the Cisbio IP-One cynomolgus PAR2 or rhesus PAR2 receptor assays.

[0332]

[0333] Functional assay: Cell-based IP-One Gαq assay for human PAR1

[0334] use IP-One Gαq assay Functional characterization was performed in duplicate at two concentrations to test the lead clone cross-species antagonist activity against the human PAR1 receptor in response to challenge with the synthetic agonist SFLLR.

[0335] HEK293f cells were infected with 5% v / v human PAR1 BacMam virus in growth medium (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin / streptomycin) in the presence of 0.5 mM sodium butyrate and cultured in suspension for 24 hours at 37° C. in a humidified incubator with a 5% CO2 atmosphere. On the day of the experiment, cells were harvested and resuspended in assay buffer containing LiCl and 0.5% bovine serum albumin (BSA, Sigma) at a density of 1×10^6 / ml (1 part stimulation buffer (Cisbio): 5 parts ddH20).

[0336] Antibody stocks were serially diluted 1:2 using a 10-point concentration curve to determine the IC 50 Value. Antibody lead clones are tested in terms of attack for SFLLR (100nM). 5 μl aliquots of the antibody of any concentration are added in duplicate to half-area white 96-well plates (Corning), followed by 25 μl PAR2 cell suspensions (PAR2 of cellsuspension). Plates are incubated at 37°C for 30 minutes, then the SFLLR of 5 μl / well is added, so that the final agonist attack concentration is 100nM. Plates are incubated at 37°C for another 30 minutes, then the 10 μl IP-Onedetection kit (Cisbio) added in lysis buffer is added. Cell plates are placed on a plate oscillator and continue for 1 hour at room temperature (RT), then standard HTRF protocols are used to read plates on a PHERAstar FS microplate reader (BMG Labtech), wherein excitation is at 335nm and emission is read at 620nm and 665nm. HTRF ratios are calculated by equation 1. Responses were normalized to the % fold inhibition at 100 nM final assay concentration of SFLLR alone, and results were fit to a 4-parameter sigmoidal dose-response curve in GraphPad Prism version 7.04 (Equation 2).

[0337] As a positive antagonist control for the PAR1 human in vitro assay, a dose response curve of the small molecule PAR1 antagonist Vorapaxar (Axon Medchem Cat 1755) was incubated (tested in a 3-fold dilution series from 10 μM final assay concentration) and the IC was determined. 50 .

[0338] Table 11: Functional characterization of lead clones against SFLLR in the Cisbio IP-One human PAR1 selective assay

[0339]

[0340] Pilot Panel - Functional Assay: Cell-based FLIPR calcium mobilization Gαq assay using HT-29 cell line

[0341] After scale-up, the lead clone was characterized in HT-29 cells (following the method of Example 8) for antagonist activity against 0.1 nM bovine trypsin challenge of endogenously expressed PAR2 ( Fig.14 ).

[0342] In addition, they also show dual activity in the following aspects: proteolytic cleavage by trypsin in the extracellular N-terminus, and inhibition of PAR2-AP activation. The analysis in the 3P specific assay highlighted several clones with enhanced binding nonspecific antigen tendency, and thus reduced the number of dual-activity IgGs for further consideration as therapeutic candidates. In order to refine the target epitope and conduct preliminary epitope assessment, these IgG clones that also passed the 3P assay QC were analyzed in the epitope binning experiment based on ELISA. All candidates showed competition with each other and possible targeting of the proximal binding region different from the N-terminal epitope of benchmark 1. The in-depth characterization of the final candidate selection also includes the affinity measurement performed by SPR.

[0343] Based on functional assays, sequence diversity and binding profiles, the number of remaining clones was further reduced. These candidates were generated in a monovalent FabCys-AviH format and tested for cross-functionality with cynomolgus monkey PAR2. In addition, affinity determination was improved using an optimized setup.

[0344] Y022870, Y022877, and Y022883 (three derivatives of Y022065) were selected as final candidates and defined as the “lead group” or “lead set” based primarily on functional assays, SPR affinity data, production QC, and nonspecific binding to 32 different proteins in a proteomic profiling (3P) assay.

[0345] These three kinds of candidate are carried out final characterization and compared with the set of six kinds of competitor antibodies.In-depth characterization shows that the lead set is combined with the PAR2 of purification and cell surface expression with the affinity within the scope of the competitor antibody.No off-target combination with the related protein PAR1, PAR3 and PAR4 is detected.Compared with all competitors (benchmark) antibodies, the lead set of the antibody provided herein can not only inhibit protease activation, but also can inhibit the peptide activation of PAR2, thereby serving as the dual inhibitor of PAR2 activation.

[0346] Table 12: In vitro cross-species summary of SLIGKV agonist challenge

[0347]

[0348] Example 9 - Affinity and kinetics determination of affinity matured antibody clones

[0349] Label-free methods using Biacore and Octet to determine KD and k off

[0350] For high-throughput kinetic evaluation of IgG, the dissociation rate constant (k) was determined using Biacore T200 or Octet (QK384 or HTX) instruments. off ). When using monomeric antigen material (PAR2 StaR), the same basic prerequisites, principles, and considerations as described for the full kinetic characterization apply. For all 88 purified IgGs, k off This was determined by Octet and showed a net improvement compared to the parental antibody. The kd [1 / sec] estimated for truncated huPAR2 StaR and FL-huPAR2 StaR ranged from 1.1E-4 to 1.0E-05.

[0351] Generally, only one analyte concentration is used for k off Samples evaluated directly from IgG supernatants or bacterial lysates (Fab fragments) were analyzed using Octet in antibody capture format. off Determine.As a prerequisite, monomeric antigen is used as analyte.IgG sample is captured on the streptavidin sensor loaded with high-density capture ligand (biotinylated MabSelect SuRe ligand) with medium capture level (0.4nm).Monomer antigen protein human PAR2-1 variant (P15155-377, truncated) and human PAR2-2 variant (P166:1-377, full length) are used as analytes in solution.Dissociation is monitored for up to 1500 seconds.The sensorgram recorded is fitted to a 1:1 binding model using the corresponding instrument specificity evaluation software (Biacore T200 evaluation software 2.x or 3.x│Octet data analysis 9.x or 10.x).

[0352] For all 11 IgG1f_AEASS, the affinity for human PAR2 was determined by SPR. IgG was captured by its Fc-fragment, and FL-N PAR2 StaR dissolved by detergent (LMNG / CHS) was used as the analyte in the solution. 50mM HEPES pH 7.5, 150mM NaCl, 0.02% LMNG were used as assay buffer. In order to minimize the artifacts (artefacts) caused by nonspecific interactions of FL-N PAR2 StaR with the sensor, the CM3 sensor was used as the basis for fixing high-density anti-hu-Fc capture antibodies (BR-1008-39, GE Healthcare). IgG was captured at a relatively high level of about 500RU, thereby achieving a higher ratio of specific binding to nonspecific binding (while still maintaining conditions suitable for kinetic characterization). Affinity was 6.5nM to 130pM. The k of the best candidate offThe value is the lower assay limit. The minimum k off 2.00E-05 seconds -1 (corresponding to 3% dissociation in 1500 seconds). A range of affinities with KD values ​​ranging from 6.5 nM to 130 pM were observed, and most showed similar affinity to the parental clone Y022065 (K D =5.3 nM), as expected.

[0353] Table 13: How to use the Octet system and soluble PAR2 SPR kinetics of identified affinity matured clones

[0354]

[0355]

[0356] Table 14: When using the Biacore system and soluble PAR2 Determined SPR kinetics and analysis of the first three affinity matured clones of interest

[0357]

[0358] Table 15: SPR kinetics as determined using the Biacore system using human PAR2 incorporated into nanodiscs ——Comparison of three affinity matured clones with a non-affinity matured clone (Y021171).

[0359]

[0360] Using recombinant PAR2 Protein affinity was measured by SPR and the effect of temperature on binding was evaluated: Binding of human PAR2 to immobilized mAb at 37°C and 40°C

[0361] Biacore T200 instrument (GE Healthcare) is used to carry out the mensuration of PAR2 and anti-PAR2 antibody interaction.By amine coupling, anti-human IgG antibody (human antibody capture kit, GE Healthcare) is fixed in the target flow cell and the reference flow cell of sensor chip CM3 (GE Healthcare) so that surface density is 2700 to 3000 resonance units (RU).In HBS-EP+ buffer (GE Healthcare), fix at 25 ℃.Then buffer is exchanged with 50mM HEPES, pH 7.5,150mM NaCl, 0.02% LMNG, 0.002% CHS for the PAR2-antibody interaction mensuration carried out at 25 or 37 ℃.Each cycle of mensuration starts from the antibody capture in the target flow cell to the level reaching 400 to 450RU.Then PAR2 is injected into the target flow cell and the reference flow cell with three kinds of concentrations, each kind of 5 minutes, and then dissociates in 30 minutes.Then the surface is regenerated with the solution provided in the test kit (human antibody capture kit, GE Healthcare). For each antibody tested, the first cycle was a blank (three injections of running buffer) and the second cycle was a 50, 100 and 200 nM PAR2 injection. The blank-subtracted data were fit to a 1:1 interaction model to obtain the association and dissociation rate constants k a and k d and the affinity constant K D .

[0362] Table 16: Summary of binding of lead clones after affinity maturation at 25°C and 37°C

[0363]

[0364] Evaluation to determine if lead clones exhibit pH-sensitive binding

[0365] Y22883 is captured to 280 to 320RU, and 100nM PAR2 is injected for 2 minutes. About 400 seconds after injection stops, pH 6.0 buffer is injected for 5 minutes to see whether it can cause faster dissociation. The experiment is designed in such a way that the dissociation rate constant cannot be numerically evaluated. However, it is obvious that the condition of pH 6.0 does not accelerate dissociation ( Fig.15 ). Whole-cell K expression by KinExA D Sure

[0366] To determine the apparent K using “native” PAR2 (i.e., PAR2 expressed on the cell surface), D, the KinExA method was applied to evaluate the subset of the lead clones, i.e. Y022870 and Y02283. In order to determine the equilibrium KD of surface expressed antigens (rather than soluble antigens), HEK-293F cells infected with BacMam expressing wild-type full-length human PAR2 were used for titration. The cells were titrated in incubation buffer (D-PBS of Gibco, 0.5% (w / v) BSA, 0.02% (v / v) sodium azide). The experiment consisted of two titrations with low and high fixed IgG concentrations. In two titration experiments (stoichiometry and affinity controlled conditions), the maximum amount of cells must contain a sufficiently high effective ligand concentration to fully saturate IgG. The mixture of IgG and cells was incubated overnight at room temperature under very gentle stirring to allow equilibrium to form. Subsequently, the formed cell-antibody complex was removed by centrifugation. The amount of free IgG in the supernatant was determined using goat anti-human F(ab')2 fragment specific antibody (Jackson ImmunoResearch, #109-005-097) coated on PMMA (polymethyl methacrylate) beads and fluorescence detection using anti-human F(ab')2 fragment specific Alexa Fluor 647 antibody (Jackson ImmunoResearch, #109-605-097). Since IgG molecules were used in the KinExA experiment, the values ​​obtained were labeled as 'apparent K D ', to indicate that bivalent binding on antigen presenting cells cannot be excluded and that binding may be enhanced by the contribution of avidity. The recorded titration curves were analyzed using the n-curve analysis tool using KinExAPro software 4.1.11 by applying the "equilibrium, whole cell" model. Y022870 binds to wild-type human PAR2 expressed on whole cells with an affinity of about 370 pM, whereas Y022883 has an affinity of about 260 pM ( Fig.16 ). The values ​​are consistent with the SPR determined using PAR2 nanodiscs (210 and 140 pM, respectively).

[0367] Example 10 - Epitope binning and analysis of affinity matured clones by flow cytometric analysis of receptor mutations and HDX

[0368] ELISA-based epitope binning by antibody competition

[0369] Epitope binning is carried out by the mensuration based on ELISA.Anti-human PAR2 IgG1f sample IgG is directly coated on microtiter plate with the concentration of 100nM.In parallel, the labeled antigen (for example, StaR embedded in nano-disc) of constant concentration (12.5nM to 25nM) is incubated with the second anti-human PAR2 IgG1f of 25 times of molar excess, so that all epitopes (maximum concentration 312.5nM to 625nM, 7-point titration, 1:4) are saturated for 1 hour, and complex is formed.For each IgG sample evaluated, internal positive control (self-competition) is included.Antigen-IgG complex is added to the plate coated with IgG and continues for 30 minutes.Antigen-IgG complex combined with coated IgG is detected by tag specific antibody (for example, anti-StrepII or anti-His), and the tag specific antibody is combined with suitable alkaline phosphatase (AP) coupled second antibody and 'AttoPhos' fluorescent substrate to detect.Multiple washing steps have been carried out between each determination step. In the case of competition for the same epitope, the antigen-IgG complex cannot bind to the IgG-coated plate, which translates into a low signal. Targeting different epitopes allows the antigen-IgG complex to bind to both the coated IgG and the IgG in solution simultaneously, resulting in a positive detection signal.

[0370] The competitive ELISA device is used to characterize the target epitope and epitope diversity of available candidates by epitope binning. 16 kinds of bifunctional PAR2 specific candidates were tested for binding competition to each other and for the binding competition to reference antibodies (including MAB3949 (R&D Systems) and benchmark 1). The set of these 16 kinds of antibodies comprises at least one derivative of each in five parent families, i.e. the clones (Y022079, Y022069, Y022065, Y021171, Y022059) derived from parent clones obtained by affinity maturation. All affinity maturation families compete with each other, and compete with anti-huPAR2 (MAB3949 R&D Systems). This shows that all these antibodies target the proximal binding region (i.e. the extracellular domain except the N-terminal). None of the candidates compete with benchmark mAb1. Benchmark mAb 1 was the only antibody in the panel tested that bound exclusively to full-length PAR2 (i.e., bound to a linear N-terminal epitope); these results suggest that the affinity-matured candidates bind to a distinctly different epitope that is more closely associated with or directly targets the extracellular loop region ( Fig.17 ).

[0371] Additionally, binding under StaR denaturing conditions was assessed to aid classification as recognition of a linear or conformational epitope. 97% of the IgG candidates tested showed a loss of binding and were therefore assigned as binding to a conformational epitope.

[0372] Hydrogen-deuterium exchange

[0373] Hydrogen deuterium exchange (HDX) is one of the methods that can be used to query and identify binding interfaces. Protein-protein interaction sites are probed by cataloging the hydrogen / deuterium exchange rates of amide hydrogens in the protein backbone. How fast this exchange occurs depends on the accessibility of those amide hydrogens to the solvent. Therefore, amides in exposed areas should have higher exchange rates than amides buried in the protein-protein interface because they have higher accessibility to the solvent environment.

[0374] The following apparatus and materials were used in the examples. Liquid chromatography separation was performed on a Thermo Vanquish UHPLC (Thermo Scientific) equipped with two temperature-controlled column chambers. Online digestion was performed on an Enzymate BEH pepsin column 2.1×50 mm (Waters) at 15°C, and the obtained peptides were separated on a Kinetex Evo C18 2.6μm 50×2.1 mm (Phenomenex) at 4°C. The mobile phase for peptide separation consisted of acetonitrile / 0.1% formic acid (FA) and water / 0.1% FA (Fisher Chemical). A 17.5-minute gradient of 0% to 40% acetonitrile / 0.1% FA was used to achieve separation. MS1 and MS / MS analysis was performed on the Orbitrap Fusion instrument (Thermo Scientific). Peptide analysis was performed using Proteome Discoverer 2.4 (Thermo Scientific) with a Byonic node, and HDX treatment was performed using HDExaminer 2.4 (Sierra Analytics). Genetically modified PAR2-21-377 (glycosylation sites removed) PAR2 StaR protein was purified in LMNG / CHS. Antigen-binding fragment (Fab) Y022883 was directly expressed (see Example 4) and the reference Fab was derived from papain digest of R&D IgG MAB3949.

[0375] A typical bottom-up HDX mass spectrometry (MS) procedure for HDX includes protein-Fab incubation, isotope labeling with deuterated buffer, quenching, proteolytic digestion, desalting / separation, and MS analysis. The PAR2-2 construct was diluted to 50 μM and incubated with equimolar concentrations of Fab Y022883 (or Fab MAB3949) at 22°C for 30 minutes. HDX was initiated by diluting the protein mixture in deuterated buffer (10 mM phosphate buffer pH 7.5, 150 mM NaCl) at 22°C for several time periods (continuous labeling): 1 minute, 10 minutes, 30 minutes, 2 hours. A 20-fold dilution was selected to achieve up to 90% to 95% D labeling of solvent accessible hydrogen (usually 2 μl of protein mixture was mixed with 38 μl of deuterated buffer). The PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. The deuterated PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex is then subjected to the same protocol as described above. To validate the approach, the HDX results obtained for PAR2 / MAB3949 Fab were mapped onto the published co-structure of PAR2 in complex with MAB3949 Fab (Cheng et al., Nature, 2017). The interactions inferred from HDX matched well with those observed in the crystal co-structure. PAR2-2 / FabY022883 HDX was then queried, and the data indicated interactions that were different from those observed for Fab MAB3949 (Cheng et al., Nature, 2017). Figures 18 to 20 ). The N-terminal region of PAR2 showed a unique deuterium-hydrogen exchange pattern, which may indicate that this region is involved in binding.

[0376] Table 17: List of PAR2-2 peptides identified by HDX that were determined to be located in the interaction region and therefore comprise the Y022883 antibody epitope.

[0377]

[0378] These HDX data suggest that ECL1 or ECL2 of PAR2 are much less likely to be involved in direct interactions with the Y022883PAR2 antibody; direct interactions observed using this approach are localized to the following regions: segment 1, helix 0 / 1 (close to the transmembrane domain and in contrast to other PAR2-targeting antibodies that bind to relatively distal regions of the receptor), and ECL3.

[0379] Mutation analysis using flow cytometry

[0380] PAR2 binding epitope mutations were evaluated by flow cytometry on HEK293F cells expressing human PAR2 as follows. Benchmark 1 antibody was used to monitor expression levels. Mutations introduced in WT PAR2 background targeted segment 1, spiral 0 / 1 (single mutation F59A, S60W, D62G, D62F, E63K, E63A and double mutation D62G / E63G) or ECL3 (single mutation Q317A, G318F, Q319A, Q319I). Various forms of PAR2 were introduced into HEK293F cells by BacMam infection.

[0381] 200 μL HEK293F-cells at 4×10^6 cells / mL were stirred at 4°C for 1 hour with 20 nM Y022883 or Benchmark 1 in FACS-buffer (PBS (Sigma, #F9665), 1% BSA (Sigma, #A9647) and Roche Complete Protease Inhibitor (#11836145001)). The cells were pelleted and washed three times with 200 μL FACS buffer. Subsequently, the cells were stirred at 4°C for 1 hour with 200 μL secondary phycocyanin (APC)-conjugated anti-human IgG antibody (20 nM) and washed again three times with 200 μL FACS buffer.

[0382] When tested with Benchmark 1, mutant PAR2 and WT PAR2 showed very similar expression profiles, excluding D62F, which showed a partially reduced expression level, whereas S60W, D62F, G318F and the double mutation D62G / E63G all showed reduced expression profiles compared to WT when tested with Y022883, indicating that the region identified by HDX analysis is involved in the binding of Y022883 to human PAR2. The reduction in binding observed with the selected mutations was stronger for segment 1 / helix 0 / 1 than for ECL3 ( Fig.21 ).

[0383] The effects of these mutations may confer conformational changes that alter the antibody epitope and may also result in changes in the functional activity of the receptor.

[0384] Example 11 - Pharmacokinetics in rats

[0385] The pharmacokinetics of Y022883 were explored in adult male Sprague Dawley rats. Three rats were injected intravenously with 10 mg / kg Y022883 and blood samples were obtained at different time points over 2 weeks. The blood samples were used to generate serum, and these sera were analyzed by a qualified non-GLP ELISA method on the Gyrolab platform (universal PK kit), where PhoenixTM WinNonlin was used to calculate PK parameters by non-compartmental analysis (NCA). The summary data are shown in Fig. 22 and Table 18. These data are consistent with the expectation for a human mAb that it does not have a significant antigen sink, which is expected as Y022883 does not bind rat PAR2.

[0386] Table 18: Group PK parameters of Y022883 in rats (n=3). Data are shown as mean average with standard deviation in parentheses.

[0387]

[0388] Example 12 - Pharmacokinetics and Pharmacodynamics in Cynomolgus Monkeys

[0389] The pharmacokinetics of Y022883 were explored in adult male cynomolgus monkeys. Three groups of three adult male cynomolgus monkeys were injected intravenously with 10 mg / kg, 3 mg / kg, or 1 mg / kg of Y022883, and blood samples were obtained at different time points over 4 weeks. Blood samples were used to evaluate pharmacodynamics (including Figures 24 to 26 ) or to generate serum for pharmacokinetics (see Fig.23 ).

[0390] Serum was analyzed by a qualified non-GLP ELISA method on the Gyrolab platform (Universal PK Kit), with PK parameters calculated by non-compartmental analysis (NCA) using PhoenixTM WinNonlin. Summary data are shown in Fig.23 and Table 19. These data are consistent with expectations for human mAbs, which do not have significant antigen silencing.

[0391] Table 19: Group PK parameters of Y022883 in cynomolgus monkeys (n=3). Data are shown as mean means with standard deviations in parentheses.

[0392]

[0393] Pharmacodynamics was evaluated under sterile conditions at 4 time points (before administration, 1, 13 and 29 days after administration). 1 volume of PBS was added to each blood sample, and 6% dextran T-500 was added to a final concentration of 2%. After 25 to 40 minutes of incubation (when the 2 phases rich in leukocytes and rich in erythrocytes were each 50% when the reaction stopped), the supernatant containing leukocytes was removed, washed in PBS, and resuspended in culture medium (RPMI-1640, 2% FCS, 1% penicillin-streptomycin, 1% L-glutamine, 1% non-essential amino acids) with the original blood volume. Then 1mL cell sample was attacked with PBS, 100mcM 2f-LIGRLO, 100nM trypsin or 100pg / mL LPS, and incubated at 37°C for 6 hours. Then the sample was precipitated (306g, 5 minutes, room temperature), the precipitate was resuspended and mixed with 0.48mL RNAlater, and then frozen at -70°C. Sample RNA was then extracted using standard methods and the transcriptome was assessed using genome-based read mapping at a read depth of 50×.

[0394] Sequencing reads (FASTQ files) are compared with reference genomes, and the counted number of reads mapped to each gene is in turn used to generate a gene count table. Then differential gene expression analysis is performed between each stimulation of each administration group at each time point, such as 1mg / kg PBS before administration vs 1mg / kg PAR2AP before administration, 1mg / kg PBS before administration vs 1mg / kg trypsin before administration, 1mg / kg PBS before administration vs 1mg / kg LPS before administration. Then differential gene expression is determined by generating a volcano map (scatter plot of gene expression fold change vs statistical significance (P value)). Genes that are greater than or equal to 2 times up-regulated or down-regulated and significance is less than or equal to 0.05 are considered to be differentially expressed. Therefore, a set of differentially expressed genes is generated for each stimulation (from pre-administration samples); by determining whether the differentially expressed gene set before administration of each stimulation is still differentially expressed at each time point, the effect of the treatment performed with mAb is evaluated. For example, in the 1 mg / kg dosing group, 1816 genes were differentially regulated by LPS at the pre-dose time point (vs pre-dose PBS); at 24 hours, 1379 of these genes were still differentially regulated by LPS (vs 24 hour PBS), so that in the 1 mg / kg LPS dosing group, 76% of the pre-dose LPS gene-signature was present at 24 hours post-dose.

[0395] Using this approach, it was found that a single IV infusion of 10 mg / kg Y022883 suppressed both the PAR2AP-induced and trypsin-induced gene signatures at all time points, whereas the LPS gene signature was suppressed by 36% at 24 hours and then recovered to 23% suppression by 29 days ( Fig.24 A similar set of observations was made following 3 mg / kg Y022883 ( Fig.25 ), although trypsin gene signatures began to recover by day 29 in the presence of 1 mg / kg Y022883 (96%, 92%, 91% inhibition at 24 h, day 13, day 29; Fig.26 ). These data are consistent with Y022883 providing blockade of PAR2 on peripheral blood leukocytes for one month following a single infusion of at least 1 mg / kg and specifically antagonizing PAR2 (at least relative to TLR4). The partial inhibition of the LPS-induced gene signature by PAR2 antagonism is consistent with literature reports linking the TLR4 and PAR2 signaling cascades (Yamaguchi et al 2016).

[0396] Example 13 Effects of SH-C and Benchmark II on Trypsin- and Par2-AP-induced p38 MAPK and pERK phosphorylation in T84 cell lines

[0397] T84 cells were treated with trypsin (1 μM) or PAR2-AP (2fu-LIGRLO) (10 μM) in growth medium. Antibodies Y022883 (SH-C) and Benchmark 2 (SH-D) were tested at concentrations ranging from 10 nM to 3 μM. Cells were lysed in lysis buffer (PBS + 1% TritonX-100, protease and phosphatase inhibitors) 5 or 30 minutes after treatment. Total protein concentration was assessed by BCA prior to phosphoprotein analysis. Protein concentration was adjusted to 0.3 mg / ml and samples were loaded onto Protein Simple's 12-230 kDa Wes Separation Module 8×25 capillary cartridge. Rabbit antibodies were used to detect pERK and ERK (pERK, Cell signalling, cat: 9101S; ERK, Cell signalling, cat: 4695S) and phosphorylated p38 MAPK and p38 MAPK (Cell signalling, cat: 9211L; p38, Cell signalling, cat: 9212S). Anti-rabbit detection module (Protein Simple, cat: DM-001) was used as a secondary antibody. Data for phosphorylated-ERK or phosphorylated p38 were normalized to data for total ERK or total p38; biological replicates were analyzed. The percentage of the maximum ratio was used for normalization between plates ( Figures 27 to 29 ).

[0398] Treatment with trypsin or PAP2-AP caused a >8-fold increase in pERK and a >3-fold increase in phosphorylated p38-MAPK. Both SH-C and Benchmark II showed inhibition of ERK and p38-MAPK phosphorylation in cells treated with trypsin. Only SH-C showed significant inhibition of phosphorylation in cells treated with PAR2-AP.

[0399] sequence

[0400] Tables 20 to 23: Antibody clone sequences of clones Y022065, Y022870, Y022877 and Y022883 with CDRs identified according to Kabat.

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410] References

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[0441] Van den Brulle J,Fischer M,Langmann T,Horn G,Waldmann T,Arnold S,Fuhrmann M,Schatz O,O′Connell T,O′Connell D,Auckenthaler A,Schwer H.2008.Anovel solid phase technology for high-throughput genesynthesis.Biotechniques.45(3),pp.340-3.

[0442] Wagner F,Lange CF,Nowak M,Ignatenko S.2014.FRI0315 First Human Doseof the Anti-C5a Receptor-Targeting,Human Monoclonal Antibody NNC0215-0384inPatients with Rheumatoid Arthritis:A Phase 1,Randomised,Double-Blind,Single-Dose,Dose-Escalation Trial.Annals of the Rheumatic Diseases,73(Supplement 2),abstract FRI0315. http: / / dx.doi.org / 10.1136 / annrheumdis-2014-eular.2156

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[0446] The scope of the present invention is not limited by the specific aspects described herein. In fact, various modifications of the present invention (except those described) will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All references cited herein (such as publications or patents or patent applications) are incorporated herein by reference in their entirety.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to PAR2 and inhibits PAR2 activity, wherein the antibody or fragment thereof binds to a discontinuous epitope comprising ECL3, segment 1, and helix 0 / 1 of PAR2.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody binds to regions V55-F77 and L306-Y326 of PAR2 when numbered according to SEQ ID NO:

1.

3. Optionally, an antibody or an antigen-binding fragment thereof according to claim 1 or claim 2, which specifically binds to PAR2 and inhibits PAR2 activity, and comprises a V region containing HCDR3. H Domain, the HCDR3 is selected from: (a) a HCDR3 comprising or consisting of the following amino acid sequence: SEQ ID NO: 5, 22 or 30; or SEQ ID NO: 5, 22 or 30 having 3, 2 or 1 amino acid substitutions therein; (b) a HCDR3 comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, 22 or 30; or (c) a HCDR3 amino acid sequence defined according to Kabat or Chothia and derived from a V sequence selected from SEQ ID NO: 2, 10, 13, 16, 19 or 27. H Structural domain.

4. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein said V H The domains include: i. a HCDR1 amino acid sequence selected from SEQ ID NO: 3, 11, 14, 17, 20 or 28, optionally with 3, 2 or 1 amino acid substitutions therein; and / or ii. A HCDR2 amino acid sequence selected from SEQ ID NO: 4, 12, 15, 18, 21 or 29, optionally having 3, 2 or 1 amino acid substitutions therein.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, further comprising V L A domain, optionally a V domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6, 23 or 31. L Structural domain.

6. The antibody or fragment thereof according to any preceding claim, comprising LCDR3, wherein: a. LCDR3 amino acid sequence selected from SEQ ID NO: 9, 26 or 33, optionally with 3, 2 or 1 amino acid substitutions; or b. LCDR3 amino acid sequence according to Chothia or Kabat definition and derived from V according to SEQ ID NO: 6, 23 or 31. L domain, optionally wherein the LCDR3 sequence comprises 3, 2 or 1 amino acid substitutions.

7. The antibody or antigen-binding fragment thereof according to claim 5 or claim 6, wherein the V L The domains include: a) i) the LCDR1 amino acid sequence of SEQ ID NO: 7, 24 or 32, optionally having 3, 2 or 1 amino acid substitutions therein; or ii) a LCDR1 amino acid sequence defined according to Chothia or Kabat from V according to SEQ ID NO: 6, 23 or 31 L domain, optionally wherein the LCDR1 sequence comprises 3, 2 or 1 amino acid substitutions; and / or b) i) the LCDR2 amino acid sequence of SEQ ID NO: 8 or 25, optionally having 3, 2 or 1 amino acid substitutions therein; or ii) a LCDR2 amino acid sequence defined according to Chothia or Kabat from V according to SEQ ID NO: 6, 23 or 31 L domain, optionally wherein the LCDR2 sequence comprises 3, 2 or 1 amino acid substitutions.

8. Optionally, an antibody or antigen-binding fragment according to any preceding claim, which specifically binds to PAR2, comprising: V H A region selected from SEQ ID NO: 2, 10, 13 and 16, 19 or 27, or an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical thereto; and V L A region according to SEQ ID NO: 6, 23 or 31, or an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical thereto.

9. The antibody or antigen-binding fragment according to any preceding claim, wherein the V H The region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

2.

10. The antibody or antigen-binding fragment according to any preceding claim, wherein the V H The region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID10.

11. The antibody or antigen-binding fragment according to any preceding claim, wherein the V H The region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID13.

12. The antibody or antigen-binding fragment according to any preceding claim, wherein the V H The region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID16.

13. The antibody or antigen-binding fragment according to any preceding claim, wherein the V H The region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID19.

14. The antibody or antigen-binding fragment according to any preceding claim, wherein the V H The region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID27.

15. The antibody or antigen-binding fragment according to any preceding claim, wherein the V L The region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID 6.

16. The antibody according to any preceding claim, wherein the V L The region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID 23.

17. The antibody according to any preceding claim, wherein the V L The region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID 31.

18. Optionally, the antibody or antigen-binding fragment according to any preceding claim, wherein the V H The V region comprises an amino acid sequence that is identical to or has at least 90% identity to SEQ ID NO: 2, and the V L The region comprises an amino acid sequence that is identical to SEQ ID NO:6 or has at least 90% identity thereto.

19. Optionally, the antibody or antigen-binding fragment according to any preceding claim, wherein the V H The V region comprises an amino acid sequence that is identical to or has at least 90% identity to SEQ ID NO: 10, and the V L The region comprises an amino acid sequence that is identical to SEQ ID NO:6 or has at least 90% identity thereto.

20. Optionally, the antibody or antigen-binding fragment according to any preceding claim, wherein the V H comprising an amino acid sequence identical to or at least 90% identical to SEQ ID NO: 13, and wherein V L The region comprises an amino acid sequence that is identical to SEQ ID NO:6 or has at least 90% identity thereto.

21. Optionally, the antibody or antigen-binding fragment according to any preceding claim, wherein the V H The V region comprises an amino acid sequence that is identical to or has at least 90% identity to SEQ ID NO: 16, and the V L The region comprises an amino acid sequence that is identical to SEQ ID NO:6 or has at least 90% identity thereto.

22. Optionally, the antibody or antigen-binding fragment according to any preceding claim, wherein the V H The V region comprises an amino acid sequence that is identical to or has at least 90% identity to SEQ ID NO: 19, and the V L The region comprises an amino acid sequence that is identical to, or at least 90% identical to, SEQ ID NO:

23.

23. Optionally, the antibody or antigen-binding fragment according to any preceding claim, wherein the V H The V region comprises an amino acid sequence that is identical or at least 90% identical to SEQ ID NO: 27, and the V L The region comprises an amino acid sequence that is identical to, or at least 90% identical to, SEQ ID NO:

31.

24. Optionally, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), wherein the V H Include: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; And wherein V L Include: (d) LCDR1 having an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 containing 3, 2 or 1 amino acid substitutions; as well as (f) LCDR3 having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 9 containing 3, 2 or 1 amino acid substitutions.

25. Optionally, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), wherein the V H Include: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 11 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 12 containing 3, 2 or 1 amino acid substitutions; and (c) HCDR3 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; And wherein V L Include: (d) LCDR1 having an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 9 containing 3, 2 or 1 amino acid substitutions.

26. Optionally, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), wherein the V H Include: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 15 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; And wherein V L Include: (d) LCDR1 having an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 9 containing 3, 2 or 1 amino acid substitutions.

27. Optionally, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), wherein the V H Include: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 17 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 18 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; And wherein V L Include: (d) LCDR1 having an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 9 containing 3, 2 or 1 amino acid substitutions.

28. Optionally, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), wherein the V H Include: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 20 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 21 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 22 containing 3, 2 or 1 amino acid substitutions; And wherein V L Include: (d) LCDR1 having an amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 24 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 25 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 26 containing 3, 2 or 1 amino acid substitutions.

29. Optionally, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), wherein the V H Include: (a) HCDR1 having an amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 28 containing 3, 2 or 1 amino acid substitutions; (b) HCDR2 having an amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 29 containing 3, 2 or 1 amino acid substitutions; (c) HCDR3 having an amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 30 containing 3, 2 or 1 amino acid substitutions; And wherein V L Include: (d) LCDR1 having an amino acid sequence of SEQ ID NO:32 or SEQ ID NO:32 containing 3, 2 or 1 amino acid substitutions; (e) LCDR2 having an amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 25 containing 3, 2 or 1 amino acid substitutions; and (f) LCDR3 having the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 33 containing 3, 2 or 1 amino acid substitutions.

30. The antibody or antigen binding fragment of any one of the preceding claims, wherein the antibody or antigen binding fragment thereof inhibits activation of PAR2 by a PAR2 peptide.

31. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody binds to an epitope comprising or consisting of the amino acid sequence of SEQ ID NOs: 41, 42, 43 and 44.

32. The antibody of any of the preceding claims, wherein inhibiting PAR2 activation comprises inhibiting PAR2 bound ligand binding.

33. Optionally, the antibody or antigen binding fragment thereof according to any preceding claim, which specifically binds to PAR2 and inhibits the binding of a PAR2 activating peptide to PAR2.

34. Optionally, an antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or fragment binds to an epitope that is the same as the epitope that specifically binds to an antibody or fragment selected from clones Y022065, Y022870, Y022877, Y022883, Y022054 and / or Y021171.

35. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the epitope to which the antibody or fragment binds is identified by hydrogen deuterium exchange (HDX) and / or by site-directed mutagenesis and flow cytometry.

36. Optionally, an antibody or antigen-binding fragment thereof according to any preceding claim, which specifically binds to PAR2 and inhibits the binding of a PAR2 activating peptide to PAR2, wherein the antibody or fragment has an IC of 1 to 100 nM. 50 Inhibiting PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation, optionally wherein PAR2 peptide-mediated IP accumulation is determined using a PAR2 peptide-stimulated IP signaling assay.

37. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or fragment has an IC of 1 to 300 nM. 50 Inhibiting trypsin-mediated IP accumulation, optionally wherein trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

38. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or fragment thereof has an IC of 1 to 100 nM. 50 Inhibiting PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation, optionally wherein PAR2 peptide inhibition is determined using a HTRF assay.

39. Optionally, an antibody or antigen-binding fragment thereof according to any preceding claim, which specifically binds to PAR2, wherein the antibody or fragment inhibits calcium mobilization mediated by the PAR2 activation peptide, optionally with an IC of 1 to 100 nM. 50 The inhibition, and optionally wherein the calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

40. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or fragment inhibits trypsin-mediated calcium mobilization, optionally with an IC of 1 to 200 nM. 50 The inhibition, and optionally wherein the calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

41. Optionally, an antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or fragment is not internalized into the cell when bound to PAR2 on the cell surface, optionally wherein internalization is determined by quantifying antibody or fragment binding using FAC.

42. Optionally, the antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or fragment does not inhibit ligand SFLLR-mediated PAR1 activation, wherein PAR1 activation is determined by an IP signaling assay using ligand SFLLR stimulation.

43. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or fragment has an EC of 600 pM to 5 nM or less. 50 Binding to cynomolgus monkey PAR2, optionally wherein cynomolgus monkey PAR2 binding is determined using flow cytometry.

44. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or fragment has a K of 100 pM to 10 nM. D Binding to human PAR2, optionally wherein the binding affinity is determined using surface plasmon resonance (SPR) or KinExA.

45. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the binding of the antibody or fragment to PAR2 is pH independent between pH 7.5 and 6.

0.

46. ​​Optionally according to the antibody or antigen binding fragment thereof described in any preceding claim, said antibody or antigen binding fragment thereof specifically binds to PAR2, wherein said antibody or fragment thereof does not bind to PAR1, optionally wherein PAR1 binding is determined using flow cytometry or ELISA.

47. Optionally the antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or fragment does not bind to PAR3, optionally wherein PAR3 binding is determined using flow cytometry or ELISA.

48. Optionally the antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or fragment does not bind to PAR4, optionally wherein PAR4 binding is determined using flow cytometry or ELISA.

49. Optionally, an antibody or antigen-binding fragment thereof according to any preceding claim, wherein 3 mg / kg of the antibody or fragment inhibits a PAR2 stimulator-induced leukocyte response by >95% over a 30 day period, wherein the inhibition is measured by determining a gene signature induced by the stimulator.

50. Optionally, the antibody or antigen-binding fragment thereof according to any preceding claim, wherein 1 mg / kg of the antibody or fragment inhibits PAR2 peptide-induced leukocyte responses by >90% over a period of 30 days, wherein inhibition is measured by determining a gene signature induced by the stimulator.

51. The antibody or fragment of any preceding claim, wherein the antibody or fragment binds to PAR2 and inhibits cross-activation of PAR2 by PAR1 -bound ligands.

52. An antibody or fragment as defined in any preceding claim for use in therapy.

53. The antibody or fragment as defined in any one of claims 1 to 52 for use in treating a PAR2-mediated disease or condition, such as atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fractures, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis and lymphangioleiomyomatosis.

54. Use of an antibody or fragment as defined in any one of claims 1 to 53 in the preparation of a medicament for the treatment of a PAR2-mediated disease or condition, such as atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fractures, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis and lymphangioleiomyomatosis.

55. A method for treating a PAR2-mediated disease or condition (e.g., pain, optionally wherein the pain is independently selected from chronic pain, inflammatory pain, postoperative pain, neuropathic pain, fracture-related pain, gout-related pain, cancer-related pain, gastrointestinal pain associated with inflammatory bowel disease, etc.), comprising administering a therapeutically effective amount of an antibody or fragment thereof as defined in any one of claims 1 to 53 to the patient (e.g., a human), wherein the PAR2-mediated disease or condition is thereby treated.

56. The antibody or fragment of claim 53, the use of claim 54, or the method of claim 55, further comprising administering an additional therapy, optionally wherein the additional therapy comprises one or more additional therapeutic agents independently selected from the group consisting of anti-inflammatory drugs, analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, hyaluronic acid, acetaminophen, codeine, lorcet, lortab, vicodin, hydrocodone, morphine, oxycontin, rosicodone, percocet, aspirin, celecoxib, pregabalin, abatacept, adalimumab, anakinra, certolizumab pegol, etanercept, golimumab, infliximab, rituximab, tocilizumab, and tofacitinib.

57. A pharmaceutical composition comprising an antibody or fragment as defined in any one of claims 1 to 53 and a pharmaceutically acceptable excipient, diluent or carrier, and optionally further comprising one or more additional therapeutic agents independently selected from the group consisting of analgesics, including anti-inflammatory drugs (e.g. NSAIDS, including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g. codeine, morphine, oxycodone, fentanyl, buprenorphine), amitriptyline, gabapentin; anticancer drugs, including alkylating agents (e.g. nitrogen mustard, nitrosamines, sedatives, nicotine, phenoxyethanol ... urea), antimetabolites (e.g., folate analogs, pyrimidine and purine analogs), antibiotics and enzymes (e.g., actinomycin D, daunorubicin, doxorubicin, L-asparaginase), natural agents (e.g., vinca alkaloids, taxens, tecans), hormones and antagonists (e.g., progestins, estrogens, GnRH, antiestrogens), hydroxyurea, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecular targeted therapies (e.g., antibody-conjugated drugs), platinum-based therapies (e.g., cisplatin, carboplatin, oxaliplatin).

58. A pharmaceutical composition according to claim 57, or a kit comprising a pharmaceutical composition as defined in claim 57, wherein the composition is used to treat a PAR2-mediated disease or condition, for example selected from atopic dermatitis, asthma, cancer (various cancers include breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, postoperative, neuropathic, fractures, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis and lymphangioleiomyomatosis.

59. A pharmaceutical composition according to claim 57 or claim 58, or a medicine kit according to claim 58, in combination with a label or instructions for treating the disease or condition in a patient; optionally wherein the label or instructions comprises a marketing authorization number (e.g., an FDA or EMA authorization number); optionally wherein the medicine kit comprises an IV or injection device containing the antibody or fragment.

60. The antibody or fragment according to any preceding claim, wherein the V H The domains include: a. having a HCDR3 amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO: 3 or SEQ ID NO: 3 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO: 4 or SEQ ID NO: 4 containing 3, 2 or 1 amino acid substitutions; b. a HCDR3 amino acid sequence having SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO: 11 or SEQ ID NO: 11 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO: 12 or SEQ ID NO: 12 containing 3, 2 or 1 amino acid substitutions; c. a HCDR3 amino acid sequence having SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO: 14 or SEQ ID NO: 14 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO: 15 or SEQ ID NO: 15 containing 3, 2 or 1 amino acid substitutions; d. a HCDR3 amino acid sequence having SEQ ID NO: 5 or SEQ ID NO: 5 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO: 17 or SEQ ID NO: 17 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO: 18 or SEQ ID NO: 18 containing 3, 2 or 1 amino acid substitutions; e. a HCDR3 amino acid sequence having SEQ ID NO: 22 or SEQ ID NO: 22 containing 3, 2 or 1 amino acid substitutions; and i. having a HCDR1 amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 20 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO: 21 or SEQ ID NO: 21 containing 3, 2 or 1 amino acid substitutions; f. a HCDR3 amino acid sequence having SEQ ID NO: 30 or SEQ ID NO: 30 containing 3, 2 or 1 amino acid substitutions; and i. a HCDR1 amino acid sequence having SEQ ID NO: 28 or SEQ ID NO: 28 containing 3, 2 or 1 amino acid substitutions; and / or ii. a HCDR2 amino acid sequence having SEQ ID NO: 29 or SEQ ID NO: 29 containing 3, 2 or 1 amino acid substitutions; g. HCDR3 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 2 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 2 H domain and contains 3, 2 or 1 amino acid substitutions; and i. HCDR1 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 2 H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 2 H domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 2 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 2 H domain and contain 3, 2, or 1 amino acid substitutions; h. HCDR3 amino acid sequence defined according to Chothia or Kabat and selected from V of SEQ ID NO: 10 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 10 H domain and contains 3, 2 or 1 amino acid substitutions; and i. HCDR1 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 10 H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 10 H domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. HCDR2 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 10 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 10 H domain and contain 3, 2, or 1 amino acid substitutions; i. HCDR3 amino acid sequence defined according to Chothia or Kabat and selected from V of SEQ ID NO: 13 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 13 H domain and contains 3, 2 or 1 amino acid substitutions; and i. HCDR1 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 13 H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 13 H domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 13 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 13 H domain and contain 3, 2, or 1 amino acid substitutions; j. HCDR3 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 16 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 16 H domain and contains 3, 2 or 1 amino acid substitutions; and i. HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 16 H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 16 H domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 16 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 16 H domain and contain 3, 2, or 1 amino acid substitutions; k. HCDR3 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 19 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 19 H domain and contains 3, 2 or 1 amino acid substitutions; and i. HCDR1 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 19 H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 19 H domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 19 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 19 H domain and contain 3, 2, or 1 amino acid substitutions; 1. HCDR3 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 27 H domain, or wherein the HCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 27 H domain and contains 3, 2 or 1 amino acid substitutions; and i. HCDR1 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 27 H domain, or wherein the HCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 27 H domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. HCDR2 amino acid sequence defined according to Chothia or Kabat and derived from V selected from SEQ ID NO: 27 H domain, or wherein the HCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 27 H domain and contain 3, 2 or 1 amino acid substitutions.

61. The antibody or fragment according to any preceding claim, wherein the V L The domains include: a. LCDR3 amino acid sequence having SEQ ID NO: 9 or SEQ ID NO: 9 containing 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence having SEQ ID NO: 7 or SEQ ID NO: 7 containing 3, 2 or 1 amino acid substitutions; and / or ii. LCDR2 amino acid sequence having SEQ ID NO: 8 or SEQ ID NO: 8 containing 3, 2 or 1 amino acid substitutions; b. LCDR3 amino acid sequence having SEQ ID NO: 26 or SEQ ID NO: 26 containing 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence having SEQ ID NO: 24 or SEQ ID NO: 24 containing 3, 2 or 1 amino acid substitutions; and / or ii. LCDR2 amino acid sequence having SEQ ID NO: 25 or SEQ ID NO: 25 containing 3, 2 or 1 amino acid substitutions; c. LCDR3 amino acid sequence having SEQ ID NO: 33 or SEQ ID NO: 33 containing 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence having SEQ ID NO: 32 or SEQ ID NO: 32 containing 3, 2 or 1 amino acid substitutions; and / or ii. LCDR2 amino acid sequence having SEQ ID NO: 25 or SEQ ID NO: 25 containing 3, 2 or 1 amino acid substitutions; d. LCDR3 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 6 L domain, or wherein the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 6 L domain and contains 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 6 L domain, or wherein the LCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 6 L domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. LCDR2 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 6 L domain, or wherein the LCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 6 L domain and contain 3, 2, or 1 amino acid substitutions; e. LCDR3 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 23 L domain, or wherein the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 23 L domain and contains 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 23 L domain, or wherein the LCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 23 L domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. LCDR2 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 23 L domain, or wherein the LCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 23 L domain and contain 3, 2, or 1 amino acid substitutions; f. LCDR3 amino acid sequence defined by Chothia or Kabat and derived from V selected from SEQ ID NO: 31 L domain, or wherein the LCDR3 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 31 L domain and contains 3, 2 or 1 amino acid substitutions; and i. LCDR1 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 31 L domain, or wherein the LCDR1 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 31 L domain and contains 3, 2 or 1 amino acid substitutions; and / or ii. LCDR2 amino acid sequence according to Chothia or Kabat definition and from V selected from SEQ ID NO: 31 L domain, or wherein the LCDR2 amino acid sequence is defined according to Chothia or Kabat and is derived from a V domain selected from SEQ ID NO: 31 L domain and contain 3, 2 or 1 amino acid substitutions.

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