Anti-PAR2 antibodies

Abstract

Antibodies and antigen-binding fragments capable of binding to human PAR2 are provided, including antibodies that can act as dual inhibitors of PAR2. The disclosure further provides for the production and use of the antibodies and antigen-binding fragments. The dual inhibitor antibodies are capable of inhibiting both protease cleavage-mediated PAR2 activation (eg, by trypsin) and peptide-mediated PAR2 activation (eg, by PAR2-AP or PAR1-AP).

Description

[Technical field]

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof capable of binding to human PAR2. The present invention further relates to antibodies that specifically bind to an epitope of the human PAR2 receptor and block, antagonize, inhibit or prevent activation of human PAR2. The present invention relates to methods for producing, using and pharmaceutical compositions of said antibodies. [Background technology]

[0002] Chronic pain and chronic inflammation are two of the greatest threats to global health. Chronic pain alone affects approximately 50 million American adults, or 20% of the population.

[0003] Chronic pain is a debilitating condition defined as persistent pain experienced most or every day for six months or more (https: / / uspainfoundation.org / wp-content / uploads / 2018 / 09 / Chronic-pain-facts-infographic.pdf).

[0004] Chronic inflammation plays a central role in many deadly diseases, including cancer, cardiovascular disease and diabetes.

[0005] It is predicted that by 2020, chronic diseases will account for approximately three-quarters of all deaths worldwide (Helamo, Delil and Dileba, 2017).

[0006] Despite the global burden of chronic pain, currently available treatments only provide 30% pain relief to patients (Rice, Smith and Blyth, 2016).

[0007] Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor that belongs to the protease-activated receptor (PAR) family.

[0008] PAR2 has been implicated as playing an important role in inflammation, pain, and other pathophysiological responses in which proteases are elevated.

[0009] PAR2 is widely expressed, with particularly high levels in the pancreas, liver, kidney, small intestine and large intestine.

[0010] Moderate expression is detected in many epithelial and endothelial cells and organs, with limited evidence of expression in brain or skeletal muscle.

[0011] In addition, PAR2 is expressed in immune and inflammatory cells such as T cells, monocytes, macrophages, neutrophils, mast cells and eosinophils.

[0012] The literature suggests that blockade of PAR2 is likely to provide clinical benefit in atopic dermatitis, asthma, cancer (various cancers including breast, melanoma, head and neck), pain (inflammatory, post-operative, neuropathic, fracture, gout, gastrointestinal related to cancer, 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 (see reviews in Yau et al., 2013; Heuberger and Schuepbach, 2019).

[0013] It is therefore believed that PAR2 antagonists may benefit a wide range of patients and reduce pain and / or inflammation-related pathologies.

[0014] Therefore, PAR2 is considered as a valuable therapeutic target for treating several disease indications.

[0015] There is a need to identify therapeutic moieties that can specifically inhibit PAR2.

[0016] Such agents would be particularly desirable if they could inhibit all mechanisms of PAR2 activation. Summary of the Invention [Problem to be solved by the invention]

[0017] Provided herein are antibodies and antigen-binding fragments thereof that bind to PAR2. The antibodies and antigen-binding fragments of the 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.

[0018] The antibodies or antigen-binding fragments thereof provided herein specifically bind to and inhibit the activity of PAR2, where the antibodies or fragments thereof bind to an epitope comprising the extracellular loop (ECL) and the N-terminal segment of PAR2, including helices 0 and 1.

[0019] Binding to both of these regions could lead to global functional inhibition of PAR2 activity.

[0020] Thus, the antibodies provided herein are dual active in that they can inhibit both PAR2 activation via protease cleavage (e.g., by trypsin) and peptide-mediated PAR2 activation (e.g., by PAR2-AP or PAR1-AP).

[0021] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to a discontinuous epitope of PAR2, the epitope comprising one or more regions of non-helical segment 1 preceding helix 0 / 1, the helix 0 / 1 region, and ECL3, optionally the regions of segment 1, helix 0 / 1, and ECL3 are selected from V55-F77, L306-Y311, and F312-Y326 of PAR2, as numbered according to the human PAR2 sequence of SEQ ID NO:1.

[0022] In an embodiment, an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of PAR2 comprises a VH domain comprising the following HCDR3: (a) an HCDR3 consisting of or comprising the amino acid sequence of SEQ ID NO: 5, 22 or 30; or a sequence having 3, 2 or 1 amino acid substitutions in SEQ ID NO: 5, 22 or 30; (b) an HCDR3 comprising an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, 22 or 30; or d) an HCDR3 amino acid sequence as defined by Kabat or Chothia from a VH domain selected from SEQ ID NO: 2, 10, 13, 16, 19 or 27.

[0023] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain, wherein the VH domain comprises: (i) an HCDR1 amino acid sequence selected from SEQ ID NO: 3, 11, 14, 17, 20 or 28 (optionally containing three, two or one amino acid substitutions); and / or (ii) an HCDR2 amino acid sequence selected from SEQ ID NO: 4, 12, 15, 18, 21 or 29 (optionally containing three, two or one amino acid substitutions).

[0024] In embodiments, the antibody or antigen-binding fragment thereof comprises a VL domain, and optionally the VL domain comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6, 23 or 31.

[0025] In embodiments, the antibody or antigen-binding fragment thereof comprises an LCDR3, wherein a) the LCDR3 amino acid sequence is selected from SEQ ID NO: 9, 26 or 33 (optionally comprising 3, 2 or 1 amino acid substitutions); or b) the LCDR3 amino acid sequence is from a VL domain as defined by Chothia or Kabat and according to SEQ ID NO: 6, 23 or 31. Optionally, the LCDR3 sequence comprises 3, 2 or 1 amino acid substitutions.

[0026] In an embodiment, the antibody or antigen-binding fragment thereof comprises a VL domain, wherein the VL domain comprises: a) i) an LCDR1 amino acid sequence of SEQ ID NO: 7, 24 or 32 (optionally comprising 3, 2 or 1 amino acid substitutions); or ii) an LCDR1 amino acid sequence as defined by Chothia or Kabat, which is from a VL domain according to SEQ ID NO: 6, 23 or 31 (optionally comprising 3, 2 or 1 amino acid substitutions); and / or b) i) an LCDR2 amino acid sequence of SEQ ID NO: 8 or 25 (optionally comprising 3, 2 or 1 amino acid substitutions); or ii) an LCDR2 amino acid sequence as defined by Chothia or Kabat, which is from a VL domain according to SEQ ID NO: 6, 23 or 31 (optionally comprising 3, 2 or 1 amino acid substitutions).

[0027] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds 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.

[0028] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2.

[0029] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10.

[0030] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:13.

[0031] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16.

[0032] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:19.

[0033] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27.

[0034] In embodiments, the antibody or antigen-binding fragment thereof comprises a VL region, wherein the VL region comprises an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:23.

[0035] In embodiments, the antibody or antigen-binding fragment thereof comprises a VL region, wherein the VL region comprises an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31.

[0036] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:2, and the VL region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:6.

[0037] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:10, and the VL region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:6.

[0038] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:13, and the VL region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:6.

[0039] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:16, and the VL region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:6.

[0040] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO: 19, and the VL region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:23.

[0041] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:27, and the VL region comprises an amino acid sequence identical or at least 90% identical to SEQ ID NO:31.

[0042] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: (a) HCDR1 comprising the sequence of SEQ ID NO:3 or SEQ ID NO:3 with 3, 2 or 1 amino acid substitutions; (b) HCDR2 comprising the sequence of SEQ ID NO:4 or SEQ ID NO:4 with 3, 2 or 1 amino acid substitutions; (c) HCDR3 comprising the sequence of SEQ ID NO:5 or SEQ ID NO:5 with 3, 2 or 1 amino acid substitutions; and the VL comprises: (d) LCDR1 comprising the sequence of SEQ ID NO:7 or SEQ ID NO:7 with 3, 2 or 1 amino acid substitutions; (e) LCDR2 comprising the sequence of SEQ ID NO:8 or SEQ ID NO:8 with 3, 2 or 1 amino acid substitutions; and (f) LCDR3 comprising the sequence of SEQ ID NO:9 or SEQ ID NO:9 with 3, 2 or 1 amino acid substitutions.

[0043] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: (a) an HCDR1 comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 11 with 3, 2 or 1 amino acid substitutions; (b) an HCDR2 comprising the sequence of SEQ ID NO: 12 or SEQ ID NO: 12 with 3, 2 or 1 amino acid substitutions; and (c) an HCDR3 comprising the sequence of SEQ ID NO: 5 or SEQ ID NO: 5 with 3, 2 or 1 amino acid substitutions. And the VL comprises: (d) an LCDR1 comprising the sequence of SEQ ID NO: 7 or SEQ ID NO: 7 with 3, 2 or 1 amino acid substitutions; (e) an LCDR2 comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with 3, 2 or 1 amino acid substitutions; and (f) an LCDR3 comprising the sequence of SEQ ID NO: 9 or SEQ ID NO: 9 with 3, 2 or 1 amino acid substitutions.

[0044] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: (a) HCDR1 comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 14 with 3, 2 or 1 amino acid substitutions; (b) HCDR2 comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 15 with 3, 2 or 1 amino acid substitutions; (c) HCDR3 comprising the sequence of SEQ ID NO: 5 or SEQ ID NO: 5 with 3, 2 or 1 amino acid substitutions; and the VL comprises: (d) LCDR1 comprising the sequence of SEQ ID NO: 7 or SEQ ID NO: 7 with 3, 2 or 1 amino acid substitutions; (e) LCDR2 comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with 3, 2 or 1 amino acid substitutions; and (f) LCDR3 comprising the sequence of SEQ ID NO: 9 or SEQ ID NO: 9 with 3, 2 or 1 amino acid substitutions.

[0045] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: (a) an HCDR1 comprising the sequence of SEQ ID NO: 17 or SEQ ID NO: 17 with 3, 2 or 1 amino acid substitutions; (b) an HCDR2 comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 18 with 3, 2 or 1 amino acid substitutions; (c) an HCDR3 comprising the sequence of SEQ ID NO: 5 or SEQ ID NO: 5 with 3, 2 or 1 amino acid substitutions; and the VL comprises: (d) an LCDR1 comprising the sequence of SEQ ID NO: 7 or SEQ ID NO: 7 with 3, 2 or 1 amino acid substitutions; (e) an LCDR2 comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with 3, 2 or 1 amino acid substitutions; and (f) an LCDR3 comprising the sequence of SEQ ID NO: 9 or SEQ ID NO: 9 with 3, 2 or 1 amino acid substitutions.

[0046] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: (a) HCDR1 comprising the sequence of SEQ ID NO:20 or SEQ ID NO:20 with 3, 2 or 1 amino acid substitutions; (b) HCDR2 comprising the sequence of SEQ ID NO:21 or SEQ ID NO:21 with 3, 2 or 1 amino acid substitutions; (c) HCDR3 comprising the sequence of SEQ ID NO:22 or SEQ ID NO:22 with 3, 2 or 1 amino acid substitutions; and the VL comprises: (d) LCDR1 comprising the sequence of SEQ ID NO:24 or SEQ ID NO:24 with 3, 2 or 1 amino acid substitutions; (e) LCDR2 comprising the sequence of SEQ ID NO:25 or SEQ ID NO:25 with 3, 2 or 1 amino acid substitutions; and (f) LCDR3 comprising the sequence of SEQ ID NO:26 or SEQ ID NO:26 with 3, 2 or 1 amino acid substitutions.

[0047] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: (a) HCDR1 comprising the sequence of SEQ ID NO:28 or SEQ ID NO:28 with 3, 2 or 1 amino acid substitutions; (b) HCDR2 comprising the sequence of SEQ ID NO:29 or SEQ ID NO:29 with 3, 2 or 1 amino acid substitutions; (c) HCDR3 comprising the sequence of SEQ ID NO:30 or SEQ ID NO:30 with 3, 2 or 1 amino acid substitutions; and the VL comprises: (d) LCDR1 comprising the sequence of SEQ ID NO:32 or SEQ ID NO:32 with 3, 2 or 1 amino acid substitutions; (e) LCDR2 comprising the sequence of SEQ ID NO:25 or SEQ ID NO:25 with 3, 2 or 1 amino acid substitutions; and (f) LCDR3 comprising the sequence of SEQ ID NO:33 or SEQ ID NO:33 with 3, 2 or 1 amino acid substitutions.

[0048] In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:77.

[0049] In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:77.

[0050] In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:78.

[0051] In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:78.

[0052] In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:79.

[0053] In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:79.

[0054] In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:80.

[0055] In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:80.

[0056] In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:83.

[0057] In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:83.

[0058] In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:84.

[0059] In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:84.

[0060] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain, wherein the VH domain comprises: (a) the HCDR3 amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing three, two or one amino acid substitutions; and i) the HCDR1 amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 containing 3, 2 or 1 amino acid substitutions; and / or ii) the HCDR2 amino acid sequence of SEQ ID NO:4 or SEQ ID NO:4 containing 3, 2 or 1 amino acid substitutions; (b) the HCDR3 amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing three, two or one amino acid substitutions; and i) the HCDR1 amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 11 containing 3, 2 or 1 amino acid substitutions; and / or ii) the HCDR2 amino acid sequence of SEQ ID NO:12 or SEQ ID NO:12 containing 3, 2 or 1 amino acid substitutions; (c) the HCDR3 amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing three, two or one amino acid substitutions; and i) the HCDR1 amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 containing 3, 2 or 1 amino acid substitutions; and / or ii) the HCDR2 amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 15 containing 3, 2 or 1 amino acid substitutions; (d) the HCDR3 amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 containing three, two or one amino acid substitutions; and i) the HCDR1 amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 17 containing 3, 2 or 1 amino acid substitutions; and / or ii) the HCDR2 amino acid sequence of SEQ ID NO:18 or SEQ ID NO:18 containing 3, 2 or 1 amino acid substitutions; (e) the HCDR3 amino acid sequence of SEQ ID NO:22 or SEQ ID NO:22 containing three, two or one amino acid substitutions; and i) the 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) the HCDR2 amino acid sequence of SEQ ID NO:21 or SEQ ID NO:21 containing 3, 2 or 1 amino acid substitutions; (f) the HCDR3 amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 30 containing three, two or one amino acid substitutions; and i) the HCDR1 amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 28 containing 3, 2 or 1 amino acid substitutions; and / or ii) the HCDR2 amino acid sequence of SEQ ID NO:29 or SEQ ID NO:29 containing 3, 2 or 1 amino acid substitutions; (g) an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 2; or an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 2 containing 3, 2 or 1 amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 2; or an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 2 containing 3, 2 or 1 amino acid substitutions; and / or ii) an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 2; or an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 2 containing 3, 2 or 1 amino acid substitutions; (h) an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 10; or an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 10 containing 3, 2 or 1 amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 10; or an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 10 containing 3, 2 or 1 amino acid substitutions; and / or ii) an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 10; or an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 10 containing 3, 2 or 1 amino acid substitutions; (i) an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 13; or an HDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 13 containing 3, 2 or 1 amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 13; or an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 13 containing 3, 2 or 1 amino acid substitutions; and / or ii) an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 13; or an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 13 containing 3, 2 or 1 amino acid substitutions; (j) an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 16; or an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 16 containing 3, 2 or 1 amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 16; or an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 16 containing 3, 2 or 1 amino acid substitutions; and / or ii) an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 16; or an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 16 containing 3, 2 or 1 amino acid substitutions; (k) an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 19; or an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 19 containing 3, 2 or 1 amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 19; or an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 19 containing 3, 2 or 1 amino acid substitutions; and / or ii) an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 19; or an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 19 containing 3, 2 or 1 amino acid substitutions; (l) an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 27; or an HCDR3 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 27 containing 3, 2 or 1 amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 27; or an HCDR1 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 27 containing 3, 2 or 1 amino acid substitutions; and / or ii) an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 27; or an HCDR2 amino acid sequence derived from a VH domain as defined by Chothia or Kabat and selected from SEQ ID NO: 27 containing 3, 2 or 1 amino acid substitutions.

[0061] In embodiments, the antibody or antigen-binding fragment thereof comprises a VL domain, wherein the VL domain comprises: (a) the LCDR3 amino acid sequence of SEQ ID NO:9 or SEQ ID NO:9 containing three, two or one amino acid substitutions; and i) the LCDR1 amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 containing 3, 2 or 1 amino acid substitutions; and / or ii) the LCDR2 amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 containing 3, 2 or 1 amino acid substitutions; (b) the LCDR3 amino acid sequence of SEQ ID NO:26 or SEQ ID NO:26 containing three, two or one amino acid substitutions; and i) the LCDR1 amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 24 containing 3, 2 or 1 amino acid substitutions; and / or ii) the LCDR2 amino acid sequence of SEQ ID NO:25 or SEQ ID NO:25 containing 3, 2 or 1 amino acid substitutions; (c) the LCDR3 amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 33 containing three, two or one amino acid substitutions; and i) the LCDR1 amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 32 containing 3, 2 or 1 amino acid substitutions; and / or ii) the LCDR2 amino acid sequence of SEQ ID NO:25 or SEQ ID NO:25 containing 3, 2 or 1 amino acid substitutions; (d) an LCDR3 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 6; or an LCDR3 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 6 containing 3, 2 or 1 amino acid substitutions; and i) an LCDR1 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 6; or an LCDR1 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 6 containing 3, 2 or 1 amino acid substitutions; and / or ii) an LCDR2 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 6; or an LCDR2 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 6 containing 3, 2 or 1 amino acid substitutions; (e) an LCDR3 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23 as defined by Chothia or Kabat; or an LCDR3 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23 as defined by Chothia or Kabat and containing 3, 2 or 1 amino acid substitutions; and i) an LCDR1 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 23; or an LCDR1 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 23 containing 3, 2 or 1 amino acid substitutions; and / or ii) an LCDR2 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 23; or an LCDR2 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 23 containing 3, 2 or 1 amino acid substitutions; (f) an LCDR3 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 31; or an LCDR3 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 31 containing 3, 2 or 1 amino acid substitutions; and i) an LCDR1 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 31; or an LCDR1 amino acid sequence derived from a VL domain as defined by Chothia or Kabat and selected from SEQ ID NO: 31 containing 3, 2 or 1 amino acid substitutions; and / or ii) an LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 31 as defined by Chothia or Kabat; or an LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 31 as defined by Chothia or Kabat and containing 3, 2 or 1 amino acid substitutions.

[0062] In a preferred embodiment, the antibodies of the invention are isolated or purified.

[0063] In embodiments, the antibody or antigen-binding fragment thereof inhibits activation of the PAR2 peptide.

[0064] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits PAR2 activity, including binding to segment 1, helix 0 / 1, ECL3 of the PAR2 receptor.

[0065] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits PAR2 activation. Inhibiting PAR2 activation includes inhibiting PAR2 tethered ligand binding.

[0066] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits PAR2 activation, including inhibiting cross-activation by a PAR1 tethered ligand in a PAR1-PAR2 heterodimer.

[0067] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of a PAR2-activating peptide to PAR2.

[0068] In embodiments, the antibodies provided herein can inhibit protease cleavage-mediated activation of PAR2 (e.g., by trypsin) and peptide-mediated activation of PAR2 (e.g., by PAR2-AP or PAR1-AP).

[0069] In embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope as an antibody or fragment selected from clones Y022065, Y022870, Y022877, Y022883, Y022054 and / or Y021171.

[0070] In embodiments, the antibody or antigen-binding fragment thereof binds to an epitope to which the antibody or fragment binds, the epitope being identified by hydrogen deuterium exchange (HDX) and / or site-directed mutagenesis and flow cytometry.

[0071] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of a PAR2-activating peptide to PAR2. The antibody or fragment inhibits PAR2-activating peptide-mediated inositol monophosphate (IP) accumulation with an IC50 of 1-100 nM, and optionally, PAR2 peptide-mediated IP accumulation is determined using a PAR2 peptide-stimulated IP signaling assay.

[0072] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated PAR2 activation.

[0073] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated IP accumulation with an IC50 of 1-300 nM; optionally, trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

[0074] In embodiments, the antibody or antigen-binding fragment thereof inhibits PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation with an IC50 of 1-100 nM, optionally where PAR2 peptide inhibition is determined using an HTRF assay.

[0075] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, and the antibody or fragment inhibits PAR2-activating peptide-mediated calcium mobilization, optionally with an IC50 of 1-100 nM. Optionally, calcium mobilization is determined using a PAR2-activating peptide-stimulated calcium mobilization assay.

[0076] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization, optionally with an IC50 of 1-200 nM, and optionally calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0077] In embodiments, the antibody or antigen-binding fragment thereof is not internalized into the cell upon binding to PAR2 on the cell surface. Optionally, internalization is determined by quantifying antibody or fragment binding using FACS.

[0078] In embodiments, the antibody or antigen-binding fragment thereof does not inhibit activation of PAR1 via the ligand SFLLR, and activation of PAR1 is determined using a ligand SFLLR-stimulated IP signaling assay.

[0079] In embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey PAR2 with an EC50 of 600 pM to 5 nM or less. Optionally, cynomolgus monkey PAR2 binding is determined using flow cytometry.

[0080] In embodiments, the antibody or antigen-binding fragment thereof binds to human PAR2 with a KD of 100 pM to 10 nM. Optionally, the binding affinity is determined using surface plasmon resonance (SPR) or KinExA.

[0081] In an embodiment, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2 peptide to PAR2. The binding of the antibody or fragment to PAR2 is pH-independent between pH 7.5 and 6.0.

[0082] In embodiments, the antibody or antigen-binding fragment thereof does not bind to PAR1. Optionally, PAR1 binding is determined using flow cytometry or ELISA.

[0083] In embodiments, the antibody or antigen-binding fragment thereof does not bind to PAR3. Optionally, PAR3 binding is determined using flow cytometry or ELISA.

[0084] In embodiments, the antibody or antigen-binding fragment thereof does not bind to PAR4. Optionally, PAR4 binding is determined using flow cytometry or ELISA.

[0085] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of PAR2 peptide to PAR2. 3 mg / kg of the antibody or fragment suppresses PAR2 stimulation-induced responses in leukocytes by more than 95% over a 30 day period, as measured by determining a stimulation-induced gene signature.

[0086] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits binding of PAR2 peptide to PAR2. 1 mg / kg of the antibody or fragment inhibits PAR2 peptide-induced responses in leukocytes by more than 90% over a 30 day period, as measured by determining a stimulus-induced gene signature.

[0087] In an embodiment, the antibody or antigen-binding fragment thereof competes with the functional ligand AZ8838 for binding to PAR2.

[0088] In an embodiment, the antibody or antigen-binding fragment thereof competes directly with AZ8838 for binding to PAR2.

[0089] In embodiments, the antibody or antigen-binding fragment thereof binds to a PAR2 homodimer.

[0090] In embodiments, the antibody or antigen-binding fragment thereof binds to a PAR2-PAR1 heterodimer. Optionally, the binding inhibits cross-activation of PAR2 by a PAR1 tethered ligand.

[0091] Provided herein are antibodies and antigen-binding fragments thereof that bind to PAR2 for therapeutic use.

[0092] In embodiments, the antibodies or antigen-binding fragments thereof are used to treat a PAR2-mediated disease or condition, such as, for example, atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fracture, gout, cancer, gastrointestinal related to 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.

[0093] In embodiments, the antibody or antigen-binding fragment thereof is used in the manufacture of a medicament for the treatment of a PAR2 mediated disease or condition, such as, for example, atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, post-operative, neuropathic, fracture, gout, cancer, gastrointestinal related 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, etc.

[0094] Provided herein is a method of treating a PAR2-mediated disease or condition in a patient, e.g., pain (optionally, pain independently selected from chronic pain, inflammatory pain, post-operative pain, neuropathic pain, fracture-related pain, gout-related pain, cancer-related pain, gastrointestinal pain associated with inflammatory bowel disease, etc.). The method comprises administering to the patient (e.g., human) a therapeutically effective amount of an antibody or fragment thereof of the present disclosure, thereby treating the PAR2-mediated disease or condition.

[0095] In embodiments, the antibodies of the present disclosure may be combined or administered with additional therapies. Optionally, the additional therapies include analgesics (e.g., anti-inflammatory agents (e.g., NSAIDS including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g., codeine, morphine, oxycodone, fentanyl, buprenorphine)), amitriptyline, gabapentin, anti-cancer drugs (e.g., alkylating agents (e.g., nitrogen mustards, nitroureas), antimetabolites (e.g., folic acid analogs, pyrimidine and purine analogs), antibiotics and enzymes (e.g., dactinomycin, daunorubicin, doxorubicin, , L-asparaginase), natural products (e.g., vinca alkaloids, taxanes, tecans), hormones and antagonists (e.g., progestins, estrogens, GnRH, antiestrogens), hydroxyurea, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecular targeted therapies (e.g., antibody conjugates), platinum-based therapies (e.g., cisplatin, carboplatin, oxaliplatin); and / or optionally, the additional therapy is selected from radiation therapy and / or surgical resection of the tumor.

[0096] Provided herein is a pharmaceutical composition comprising an antibody or fragment of the disclosure and a pharma- ceutically acceptable excipient, diluent or carrier. The pharmaceutical composition may optionally contain an analgesic (e.g., an anti-inflammatory agent (e.g., NSAIDS including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, an opioid (e.g., codeine, morphine, oxycodone, fentanyl, buprenorphine)), amitriptyline, gabapentin, an anti-cancer agent (e.g., alkylating agents (e.g., nitrogen mustards, nitroureas), antimetabolites (e.g., folic acid analogs, pyrimidine and purine analogs), antibiotics and enzymes (e.g., dactylic acid analogs, pyrimidine and purine analogs), and the like). and / or one or more additional therapeutic agents independently selected from the group consisting of: cyclosporine, daunorubicin, doxorubicin, L-asparaginase), natural products (e.g., vinca alkaloids, taxanes, tecans), hormones and antagonists (e.g., progestins, estrogens, GnRH, antiestrogens), hydroxyurea, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecular targeted therapies (e.g., antibody conjugates), platinum-based therapies (e.g., cisplatin, carboplatin, oxaliplatin).

[0097] Provided herein is a pharmaceutical composition of the present disclosure or a kit comprising said pharmaceutical composition.The composition is for treating PAR2-mediated disease or condition selected from atopic dermatitis, asthma, cancer (various cancers such as breast cancer, melanoma, head and neck cancer), pain (chronic, inflammatory, postoperative, neuropathic, fracture, gout, cancer, gastrointestinal system related to inflammatory bowel disease), rheumatoid arthritis and related uveitis, scleroderma, systemic lupus erythematosus, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, Reiter's disease, psoriatic arthritis, chronic Lyme's arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

[0098] Provided herein are kits of the disclosure in combination with a pharmaceutical composition of the disclosure or a label or instructions for use in treating a disease or condition in a patient. Optionally, the label or instructions includes a marketing approval number (e.g., an FDA or EMA approval number); optionally, the kit includes an IV or injection device that includes the antibody or fragment.

[0099] In an embodiment, the amino acid substitution comprises a homologous substitution.

[0100] These amino acid substitutions may be conservative substitutions.

[0101] A conservative amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain.

[0102] Families of amino acid residues that have similar side chains have been defined in the art.

[0103] These families include amino acids with 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0104] In some embodiments, a predicted nonessential amino acid residue in the anti-PAR-2 ​​antibody is replaced with another amino acid residue from the same side chain family.

[0105] Methods for identifying conservative amino acid substitutions 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)).

[0106] In embodiments, the amino acid substitutions reduce the binding affinity of the antibody or antigen-binding fragment thereof to human PAR2 by 1000-fold, 800-fold, 700-fold, 500-fold, 400-fold, 300-fold, 200-fold, 100-fold, 50-fold, or 10-fold or less, compared to an antibody or antigen-binding fragment thereof having a VH having the amino acid sequence of SEQ ID NO: 16 and a VL having the amino acid sequence of SEQ ID NO: 6, when tested in a PAR2 binding assay (e.g., an SPR or Kinexa assay) at pH 7.4 to 7.6.

[0107] The antibodies provided herein can be human.

[0108] In an embodiment, the antibody is a monoclonal antibody.

[0109] In an embodiment, the antibody is an IgG antibody.

[0110] In embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0111] In an embodiment, the antigen-binding fragment is an scFv.

[0112] In an embodiment, the antigen-binding fragment is a Fab.

[0113] In embodiments, the antibody or antigen-binding fragment thereof is humanized.

[0114] The antibodies provided herein bind to human PAR2, and may also bind to cynomolgus monkey PAR2 and rhesus monkey PAR2, but not to mouse and / or rat PAR2.

[0115] In embodiments, the antibody or antigen-binding fragment thereof blocks trypsin, tryptase and / or matriptase from interacting with PAR2.

[0116] In embodiments, the antibody or antigen-binding fragment thereof inhibits activation of PAR2 by trypsin.

[0117] In embodiments, the antibody or antigen-binding fragment thereof inhibits exposure of the tethered ligand.

[0118] In embodiments, the antibody or antigen-binding fragment thereof blocks the tethered ligand from interacting with PAR2.

[0119] In embodiments, provided herein are nucleic acids capable of expressing an antibody or antigen-binding fragment thereof.

[0120] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 90% identical to any of SEQ ID NOs:34, 70 and 72.

[0121] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 95% identical to any of SEQ ID NOs:34, 70 and 72.

[0122] In an embodiment, the nucleic acid comprises the nucleic acid sequence of any of SEQ ID NOs: 34, 70 and 72.

[0123] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 90% identical to any of SEQ ID NOs: 35, 36, 37, 38, 71 and 73.

[0124] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 95% identical to any of SEQ ID NOs: 35, 36, 37, 38, 71 and 73.

[0125] In an embodiment, the nucleic acid comprises the nucleic acid sequence of any of SEQ ID NOs: 35, 36, 37, 38, 71 and 73.

[0126] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 90% identical to SEQ ID NO:34.

[0127] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 95% identical to SEQ ID NO:34.

[0128] In an embodiment, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO:34.

[0129] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 90% identical to SEQ ID NO:38.

[0130] In an embodiment, the nucleic acid comprises a nucleic acid sequence at least 95% identical to SEQ ID NO:38.

[0131] In an embodiment, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO:38.

[0132] In a preferred embodiment, the nucleic acids of the invention are isolated or purified.

[0133] In embodiments, the disclosure provides a vector comprising any of the nucleic acids disclosed herein.

[0134] In embodiments, the present disclosure provides a set of vectors comprising one or more of the nucleic acids disclosed herein.

[0135] In embodiments, the present disclosure provides a host cell comprising one or more of the vectors disclosed herein.

[0136] In embodiments, the present disclosure provides a composition comprising a pharma- ceutically acceptable carrier and any of the antibodies or antigen-binding fragments disclosed herein.

[0137] In embodiments, the present disclosure provides a lyophilized composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0138] In embodiments, the present disclosure provides a reconstituted lyophilized composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0139] In embodiments, the composition is formulated for administration by lozenge, spray, oral administration, sustained or extended release, transmucosal administration, syrup, mucoadhesive, buccal tablet, mucoadhesive tablet, topical administration, parenteral administration, injection, subcutaneous administration, oral solution, rectal administration, buccal administration, or transdermal administration.

[0140] In embodiments, the present disclosure provides a kit comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, or any of the compositions disclosed herein.

[0141] In embodiments, the present disclosure provides a method of treating pain in a subject in need thereof, comprising administering to the subject a pharma- ceutical effective amount of any of the compositions disclosed herein.

[0142] In some embodiments, the pain is selected from the group consisting of nociceptive, neuropathic and mixed pain.

[0143] In some embodiments, the pain is associated with headache, chronic headache, migraine, cancer, a viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease, liver disease, multiple sclerosis, spinal cord injury, post-herpetic neuralgia, diabetic neuropathy, back pain, inflammatory heart disease, kidney disease, gastritis, gingivitis, periodontal disease, asthma, chronic obstructive pulmonary disease, autoimmune diseases, irritable bowel syndrome, fibromyalgia, leg pain, restless legs syndrome, diabetic neuropathy, allergic conditions, surgical procedures, acute or chronic physical trauma, fractures or crush injuries, spinal cord injuries, inflammatory diseases, non-inflammatory neuropathic or dysfunctional pain conditions, or combinations thereof.

[0144] In some embodiments, the pain is osteoarthritis pain.

[0145] In some embodiments, the subject is a human.

[0146] In some embodiments, the disclosure provides a method of producing any of the antibodies or antigen-binding fragments disclosed herein, comprising the steps of expressing any of the nucleic acids disclosed herein in a cultured cell and purifying the antibody or antigen-binding fragment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0147] The present invention provides an antibody or antigen-binding fragment that binds to the human PAR2 receptor.

[0148] The antibodies of the invention are useful for inhibiting PAR2 and its downstream signaling cascade.

[0149] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methodology and experimental conditions described herein, as such methods and conditions may vary.

[0150] 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.

[0151] PAR2 or GPR11 (PAR2; human PAR2 UniProt protein ID: P55085) is a 44 kDa G protein-coupled receptor that belongs to the protease-activated receptor (PAR) family.

[0152] It is encoded by the F2RL1 gene (clotting factor II receptor-like 1).

[0153] PAR2 belongs to the protease-activated receptor (PAR) family that is activated by proteolytic cleavage of the extracellular N-terminus.

[0154] This family contains four members, PAR1 to PAR4, which are activated by different proteases.

[0155] PAR2 is activated primarily by the serine proteases tryptase and trypsin, whereas other PAR family members are activated primarily by thrombin, although proteinase 3, factor VIIa, and factor Xa have also been reported to be involved in PAR activation.

[0156] For reviews, see Yau et al., 2016; Mrozkova et al., 2016; Hamilton and Trejo, 2017; Kagota et al., 2016.

[0157] PAR2 is activated by three main mechanisms.

[0158] One mechanism involves cleavage of the N-terminal extracellular domain by proteases.

[0159] Tethered ligands that bind to a conserved region on extracellular loop 2 of the receptor are exposed, triggering intracellular signaling.

[0160] Alternatively, PAR2 can be activated by a synthetic short peptide known as the activation peptide (PAR2-AP), which mimics the first six amino acids at the N-terminus of the tethered ligand.

[0161] In addition, PAR2 can also be activated by cross-activation by PAR1 tethered ligands upon PAR1-PAR2 heterodimerization.

[0162] Activation of PAR2 leads to activation of Gaq and Gi proteins, which induce calcium influx, MAPK signaling and downstream inflammatory signaling.

[0163] This leads to subsequent biological responses such as proliferation or secretion of proinflammatory cytokines such as IL-6, IL-8 (also known as CXCL8) and GM-CSF.

[0164] PAR2 expression has been shown to be increased in the synovial lining, chondrocytes and tissues in human rheumatoid arthritis and animal models of arthritis (Amiable et al 2009).

[0165] PAR2 also enhances signaling through channels such as TRPV1 (Dai et al., 2007), a ligand-gated ion channel involved in inflammatory pain.

[0166] PAR2 signaling is also known to sensitize TRPV1 in vivo, resulting in thermal hyperalgesia ( Amadesi et al., 2006 ).

[0167] Activation of PAR2 has been shown to contribute to a variety of inflammatory signaling pathways.

[0168] Mice lacking the PAR2 receptor have a delayed onset of inflammation in response to inflammatory mediators (Lindner et al, 2000).

[0169] Other rodent PAR2 knockout studies have demonstrated that PAR2 plays an important role in the pathophysiology of many disease conditions, including pain, musculoskeletal inflammation including osteoarthritis, neuroinflammatory diseases, airway inflammation, pruritus, dermatitis, colitis, and related conditions (Yau et al., 2013).

[0170] PAR2 receptor antagonists such as GB88 have also been shown to block inflammatory responses in vivo, including in a collagen-induced arthritis model in rats (Lohman et al., 2012).

[0171] Known small molecule PAR2 antagonists are not ideal therapeutic agents due to their modest efficacy and broad specificity.

[0172] Given the range of diseases in which PAR2 is thought to be important, there is a need to identify potent and specific PAR2 antagonists.

[0173] The antibodies of the present invention are potent and specific PAR2 antagonists that inhibit PAR2 activation via cleavage of the N-terminal domain.

[0174] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0175] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described below.

[0176] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0177] It may be noted here that "and / or" as used in this specification is to be interpreted as specifically disclosing each of the two specified features or components, regardless of the presence or absence of the other.

[0178] For example, "A and / or B" shall be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0179] 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.

[0180] Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0181] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations.

[0182] It is to be understood that all numerical designations are preceded by the term "about", even if not expressly stated.

[0183] It is also to be understood that, unless expressly stated, the reagents described herein are merely exemplary and equivalents thereof are known to those of skill in the art.

[0184] 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), having UniProt ID number: P55085, reproduced herein as SEQ ID NO:1.

[0185] Human PAR2 includes any sequence that is at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, or a biologically active fragment thereof.

[0186] The term "tethered ligand" as used herein refers to a region of the N-terminal portion of PAR2 that is exposed following proteolytic cleavage by a protease (e.g., trypsin) and, once cleaved, binds to and activates the proximal binding site on the PAR2 receptor.

[0187] The N-terminal tethered ligand of the human PAR2 receptor comprises SEQ ID NO:39.

[0188] By "PAR2-AP," "PAR2 activating peptide," or "PAR2 activating peptide" is meant a synthetic short peptide that mimics the N-terminus of a truncated tethered ligand.

[0189] The human peptide is SEQ ID NO:39 and the mouse peptide comprises SEQ ID NO:40.

[0190] As used herein, the terms "antibody," "antibody to PAR2," or "anti-PAR2" refer to full-length antibodies or antigen-binding fragments.

[0191] The antibodies used herein interact with PAR2 (eg, by binding, steric hindrance, stabilization / destabilization, spatial distribution) and inhibit activation of PAR2.

[0192] A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.

[0193] 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.

[0194] The VH and VL regions can be further divided into hypervariable (HV) and framework (FR) regions.

[0195] Each VH and VL is composed of three complementarity determining regions (CDRs) and four FRs.

[0196] The term "antibody" can refer to, for example, a monoclonal antibody, a human antibody, a humanized antibody, a shark antibody, a camel antibody, or a chimeric antibody.

[0197] Both the light and heavy chains are divided into regions of structural and functional homology.

[0198] The terms "constant" and "variable" are used functionally.

[0199] In this regard, it will be appreciated that the variable domains of both the heavy and light chains determine antigen recognition and specificity.

[0200] Conversely, the constant domains of the light chain (CL) and the constant domains of the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement fixation.

[0201] By convention, the numbering of the constant region domains increases as they move away from the amino-terminus or antigen binding site of the antibody.

[0202] The N-terminus is the variable region and the C-terminus is the constant region. The CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0203] Antibodies (immunoglobulins) can be assigned to different classes, depending on the amino acid sequence of the constant domain of their heavy chains.

[0204] There are five major classes (isotypes) of immunoglobulins in humans: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0205] The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0206] The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are described generally in, for example, Abbas et al. (2000).

[0207] An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0208] A "complementarity determining region" ("CDR") is an amino acid sequence having boundaries determined using a number of well-known systems, such as the "Kabat" and "Chothia" numbering systems, as shown in Table 1.

[0209] For example, in the Kabat system, the CDR amino acid residues of the heavy chain, heavy chain variable domain (VH), are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), whereas in the Chothia system, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3).

[0210] Table 1: CDR definitions (numbering follows Kabat nomenclature) [Table 1]

[0211] As used herein, terms such as "antigen-binding fragment" or "epitope-binding fragment" of an antibody include naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex.

[0212] Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally the constant domains of an antibody.

[0213] Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized.

[0214] The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example to place one or more variable and / or constant domains in the appropriate configuration, to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids.

[0215] The two fragment variable (Fv) domains, VL and VH, are encoded by separate genes but can be joined by a synthetic linker using recombinant techniques to generate a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., 1988; and Huston et al., 1988).

[0216] Such single chain antibodies are also intended to be encompassed within the term "fragment," "epitope-binding fragment" or "antibody fragment."

[0217] These fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0218] Further 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) a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide).

[0219] Other engineered molecules such as domain-specific antibodies, single domain antibodies, camelid antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), adnectins, small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0220] An antigen-binding fragment of an antibody will typically comprise at least one variable domain (eg, at least one of VH or VL).

[0221] Variable domains may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to, or in frame with, one or more framework sequences.

[0222] In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to one another in any suitable orientation.

[0223] For example, the variable region may be a dimer and comprise a VH-VH, VH-VL, or VL-VL dimer.

[0224] Alternatively, an antigen-binding fragment of an antibody may comprise a monomeric VH or VL domain.

[0225] In certain embodiments of the disclosure, an antigen-binding fragment of an antibody can comprise at least one variable domain covalently linked to at least one constant domain.

[0226] Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.

[0227] In any configuration of variable and constant domains, including any of the exemplary configurations above, the variable and constant domains may be directly linked to each other or may be linked by a full-length or partial hinge or linker region.

[0228] The hinge region may be composed of at least two (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule.

[0229] In some embodiments, the hinge region comprises a glycine-serine linker.

[0230] Furthermore, antigen-binding fragments of antibodies of the present disclosure may comprise homodimers or heterodimers (or other multimers) of any of the above variable and constant domain configurations, non-covalently associated with each other and / or associated with one or more monomeric VH or VL domains (e.g., by disulfide bonds).

[0231] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific).

[0232] A multispecific antigen-binding fragment of an antibody typically comprises at least two different variable domains, each capable of specifically binding to a separate antigen, or to a different epitope on the same antigen.

[0233] Any multispecific antibody format can be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0234] As used herein, the term "biparatopic antibody" refers to a bispecific antibody that binds to two different epitopes on a single PAR2 target.

[0235] As used herein, the term "monovalent antibody" refers to an antibody that contains one epitope binding site.

[0236] As used herein, the term "bivalent antibody" refers to an antibody that contains two epitope binding sites.

[0237] The term "multivalent antibody" refers to a single binding molecule with more than one valency, where "valency" is described as the number of antigen binding sites present per molecule of the antibody construct.

[0238] Therefore, a single binding molecule can bind to more than one binding site on a target molecule.

[0239] Examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, etc., as well as multispecific and biparatopic antibodies.

[0240] For example, in the case of PAR2, a multivalent antibody (eg, a PAR2 biparatopic antibody) has two different binding sites, each for two different epitopes of PAR2.

[0241] The term "monoclonal antibody" or "mAb" as used herein refers to a polypeptide including antibodies having substantially identical amino acid sequences or derived from the same genetic source, bispecific antibodies, etc.

[0242] The term also includes preparations of antibody molecules of single molecular composition.

[0243] A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0244] The term "fragment crystallizable region" or "Fc region" as used herein refers to a polypeptide comprising the CH3, CH2, and at least a portion of the hinge region of an antibody constant domain.

[0245] Optionally, the Fc region may include a CH4 domain, which is present in some antibody classes.

[0246] The Fc region may include the entire hinge region of an antibody constant domain.

[0247] In one embodiment, the constant region is modified compared to the wild-type constant region.

[0248] That is, the polypeptides of the invention disclosed herein may contain alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and / or the light chain constant region domain (CL).

[0249] Examples of modifications include the addition, deletion or substitution of one or more amino acids in one or more domains.

[0250] Such modifications may be included to optimize effector function, half-life, and the like.

[0251] As used herein, the terms "dual-active," "dual-activity," or "dual-activity antibody" refer to an antibody described 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).

[0252] As used herein, the terms "reference antibody," "reference mAb," "reference Ab," "benchmark" or "benchmark antibody" refer to any antibody used in the experiments of the present disclosure as a reference for the anti-PAR2 antibodies of the present invention (e.g., for setting positive or negative controls and assay conditions).

[0253] Benchmark antibodies used in the experiments herein include: Benchmark 1, which binds to an N-terminal epitope of PAR2 (Giblin et al. 2011); Benchmark 2 (WO2018167322A1), also known as R001053 or PaB670129; Benchmark 3, a Regeneron antibody, also known as R001044 or H4H581P; Benchmark 4, an Amgen antibody, also known as R001048 or 1A1; MAB3949, a murine R&D systems mAb; and Benchmark 6 (Giblin et al. 2011).

[0254] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (known as the paratope) within the variable region of an antibody molecule.

[0255] A single antigen may have more than one epitope.

[0256] Thus, different antibodies may bind to different regions on an antigen and have different biological effects.

[0257] Epitopes can be either conformational (eg, discontinuous) or linear.

[0258] Conformational epitopes are generated by spatially adjacent amino acids from different segments of a linear polypeptide chain.

[0259] A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.

[0260] In certain circumstances, an epitope may comprise a sugar, phosphate, or sulfonyl moiety on an antigen.

[0261] As used herein, the term "binding site" includes the region on a PAR2 target molecule to which an antibody or antigen-binding fragment selectively binds.

[0262] In general, 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.

[0263] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site.

[0264] Within each antigenic site, the variable regions of the antibody "arms" interact with multiple sites on the antigen through weak non-covalent forces; the more interactions, the stronger the affinity.

[0265] As used herein, the term "high affinity" with respect to an IgG antibody or fragment thereof (e.g., a Fab fragment) refers to an antibody having a KD of 10-8M or less, 10-9M or less, or 10-10M or less, or 10-11M or less, or 10-12M or less, or 10-13M or less for the target antigen.

[0266] However, high affinity binding may differ for other antibody isotypes.

[0267] For example, high affinity binding of an IgM isotype refers to an antibody with a KD of 10-7 M or less, or 10-8 M or less.

[0268] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex.

[0269] This is controlled by three major factors: antibody epitope affinity; the valency of both the antigen and the antibody; and the structural arrangement of the interacting moieties.

[0270] Ultimately, these factors define the specificity of an antibody, i.e., the likelihood that a particular antibody will bind to a precise antigen epitope.

[0271] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of any length of deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0272] Polynucleotides can have any three-dimensional structure and can perform any function.

[0273] The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, 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, siRNAs, shRNAs, RNAi agents, and primers.

[0274] Polynucleotides may be modified or substituted at one or more bases, sugars and / or phosphates by any of a variety of modifications or substitutions described herein or known in the art.

[0275] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.

[0276] If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer.

[0277] The sequence of nucleotides may be interrupted by non-nucleotide components.

[0278] A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

[0279] The term refers to both double- and single-stranded molecules.

[0280] Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0281] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after transcription and translation.

[0282] Polynucleotide sequences can be used to identify larger fragments or full-length coding sequences of the genes with which they are associated.

[0283] Methods for isolating larger fragment sequences are known to those of skill in the art.

[0284] As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, the D and L optical isomers of any amino acid, and amino acid analogs.

[0285] A peptide consisting of three or more amino acids is commonly called an oligopeptide if the peptide chain is short.

[0286] If the peptide chain is long, the peptide is commonly called a polypeptide or protein.

[0287] The terms "biomarker" and "marker" are used interchangeably herein.

[0288] A biomarker is a nucleic acid, polypeptide, or other organic or inorganic molecule expressed in humans, and the presence or absence of a mutation or difference in expression of the biomarker is used to determine susceptibility to any treatment, including an anti-PAR2 antibody according to the present invention.

[0289] For example, a protein is a biomarker for a cancer cell if it is deficient, mutated, missing, or has reduced post-translational modification, production, expression, level, stability and / or activity compared to the same protein in a normal (non-cancerous) or control cell.

[0290] The terms "polypeptide," "peptide," "peptidomimetic," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0291] These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0292] The residues may be linked by peptide bonds or other bonds, such as ester bonds, ether bonds, and the like.

[0293] When a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a particular percentage (e.g., 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence, it means that that percentage of bases (or amino acids) are identical when the two sequences are aligned.

[0294] This alignment and percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. (1987).

[0295] Preferably, default parameters are used for the alignment.

[0296] A preferred alignment tool is provided on the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) webpage, using default parameters.

[0297] Other programs include BLAST, BLASTN, and BLASTP, using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; explanation=50 sequences; sort criteria=high score; database=nonredundant.

[0298] As used herein, the term "expression product" or "gene product" refers to the nucleic acid or amino acid (eg, a peptide or polypeptide) produced when a gene is transcribed and translated.

[0299] 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.

[0300] If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0301] "Differentially expressed" as applied to a gene refers to the differential production of either the mRNA transcribed from the gene or the protein product encoded by the gene.

[0302] A differentially expressed gene may be over-expressed or under-expressed compared to expression levels in normal or control cells.

[0303] However, as used herein, overexpression refers to an increase in gene expression, typically at least 1.25-fold, or alternatively at least 1.5-fold, or alternatively at least 2-fold, or alternatively at least 3-fold, or alternatively at least 4-fold, compared to that detected in a normal or control corresponding cell or tissue.

[0304] Downregulation, as used herein, is a decrease in gene expression, typically at least 1.25-fold, or alternatively at least 1.5-fold, or alternatively at least 2-fold, or alternatively at least 3-fold, or alternatively at least 4-fold, compared to that detected in a normal or control corresponding cell or tissue.

[0305] The term "differentially expressed" also refers to cases where expression is detected in a cancer cell or tissue, but not in a control cell or normal tissue (e.g., a non-cancerous cell or tissue).

[0306] A high expression level of a gene can result from overexpression of the gene or an increase in gene copy number.

[0307] A gene may also be transcribed and translated to increased protein levels due to the deregulation or absence of a negative regulator.

[0308] Finally, high expression of a gene can result in increased stabilization or decreased degradation of the protein, resulting in protein accumulation.

[0309] As used herein, the terms "inhibit" or "inhibition" and the like refer to impeding, antagonizing, or preventing activation of the PAR2 receptor.

[0310] Such terms indicate a quantitative difference between two conditions, for example referring to a statistically significant difference between two conditions.

[0311] For example, "an amount effective to inhibit PAR2 activation" means that activation of PAR2 by an agonist and a PAR2 blocking antibody is at least statistically significantly different from cells treated with the agonist alone.

[0312] As used herein, the terms "treatment," "treat," "alleviate," and the like generally mean obtaining a desired pharmacological and / or physiological effect, and may be used to refer to the amelioration, alleviation, and / or lessening of the severity of one or more symptoms of the condition being treated.

[0313] The effect may be prophylactic, in that it completely or partially delays the onset or recurrence of the disease, condition or symptom, and / or it may be therapeutic, in that it partially or completely cures the disease or condition and / or adverse effects caused by the disease or condition.

[0314] "Treatment" as used herein encompasses any treatment of a disease or condition in a mammal, particularly a human, and includes one or more of the following: (a) preventing the onset of the disease or condition in a subject who may be susceptible to, but has not yet been diagnosed as having, the disease or condition; (b) inhibiting the disease or condition (e.g., arresting its progression); or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition, resulting in the amelioration of one or more symptoms).

[0315] For example, "treatment" of pain (eg, chronic pain or neuropathic pain) includes reducing, preventing, alleviating, or eliminating pain symptoms in the subject receiving the treatment.

[0316] The population of subjects to be treated by the disease method includes those suffering from the unwanted condition or disease, as well as those at risk of developing the condition or disease.

[0317] The term "therapeutically effective dose" means a dose that produces the effect for which it is administered.

[0318] The exact dose will vary depending on the purpose of the treatment, and can be ascertained by one skilled in the art using known techniques (see, e.g., The Art, Science, and Technology of Pharmaceutical Compounding, 3rd Edition, 2008).

[0319] Once the nucleotide sequence encoding such an antibody has been determined, the chimeric antibody can be produced by recombinant methods.

[0320] Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures generally known in the art and as disclosed herein.

[0321] An "isolated" or "purified" antibody or protein is one that has been identified, separated and / or recovered from a component of its production environment (e.g., naturally or recombinantly).

[0322] For example, the antibody or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody is derived, or, if chemically synthesized, substantially free of other chemical precursors or other chemicals.

[0323] The language "substantially free of cellular material" includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced.

[0324] Thus, an antibody that is substantially free of cellular material includes antibody preparations having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins").

[0325] When the antibodies are recombinantly produced, they are also preferably substantially free of culture medium, i.e., culture medium comprises less than about 20%, 10%, or 5% of the volume of the protein preparation.

[0326] When the antibody is produced by chemical synthesis, it is preferably substantially free from chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.

[0327] Accordingly, such antibody preparations contain less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest.

[0328] In a preferred embodiment, the antibodies of the invention are isolated or purified.

[0329] Provided herein are PAR2-directed antibodies and antigen-binding fragments thereof that bind to PAR2.

[0330] In embodiments, the antibody is an antagonistic, neutralizing and / or blocking anti-PAR2 antibody or antigen-binding fragment.

[0331] As used herein, an "antagonizing," "neutralizing," or "blocking" antibody or antigen-binding fragment is intended to refer to an antibody or antigen-binding fragment whose binding to PAR2: (i) inhibits binding of a PAR2-activating peptide to PAR2 or inhibits activation of PAR2 by a PAR2-activating peptide; and / or (ii) prevents interaction of PAR2 with an exposed tethered ligand of PAR2; and / or (iii) prevents interaction of PAR2 with proteases (e.g., trypsin, tryptase, matriptase, legumain); and / or (iv) inhibits PAR2 signaling (e.g., inhibits PAR2-mediated IP accumulation or intracellular Ca2+ mobilization); and / or (vi) inhibits PAR2 activation; and / or (vii) results in inhibition of at least one biological function of PAR2 (e.g., a PAR2-mediated inflammatory response); and / or (viii) inhibits PAR2 second messenger signaling driven via activation of a dominant Gaq signaling partner.

[0332] In embodiments, the antibodies or antigen-binding fragments of the disclosure inhibit activation of PAR2.

[0333] In some embodiments, the antibody or antigen-binding fragment inhibits exposure of the tethered ligand.

[0334] In some embodiments, the antibody or antigen-binding fragment inhibits activation of the PAR2 receptor by the exposed tethered ligand.

[0335] In embodiments, the antibody or antigen-binding fragment inhibits activation of PAR2 by exposed tethered ligands.

[0336] In embodiments, the antibody or antigen-binding fragment inhibits binding of an exposed tethered ligand to the second extracellular loop (ECL2) of PAR2.

[0337] In embodiments, the antibody or antigen-binding fragment thereof binds to a discontinuous epitope, and binding to said epitope occludes binding of the exposed tethered ligand to ECL2 through steric hindrance.

[0338] Inhibition by anti-PAR2 neutralizing, blocking or antagonistic antibodies need not be complete, so long as it is detectable using an appropriate assay.

[0339] In embodiments, the antibody or antigen-binding fragment thereof inhibits PAR2 activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to uninhibited active PAR2.

[0340] Some examples of assays for detecting the activity of representative anti-PAR2 antibodies or antigen-binding fragments are described in the Examples section. Those of skill in the art will recognize additional anti-PAR2 antibody activity assays.

[0341] Those of skill in the art will be aware of additional anti-PAR2 antibody activity assays.

[0342] In embodiments, provided herein are antibodies or antigen-binding fragments that interfere with the interaction of PAR2 with proteases.

[0343] In an embodiment, the protease is trypsin.

[0344] In an embodiment, the protease is neutrophil elastase.

[0345] In an embodiment, the protease is neutrophil proteinase 3.

[0346] In an embodiment, the protease is mast cell tryptase.

[0347] In an embodiment, the protease is tissue factor / factor Vila / factor Xa.

[0348] In an embodiment, the protease is a kallikrein-related peptidase.

[0349] In an embodiment, the protease is membrane-bound serine proteinase-1 / matriptase-1.

[0350] In an embodiment, the protease is a parasitic cysteine ​​proteinase.

[0351] In embodiments, the antibody or antigen-binding fragment inhibits / alleviates inflammation-induced pain.

[0352] 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).

[0353] An antibody or antigen-binding fragment of the present disclosure may have one or more of the aforementioned biological properties, or any combination thereof.

[0354] Other biological properties of the antibodies of this disclosure will be apparent to those of skill in the art from consideration of this disclosure, including the Examples section provided herein.

[0355] In an embodiment of the disclosure, the anti-PAR2 antibodies of the disclosure are human antibodies.

[0356] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.

[0357] The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, and in embodiments in CDR3.

[0358] However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, have been grafted onto human framework sequences.

[0359] The antibodies of the present disclosure may in embodiments be recombinant human antibodies.

[0360] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are recombinantly prepared, expressed, produced or isolated, such as antibodies expressed using a recombinant expression vector transduced into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. 1992), or antibodies prepared, expressed, produced or isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences.

[0361] Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.

[0362] However, in embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived and related to human germline VH and VL sequences, are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0363] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2-activating peptide to PAR2. Optionally, the antibody or fragment inhibits the accumulation of inositol monophosphate (IP) mediated by a PAR2-activating peptide with an IC50 value of 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, the accumulation of IP mediated by a PAR2 peptide is determined using a PAR2 peptide-stimulated IP signaling assay.

[0364] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2-activating peptide to PAR2. The antibody or fragment inhibits PAR2-activating peptide-mediated accumulation of inositol monophosphate (IP) with an IC50 value of less than about 40 nM, 50 nM, 60 nM, 70 nM, or 80 nM. Optionally, PAR2 peptide-mediated accumulation of IP is determined using a PAR2 peptide-stimulated IP signaling assay.

[0365] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits the binding of a PAR2-activating peptide to PAR2. The antibody or fragment inhibits PAR2-activating peptide-mediated accumulation of inositol monophosphate (IP) with an IC50 value of 40 nM or less. Optionally, PAR2 peptide-mediated accumulation of IP is determined using a PAR2 peptide-stimulated IP signaling assay.

[0366] In an embodiment, the IP signaling assay is a Cisbio IP-One HTRF assay.

[0367] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated IP accumulation with an IC50 value of 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, trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

[0368] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated IP accumulation with an IC50 value of less than about 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, or 300 nM. Optionally, trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

[0369] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated IP accumulation with an IC50 value of 70 nM or less. Optionally, trypsin-mediated IP accumulation is determined using a trypsin-stimulated IP signaling assay.

[0370] In embodiments, the antibody or antigen-binding fragment thereof inhibits PAR2 activating peptide-mediated inositol monophosphate (IP) accumulation with an IC50 value of 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, PAR2 peptide inhibition is determined using an HTRF assay.

[0371] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, and the antibody or fragment inhibits calcium mobilization mediated by a PAR2 activating peptide. Optionally, the IC50 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, calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0372] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, and the antibody or fragment inhibits calcium mobilization mediated by a PAR2 activating peptide. Optionally, the IC50 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, or 160 nM. Optionally, calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0373] In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, and the antibody or fragment inhibits calcium mobilization mediated by a PAR2 activating peptide. Optionally, the IC50 value is 140 nM or less. Optionally, calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0374] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization. Optionally, the IC50 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, calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0375] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization. Optionally, the IC50 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 250 nM. Optionally, calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0376] In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin-mediated calcium mobilization. Optionally, the IC50 value is 200 nM or less. Optionally, calcium mobilization is determined using a PAR2 activating peptide stimulated calcium mobilization assay.

[0377] In embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus PAR2 with an EC50 of 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, cynomolgus PAR2 binding is determined using flow cytometry.

[0378] In embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey PAR2 with an EC50 of less than about 400 pM, 800 pM, 1 nM, 5 nM, or 10 nM. Optionally, cynomolgus monkey PAR2 binding is determined using flow cytometry.

[0379] In embodiments, the antibody or antigen-binding fragment thereof binds to human PAR2 with a KD of 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, the binding affinity is determined using surface plasmon resonance (SPR) or KinExA.

[0380] In embodiments, the antibody or antigen-binding fragment thereof binds to human PAR2 with a KD of less than about 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM, or 20 nM. Optionally, the binding affinity is determined using surface plasmon resonance (SPR) or KinExA.

[0381] In embodiments, the antibody or antigen-binding fragment thereof binds to human PAR2 with a KD of 10 nM or less. Optionally, the binding affinity is determined using surface plasmon resonance (SPR) or KinExA. [Brief description of the drawings]

[0382] [Figure 1] Examples of binding of specific PAR2 nanodiscs (FL-StaR ND) and empty nanodiscs (empty ND) by the positive control mAb "Benchmark 1" and the commercial PAR2 mAb "R&D anti-huPAR2 mAb (MAB3949)" are shown, along with the dose-dependent binding observed with these and the three indicated PAR2 phage-derived antibody clones. Antibodies were coated directly onto the plate at 5 μg / mL. No significant background binding was observed with the negative control MOR03207 (anti-lysozyme antibody). Data are presented as signal divided by background (S / BG). [Diagram 2] Illustrates the degree of competition (fully, partially, and non-competitive) with a commercial positive control mAb "MAB3949" (R&D anti-huPAR2) on a panel of PAR2mAbs (derived from a Ylanthia phage antibody library) using full-length PAR2 StaR nanodiscs, thereby demonstrating diversity. 50 nM candidate mAbs were titrated against the dose response of R&D anti-huPAR2 (MAB3949) in competition against full-length StaR nanodiscs. Both fully and partially competitive binding effects show a dose-dependent effect. No dose-dependent binding effect or competition with MAB3949 was observed with the reference antibodies "Benchmark 1" (N-terminal binding antibody) or "MOR03207" (anti-lysozyme antibody used as a negative control). Data are presented as signal divided by background (S / BG). [Figure 3A] The following dose-dependent binding was measured by FACS: A) Binding of Benchmark 1 mAb and MAB3949 to cell surface expressed PAR2 on CHO_V5HIS-huPAR2 cells and CHO parental cell line to show validation of the assay system. BG = background; [Figure 3B] Binding of clone Y022065 to recombinantly expressed PAR2 on CHO_V5HIS-huPAR2 cells and to CHO parental cells [Figure 3C] Binding of clone Y022065 to A549 cells endogenously expressing human PAR2 and CHO cells expressing mouse PAR2. BG=background. Data are presented as relative light units (RLU). [Figure 4] Flow cytometry analysis of the binding ability of antibody clones to full-length huPAR2 (FL-N, second line) and N-terminal truncated huPAR2 (truncate, first line). The data obtained demonstrate the ability 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 whose epitopes also include parts of the extracellular domain (e.g., Y022065, Y022071), as well as a clone that binds to truncated but not full-length receptors (Y022075). [Diagram 5] Multiplex profiling using IntelliCyt. The graph shows the binding of purified IgG1f_AEASS on human PAR2 expressing cells. Data is an example of clone "Y022065" (functional candidate). Top) HEK Flp-In TRex 293_huPAR2 induced, HEK Flp-In TRex 293_huPAR2 uninduced, Flp-In CHO_V5His_huPAR2, parental Flp-In CHO cells and Flp-In CHO_huPAR1. Bottom) HEK293F infected BacMam WT FL-huPAR2 and HEK293F uninfected. Data are presented as signal divided by background (S / BG). [Figure 6]Functional characterization of Y021171, Y022063 and Y022054 and Y022065 hIgG1f_AEASS in peptide inhibition assays using the Cisbio™ IP-One Gαq Assay at replicates of two concentrations. Statistically significant inhibition of activating peptide-induced agonist activity against human PAR2 at the highest concentrations tested was observed for four clones, ranging from 17-43% relative to the response of 6.28 nM agonist alone. Y022065 was identified as the most active candidate. Data are shown as mean responses with standard deviations from two independent experiments performed in single runs. [Figure 7] Koff ranking ELISA using Nanodisc-embedded human PAR2 StaR™ and purified soluble StaR™ protein. Parental candidates represent a lineage of 22 mature IgG candidates identified as functionally dual-active. Data are presented as signal divided by background (S / BG). [Figure 8] Binding specificity of affinity matured clones exhibited by parental lineage of FlpIn CHO-V5His-huPar2 and FlpIn CHO parental cell line. [Figure 9] Affinity matured clones formatted as Fab fragments bind to human PAR2 expressing CHO cells as assessed by flow cytometry. Data are presented as signal divided by background (S / BG). [Figure 10] Affinity matured clones formatted as IgG or fAb bind to cynomolgus (IgG) and human PAR2 (fAb) BacMam infected HEK293 cells as assessed by flow cytometry. Data are presented as signal divided by background (S / BG). [Figure 11]Confirmation of binding to human and cynomolgus PAR-2 ​​expressing cells for affinity matured lead mAb panel as Fab fragments and IgG in comparison to benchmark mAbs. Antibodies were incubated with human PAR-2 ​​and cynomolgus PAR-2 ​​overexpressing cells in the presence of sodium azide overnight at approximately 5° C. All antibodies show clear binding to cynomolgus PAR-2 ​​and human PAR-2 ​​both as Fab fragments and full IgG. Data is presented as signal divided by background (S / BG). [Figure 12] Functional characterization of lead optimized clones in inhibition assays against PAR2 activating peptides. The IP-One assay is used to measure in vitro Gαq activation and IP accumulation as a function of human PAR2 activity. Each value represents the % inhibition normalized to 10 μM MAB3949, the highest concentration tested in replicates for each individual lead clone. Lead clones are aligned to the parental IgG. Inhibition of the activating peptide was observed for all parental IgG matured lead clones. Percent inhibition ranges from 0 to approximately 250% across clones tested compared to R&D Systems anti-human PAR2 MAB3949. This suggests that a number of lead clones inhibit the activating peptide agonist activity of human PAR2 to a greater extent compared to MAB3949 at comparable concentrations. Lead clones derived from affinity maturation of parental IgGs Y021171 and Y022065 show the greatest percent activity. [Figure 13]Functional characterization of lead optimized clones in inhibition assay against bovine trypsin The IP-One assay is used to measure IP accumulation as a function of Gαq activation and human PAR2 activity in vitro. Each value represents the % inhibition normalized to 1 μM benchmark 1, the highest concentration tested in replicate for each individual lead clone. Lead clones are aligned to the parental IgG. Inhibition of bovine trypsin was observed for lead clones matured from all parental IgGs. Inhibition ranges from 0 to approximately 130% across clones tested compared to benchmark 1. The lead clone identified from maturation of IgG Y022065 has the highest percentage activity against bovine trypsin challenge. Two representatives from Y021171 show the highest inhibition percentages. [Figure 14] Graphical representation of dose-response inhibition curves of parental clone (Y022065) and affinity matured lead representatives (Y022870, Y022877, Y022883) compared to benchmark 1 and benchmark 2 as measured in a calcium mobilization assay in response to HT-29 challenge with bovine trypsin to activate endogenous PAR2. Data are presented as mean responses with standard deviations from three independent experiments performed in duplicate wells. [Figure 15] SPR assessment by Biacore to determine the effect of pH on the binding of Y022883 to PAR2. [Figure 16] Affinity determination by KinExA of lead candidates Y022870 and Y022883 on HEK-293F-human PAR2 expressing cells. [Figure 17] Epitope binning matrix of affinity matured clones from parent families. Anti-huPAR2 is MAB3949. [Figure 18] Back view of PAR2 (ECL3, segment 1 and helices 0 / 1). The region of PAR2 that interacts with Y022883, obtained by HDX, is shown as the hatched region. [Figure 19]The top view of PAR2 (ECL3, segment 1 and helices 0 / 1) is shown. The region of PAR2 that interacts with Y022883, obtained by HDX, is shown as the hatched region. [Figure 20] A side view of PAR2 (ECL3, segment 1 and helices 0 / 1) is shown. The region of PAR2 that interacts with Y022883, obtained by HDX, is shown as the hatched region. [Figure 21] Expression profile of WT and mutant (S60W, D62F, G318F, D62G and E63G) human PAR2 detected by flow cytometry in BacMam-infected HEK293F cells stained with Benchmark1 (N-terminal binding antibody) or Y022883. 2nd antibody only: secondary FACS antibody only; ni: non-infected [Figure 22] Serum mAb concentration versus time in rats following a single intravenous dose of 10 mg / kg Y022883. Detectable mAb concentrations below the BLOQ (5 ng / mL) are reported as BLOQ / 2 (dashed line). Data are shown as means (open circles) and 95% confidence intervals (solid bars) from three adult male animals. Individual animal data are also shown (filled circles). Detectable mAb concentrations below the BLOQ (5 ng / mL) are reported as BLOQ / 2 (dashed line). [Figure 23] Serum mAb concentration versus time in cynomolgus monkeys following a single intravenous dose of 10, 3, or 1 mg / kg Y022883. Data are shown as mean values ​​(10 mg / kg: open circles, 3 mg / kg: closed squares, 1 mg / kg: closed triangles) and 95% confidence intervals (solid bars) from three adult male animals. Individual animal data are also shown (10 mg / kg: closed circles, 3 mg / kg: open squares, 1 mg / kg: closed triangles). Detectable mAb concentrations below the BLOQ (5 ng / mL) are reported as BLOQ / 2 (dashed lines). [Figure 24] Ex vivo pharmacodynamics in cynomolgus monkeys following a single dose of 10 mg / kg Y022883. Data are shown as percentage of pre-dose stimulus-induced (PAR2-AP as circles, trypsin as squares, and LPS as triangles) gene signature. [Diagram 25]Ex vivo pharmacodynamics in cynomolgus monkeys following a single dose of 3 mg / kg Y022883. Data are shown as percentage of pre-dose stimulus-induced (PAR2-AP as circles, trypsin as squares, and LPS as triangles) gene signatures. [Figure 26] Ex vivo pharmacodynamics in cynomolgus monkeys following a single dose of 1 mg / kg Y022883. Data are shown as percentage of pre-dose stimulus-induced (PAR2-AP as circles, trypsin as squares, and LPS as filled triangles) gene signatures. [Figure 27] Inhibition of trypsin-induced p38-MAPK and pERK phosphorylation in T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data are shown as relative levels compared to vehicle treatment and normalized to total ERK or p38-MAPK. [Figure 28] Inhibition of PAR2-AP-induced p38-MAPK and pERK phosphorylation in T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data are shown as relative levels compared to vehicle treatment and normalized to total ERK or p38-MAPK. [Figure 29] Inhibition of trypsin- and PAR2-AP-induced pERK (left) and p38-MAPK (right) phosphorylation in T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data are presented as percentage of difference between vehicle and positive control. EXAMPLES

[0383] Certain aspects and embodiments of the present invention will now be described, by way of example only, with reference to the above-mentioned drawings.

[0384] Example 1 – Generation of full-length and N-terminally truncated human PAR2 constructs [PAR2 construct design] The gene for human protease-activated receptor 2 (PAR2) was edited using a standard site-directed mutagenesis strategy (based on Hutchison et al. 1978) to stabilize the receptor in an antagonist conformation by the introduction of nine point mutations (Cheng et al. 2017).

[0385] The stabilized receptor was either genetically truncated by 54 amino acids at the N-terminus (the first residue in the encoded construct is V55) or maintained full length (FL-N) using polymerase chain reaction (PCR, based on Saiki et al. 1985) as is well known in the art.

[0386] All four PAR2 proteins are also genetically truncated by 20 amino acids at the C-terminus (the last residue is K377) as part of a stabilizing receptor process.

[0387] The following constructs were made: PAR2-1:P151:55-377, glycosylated, pFastBacHisStrepII, truncated PAR2-2:P166:1-377, deglycosylated: N30Q, N222Q, pFastBacHis StrepII, full length PAR2-3:P157:1-377, glycosylated, pBacMamHisStrepII, full length PAR2-4:P155:55-377, glycosylated, pBacMamHisStrepII, truncated The rationale for generating an N-terminally truncated PAR2 receptor that is sufficiently stable to be used as an antigen is to enable the discovery and identification of antibodies that bind to other regions of the receptor other than the N-terminus, thereby generating novel antibodies.

[0388] In the FL-N protein PAR2-2, potential glycosylation sites N30 and N222 were replaced with glutamine residues by site-directed mutagenesis.

[0389] StrepII and deca-histidine tags were added to the C-terminus by PCR and endonuclease cloning strategies known to those skilled in the art to allow specific selection in affinity purification and ELISA assays.

[0390] The PAR2-1 and PAR2-2 genes were cloned into the pFastBac1 vector (Thermofisher, #10360014).

[0391] The multiple cloning site of the vector was modified to allow insertion of genes using NheI as the restriction endonuclease.

[0392] The pFastBac1 vector is part of the commercially available Bac-to-Bac expression system for insect cells (Smith et al. 1983, Thermofisher, #10359016) and is known and widely used in the art.

[0393] The manufacturer's instructions were followed.

[0394] The Bac-to-Bac expression system was used for the PAR2-1 and -2 proteins.

[0395] The pFastBac1 vector was modified to generate the pBacMam vector by introducing the polyhedrin promoter followed by the human cytomegalovirus promoter 3', allowing protein expression of the PAR2-3 and PAR2-4 proteins in mammalian cells.

[0396] The instructions for the Bac-to-Bac expression system were also applied to generate pBacMam virus.

[0397] [Expression] Proteins PAR2-3 and PAR2-4 were expressed in human embryonic kidney 293F cells (Gibco 293F cells, Thermofisher Scientific, #11625019) in Lonza Pro293s CDM medium (#BE02-025Q) containing 10% fetal bovine serum albumin (FBS, Sigma-Aldrich, #F9665) supplemented with 5 mM sodium butyrate (Sigma-Aldrich, #303410) at a cell density of 2.5 x 106 cells / mL by viral infection with 2.7% CO for 48 h.

[0398] Cells were harvested and processed for Sf9 cell expression as described below.

[0399] Mammalian cell lines were used for expression of the constructs to provide the option of utilizing antigens containing mammalian-like glycosylation patterns.

[0400] Proteins PAR2-1 and 2 were expressed in Spodoptera frugiperda (Sf9) cells (Thermofisher, #89070101) in Expression Systems ES921 medium (#96-001-01) containing 10% FBS (Sigma-Aldrich, #F9665).

[0401] Cells were infected at a multiplicity of infection (MOI) of 2 at a cell density of 3.5×106 / mL.

[0402] Cells were harvested after 48 hours by centrifugation, washed with 50 mM HEPES pH 7.5, 250 mM NaCl (PAR2 buffer) supplemented with Roche EDTA-free complete protease inhibitors (PI, Sigma-Aldrich, #5056489001) and stored at -80°C.

[0403] To prepare cell membranes, cells were resuspended in PAR2 buffer supplemented with a PI tablet (PAR2 / PI) and homogenized by one pass through a Microfluidics Microfluidizer Processor.

[0404] Membranes were harvested by ultracentrifugation at 135,000 g for 1 h in a Beckman 45 Ti rotor, homogenized in PAR2 / PI buffer, and stored at -80°C.

[0405] All subsequent purification steps were performed at 4°C.

[0406] [purification] Thawed membranes were solubilized by addition of 1% (w / v) LMNG (Anatrace, #NG310) / 0.1% (w / v) cholesteryl hemisuccinate (CHS, Anatrace, #210) mixture for 1 h.

[0407] Insoluble material was removed by ultracentrifugation at 205,000 g for 30 min in a Beckman 45 Ti rotor.

[0408] Proteins were batch bound to 8 mL NiNTA Superflow resin (Qiagen, #30430) in the presence of 8 mM imidazole for 2 hours.

[0409] The resin was packed into a XK16 / 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).

[0410] Proteins were eluted with buffer A2 supplemented with 300 mM imidazole and concentrated to 0.5 mL using Amicon Ultra-15 regenerated cellulose concentrators with a 100 kDa molecular weight cutoff (Millipore, #UFC900308).

[0411] Aggregated material was removed by ultracentrifugation at 220,000 g for 10 min in a Beckman Coulter tabletop centrifuge using a TLA-100.2 rotor.

[0412] Samples were then subjected to size exclusion chromatography on a Superdex 200 10 / 300 GL column (GE Healthcare, #17-5175-01) in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.02% LMNG, 0.002% CHS.

[0413] Fractions corresponding to monomeric species were pooled and concentrated to 6–8 mg / mL using VivaSpin 500 concentrators with a molecular weight cutoff of 100 kDa (Vivaproducts, #VS0242).

[0414] The purity and monodispersity of the samples were analyzed by SDS-PAGE and analytical gel filtration.

[0415] Protein concentrations were determined using molecular weight, extinction coefficient and protein absorbance at 280 nm using a Nanodrop spectrophotometer (Thermofisher Scientific).

[0416] The protein was confirmed by mass spectrometry to ensure it was the protein of interest.

[0417] [Preparation of nanodisc-embedded human PAR2 StaR protein] Preparation of nanodiscs is known in the art and is based on Banerjee et al. 2008 using zebrafish apolipoprotein-1 (ZAP1) with an N-terminal hexahistidine tag as a scaffold protein.

[0418] Proteins PAR2-1 and -2 were reconstituted into nanodiscs at 100-200 μM and used as antigens for Fab selection.

[0419] To reconstitute nanodiscs, the following lipids were first resuspended in 200 mM sodium deoxycholate (Sigma-Aldrich, #6750) in water: 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.

[0420] The final molar ratios were 1:72:48:12:264 for PAR2:POPC:POPG:cholesterol:sodium deoxycholate, respectively.

[0421] Final sodium deoxycholate concentration was kept above 15 mM and adjusted with 10 mM HEPES 7.4, 150 mM NaCl buffer.

[0422] After incubation on ice for 30 min, zebrafish apolipoprotein 1 (ZAP1) was added at a molar ratio of PAR2:ZAP1 of 1:2.

[0423] The mixture was incubated on ice for 1 hour.

[0424] The detergent was then removed by stirring the samples overnight with Bio-Beads SM-2 (Bio-Rad, #1528920) at a 1:1 ratio by weight of protein solution to dry Bio-Beads (e.g., 900 μg Bio-Beads for 900 μL protein).

[0425] The nanodiscs were collected as the supernatant and the beads were washed with 50 mM HEPES pH 7.5, 150 mM NaCl, a volume equivalent to twice the weight of the beads (eg, 1.8 mL for 900 μg of beads).

[0426] The protein solution was concentrated to less than 1 mL, centrifuged at 13,500 g for 15 min in a tabletop centrifuge, and the supernatant was subjected to size exclusion chromatography in 50 mM HEPES pH 7.5, 150 mM NaCl on a Superdex 200 10 / 300GL column (GE Healthcare) as used above.

[0427] Fractions showing the nanodisc peak were pooled and concentrated to 1-2 mg / mL in Amicon Ultra-4 regenerated cellulose concentrators (molecular weight cut-off 100 kDa, Millipore, #UFC810096) and frozen in aliquots at -80°C.

[0428] Example 2 – Screening The antibodies or antigen-binding fragments of the present disclosure were identified from a phage display library.

[0429] A variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics.

[0430] The phage display library used to identify the antibodies provided herein is the Ylanthia™ phagemid, which is based on the concept of Ylanthia (Tiller et al. 2013) and employs CysDisplay™ technology to display Fabs on the phage surface (Lohning et al. 2000).

[0431] Ten panning strategies were designed, including solid-phase panning, capture panning, and whole-cell panning using techniques known in the art, with an emphasis on solid-phase panning using full-length or N-terminally truncated Nanodisc-embedded human PAR2 StaR proteins.

[0432] To ensure the integrity and stability of Nanodisc-embedded PAR2 StaR and to reduce the risk of enrichment of specific candidates against other undesired epitopes during the panning process, various antigen presentation techniques were implemented.

[0433] Nanodisc-embedded human PAR2 StaR mutant was used as panning antigen (i.e., detergent-free).

[0434] For most panning strategies, Nanodisc-embedded human PAR2 StaR protein was presented via a tag-specific capture antibody.

[0435] To reduce the risk of enrichment of candidates specific for the capture antibody Benchmark 1 (Giblin et al. 2011), anti-histidine-Ab IgG1 (StrepMAB-Immo) or reagent (NiNTA), alternation of capture modes was performed.

[0436] In some strategies, differential whole-cell panning was performed on human PAR2-expressing Flp-In CHO and BacMam-infected HEK293F cells.

[0437] To reduce the risk of enrichment of candidates specific for other cellular targets (off-target binding), alternation of panning on StaR followed by cell panning was employed.

[0438] To amplify phage antibodies competing with functionally active ligands of human PAR2, panning was performed using nanodisc-embedded human PAR2 StaR mutants PAR2-2 P166:1-377, deglycosylated:N30Q, N222Q, pFastBacHis StrepII or hPAR2 (55-377_10xH_Sx2 (truncated) PAR2-1:P151:55-377, glycosylated, pFastBacHisStrepII) saturated with commercially available small molecule ligands AZ8838 and AZ3451 (Cheng et al. 2017).

[0439] These pannings identified a number of Ylanthia™ candidates, most with good specificity for the human PAR2 receptor expressed in many different cell backgrounds.

[0440] For more stringent selection out of non-specifically binding phages, each round of solid-phase panning was preceded by pre-adsorption with an irrelevant antigen component.

[0441] Irrelevant antigen components included Ni-NTA, empty nanodiscs, and the capture antibodies anti-StrepII mAb (StrepMAB-Immo) and anti-poly6xhistidine-Ab IgG1 (R&D MAB050) or nanodisc-embedded surrogate GPCR (used exclusively in maturation panning).

[0442] [Phage preparation] Production of Fab-displaying phage particles: For each selection round, new phage particles were produced that displayed Fab fragments on their surface.

[0443] Thereby, E. coli TG1 cultures were infected with phages derived from the previous selection round.

[0444] The bacterial pellet was resuspended in fresh medium by centrifugation and plated on an agar plate.

[0445] After growth, colonies were scraped from the plates and used for phage rescue, polyclonal amplification of selected clones and phage production.

[0446] The purified phages were used to initiate the next panning round.

[0447] After the final panning round, single clones were picked from the agar plates into wells of sterile microtiter plates pre-filled with medium.

[0448] During growth, glycerol-containing medium was added to the wells of each master plate, and the plates were sealed in aluminum foil and stored at -80°C.

[0449] Example 3 – Identification of hits by ELISA and whole cell binding by FACS and IntelliCyt screening [ELISA] ELISA techniques were used both for screening of single Fab clones identified from the panning output of the target antigen as well as for characterization of purified antibodies.

[0450] Optimal antigen and antibody concentrations as well as blocking conditions were determined according to state-of-the-art methods.

[0451] [Direct coating of antigens] The antigen was immobilized on a microtiter plate.

[0452] Plates were blocked and incubated with antibodies such as crude E. coli lysates containing Fab or purified Fab or IgG samples.

[0453] Bound antibodies were detected using alkaline phosphatase (AP)-conjugated secondary antibodies in combination with the "AttoPhos" fluorescent substrate.

[0454] Multiple washing steps were performed between the individual assay steps.

[0455] [Antigen capture] In other ELISA settings, antigens were captured to the plate via tag-specific antibodies (eg, anti-StrepII, anti-His or Benchmark 1 reference mAb) coated onto microtiter plates.

[0456] Bound antibodies were detected using respective alkaline phosphatase (AP)-conjugated secondary antibodies in combination with the "AttoPhos" fluorescent substrate.

[0457] Multiple washing steps were performed between the individual assay steps.

[0458] [Fab expression check by anti-Fd ELISA] To verify Fab expression in crude bacterial lysates, plates were coated with Fd fragment-specific antibody.

[0459] Bound Fab was detected using respective alkaline phosphatase (AP)-conjugated anti-Fab specific antibodies in combination with the "AttoPhos" fluorescent substrate.

[0460] Multiple washing steps were performed between the individual assay steps.

[0461] [Koff Ranking ELISA] For Koff estimation, ELISA plates were washed frequently and rigorously during the first readout and a second round of detection was performed to identify candidates with slow kd.

[0462] Washing conditions were as follows: 5 washes with 10 min incubation, overnight incubation, 5 washes with 10 min incubation, 1 h incubation, 5 washes with 5 min incubation, followed by detection.

[0463] Bound antibodies were detected using respective alkaline phosphatase (AP)-conjugated secondary antibodies in combination with the "AttoPhos" fluorescent substrate.

[0464] [ELISA method implementation] ELISA techniques were used both for screening of single Fab clones identified from the panning output of the target antigen as well as for characterization of purified antibodies.

[0465] Optimal antigen and antibody concentrations as well as blocking conditions were evaluated and settings were adjusted.

[0466] After panning selection, 368 clones from each third panning round output were processed in the primary screen as bacterial lysates.

[0467] Panning outputs from solid-phase panning were screened by FACS on StaR-coated magnetic beads or by ELISA using tagged StaR mutants or nanodisc-embedded StaR.

[0468] Screening was primarily performed with the PAR2-1 P151 mutant (55-377, glycosylated, pFastBacHisStrepII).

[0469] The counterscreen included primarily empty nanodiscs, as well as anti-Fd antibody and mouse gamma globulin.

[0470] The secondary screening was mainly performed by ELISA.

[0471] The antigen panel included PAR2-1:P151:55-377, glycosylated, pFastBacHisStrepII, and Nanodisc-embedded PAR2-2:P166:1-377, deglycosylated:N30Q, N222Q, pFastBacHis StrepII saturated with ligands AZ8838 and AZ3451.

[0472] Similarly, the counterscreen was extended to the capture antibodies StrepMAB-Immo and bRIL-His-StrepII.

[0473] Although the binding signals on cells were low, all of the clones of interest showed good correlation between the binding profiles obtained on recombinant Nanodisc-embedded StaR material and cell binding.

[0474] These were selected for sequencing and IgG conversion to the silent h_IgG1f_AEASS format (IgG1 L234A L235E G237A A330S P331S).

[0475] This format has been clinically validated in Novo Nordisk's second generation anti-C5aR antibody NNC0215-0384 (Wagner et al. 2014)) and has been expressed and purified at advanced micro- and / or discovery scales for characterization.

[0476] Prior to functional characterization, IgGs were rescreened for cell binding by ELISA (FIGS. 1 and 2) and IntelliCyt to reconfirm binding specificity.

[0477] [FACS and IntelliCyt] [Screening for positive identification of PAR2 binders using cell binding by FACS and IntelliCyt] Binding events to cell surface expressed antigens were identified by flow cytometry using crude E. coli lysates or purified antibodies from the panning output.

[0478] High-throughput primary screening of panning output from differential whole-cell panning (DWCP) was performed primarily using Flp-In CHO cells expressing human wild-type PAR2 (Flp-In CHO_huPAR2), GNTI BacMam wild-type PAR2 cells and GNTI BacMam truncated PAR2 StaR, together with parental Flp-In CHO cells (PAR2 negative) or uninfected HEK293GNTI cells.

[0479] Candidates showing elevated background binding to the parental Flp-In CHO cells were considered to be either non-specific or off-target binders.

[0480] All steps were performed in FACS buffer containing FCS and sodium azide to prevent potential receptor internalization.

[0481] The cell suspension was transferred to a microtiter plate, the antibody samples were added, and the plate was then incubated at 4°C.

[0482] Sample volume and cell number were adjusted depending on the type of plate used.

[0483] After incubation, the cells were spun down and washed with FACS buffer.

[0484] A fluorescently labeled secondary reagent was used to detect bound antibody.

[0485] Plates were run on a BD FACS array or IntelliCyt HTFC / iQue system and data was analyzed using FlowJo, ForeCyt or proprietary software tools (Figure 3).

[0486] [Assessment of whole cell binding / specificity properties (FACS, IntelliCyt)] [Evaluation of binding specificity to human PAR2-expressing Sf9 cells by flow cytometry] FACS-based analysis of initial PAR2 candidates was performed using PAR2-expressing Sf9 insect cells.

[0487] To understand whether the antibodies preferentially bind to the N-terminus or other extracellularly exposed portions of the receptor, cells were infected with either the full-length N-terminally expressed PAR-2 ​​StaR (PAR2-3, 1-377, here designated FL-N StaR) or an N-terminally truncated receptor (PAR2-4, 55-377, here designated NΔ53 StaR, or designated "truncated" in Figures 4 and 7).

[0488] 200 μL of Sf9 cells at 4×106 cells / mL were incubated with 20 nM of the antibody of interest for 1 hour at room temperature in FACS buffer (PBS (Sigma, #F9665), 1% BSA (Sigma, #A9647) and Roche Complete protease inhibitor (#11836145001)).

[0489] Cells were pelleted and washed three times with 200 μL of FACS buffer.

[0490] Subsequently, cells were incubated with 200 μL of secondary allophycocyanin (APC)-conjugated anti-human IgG at 20 nM for 1 h and again washed three times with 200 μL of FACS buffer.

[0491] Readout was monitored using a BD FACS Canto II FACS instrument.

[0492] A series of binding antibodies was generated with distinct binding profiles as determined by flow cytometry, with Y022075 preferentially binding to the truncated receptor and Y022065 binding to both the truncated and full-length receptor.

[0493] In contrast, the antibody clone Y22066 binds almost exclusively to the FL-N construct.

[0494] These observations reflect the diversity of epitopes (Fig. 4 ).

[0495] [Binding specificity determined by IntelliCyt] Binding specificity was confirmed on purified IgG, performed as duplicate 16-point titrations on human PAR2-expressing cells with n=2 experiments.

[0496] The following cell lines were included in the analysis: - Flp-In CHO_V5His_huPAR2 and parental Flp-In CHO cells - Flp-In CHO_huPAR1 and parental Flp-In CHO cells - HEK Flp-In TRex 293_huPAR2 expression induced and uninduced cells - HEK293F infected BacMam WT FL-huPAR2 and uninfected cells - HEK293F infected BacMam WT FL-marmoset PAR2 and non-infected cells.

[0497] All clones of interest were derived from a PAR2 nanodisc panning strategy, which allows directed presentation by tagged receptors and / or antibody capture via the nanodisc scaffold, and demonstrated selective binding to PAR2 expressed in different cell backgrounds.

[0498] Preparation of PAR2 StaR nanodiscs presented high-quality PAR2 receptor in a detergent-free environment comparable to the native receptor expressed on cells and led to the identification of highly promising functionally active candidates.

[0499] Furthermore, panning stringency can be easily controlled using recombinant materials such as StaR nanodiscs.

[0500] [Multiplexing with IntelliCyt] To simultaneously assess species cross-reactivity and / or binding to unwanted counter-targets, screening was performed in a 384-well plate format using IntelliCyt's HTFC / iQue screening platform.

[0501] The HTFC / iQue screening system was also used for parallel assessment of binding to multiple target cell lines or unwanted / non-specific binding, in other words, multiplexing.

[0502] Different cell populations were distinguished by pre-labeling with different amounts of fluorescent dyes, such as Calcein or Cell-Tracker Green, to establish a unique signature of fluorescence intensity for each cell population, thereby creating a fluorescent coding system.

[0503] The color-coded cell lines were physically mixed and then mixed with the antibody to be tested.

[0504] Individual cell lines were identifiable via the fluorescence of each pre-labeled cell line.

[0505] Crude bacterial cell lysate was mixed with the cells and incubated for 1 hour at room temperature in the dark with gentle shaking.

[0506] Fluorescence measurements were performed on an IntelliCyt HTFC / iQue instrument.

[0507] No washing was necessary between incubation steps.

[0508] The raw data was evaluated using "ForeCyt" software.

[0509] After data acquisition, the cell lines in each sample were identified according to their fluorescent signatures and individually assessed for antibody binding.

[0510] The staining conditions for each cell line were optimized to find an assay setup that allowed separation of the different cell lines (Figure 5).

[0511] The parent of the final read set, Y022065, was derived from a panning strategy employing captured Nanodisc-embedded deglycosylated full-length hPAR2 (PAR2-2, 1-377, N30Q, N222Q)_10xH_Sx2.

[0512] In the first and third rounds, the antigen was captured with an anti-StrepII antibody.

[0513] In the second round, antigen was either captured with Benchmark 1 or coated onto Ni-NTA plates.

[0514] Briefly, 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; assessment of binding to PAR2 expressing cell lines (n>2): CHO V5His_huPAR2, induced HEK Flp-In TRex 293_huPAR2, HEK293F infected BacMam WT FL-huPAR2, A549, Flp-In CHO_maPAR2 (Rhesus monkey, Macaca mulatta), Flp-In CHO_moPAR2 (mouse) and PAR2 negative cell lines (parental Flp-In CHO, Jurkat, Flp-In CHO_huPAR1).

[0515] Example 4 – Expression and purification of Fab fragments and IgG [Subcloning into Fab expression vector] Optionally, and to facilitate rapid expression of soluble Fabs in E. coli, the Fab-encoding inserts of selected Ylanthia™ phages were subcloned from the pYPdis10 display vector into a Fab expression vector (pYBex10_h_Fab-FH) containing the desired characteristics.

[0516] Subcloning was performed by triple digestion mediated by XbaI│EcoRI-HF│PstI-HF.

[0517] Selected candidates were also cloned into the pYMex10_h_FabCys-AviH expression vector for full-length FabCys expression in HKB11 cells.

[0518] Subcloning was performed by excising the antibody-encoding fragment from the pYMex_h_IgG1f_AEASS feeder vector with restriction enzymes NdeI│XhoI and ligating into the target vector pYMex10_h_FabCys-Avi-His.

[0519] [Subcloning into IgG expression vector] Selected candidates were cloned into the pYMex10_h_IgG1f_AEASS expression vector for full-length IgG expression in HKB11 cells.

[0520] Subcloning was performed using a method that conveniently and efficiently converts large amounts of sequence-specific Fab clones into an IgG format.

[0521] Briefly, the VH-encoding fragment (flanked by restriction sites NheI│XhoI), the VL-encoding fragment (flanked by restriction sites NdeI│KpnI) and the kappa- or lambda-specific eukaryotic pYMin expression cassettes (flanked by restriction sites KpnI│NheI) were cloned in one or two cloning steps into an acceptor vector for expression in mammalian cells (digested with NdeI│XhoI).

[0522] After transformation of E. coli XL-1 blue cells, quality control of single clones was performed by colony PCR and sequencing of the entire insert.

[0523] For large-scale expression in HKB11 cells, selected candidates were cloned into pCMex003 / 004_kan_h_IgG1f_AEASS expression vector containing a Zeocin resistance gene for generation of stable pools.

[0524] 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.

[0525] [Generation of bacterial lysates containing Fab fragments] 96-well / 384-well microtiter plates pre-filled with growth medium (2xYT containing chloramphenicol, IPTG and 0.1% glucose) were inoculated with the glycerol stocks from the master plate.

[0526] Plates were incubated at 37°C to allow bacterial growth and then shaken overnight at 22°C for Fab expression.

[0527] The next day, the expression cultures were lysed by the addition of BEL buffer containing borate buffer, EDTA and lysozyme.

[0528] Volumes were adjusted depending on the plate format and application chosen and blocking protocols were adapted accordingly.

[0529] If the lysates were used for sensitive cell screens (eg functional assays), EDTA was omitted.

[0530] [Exploratory scale production of Fab fragments] Expression of the Fab fragments encoded by the bacterial expression vector in E. 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.

[0531] The culture was shaken until the OD600 reached 0.5.

[0532] IPTG (isopropyl-β-D-thiogalactopyranoside) was added, and the cells were further cultured for 20 hours to induce Fab expression.

[0533] The cells were harvested and disrupted with lysozyme.

[0534] His6-tagged Fab fragments were isolated via IMAC (Bio-Rad | Germany) and eluted with imidazole.

[0535] The buffer was replaced with 1x Dulbecco's PBS (pH 7.2) using a "PD10" column (GE Healthcare | Germany).

[0536] The samples were sterile filtered (0.2 μm).

[0537] Protein concentration was measured by UV spectrophotometry.

[0538] The purity of the samples was analyzed by denaturing non-reducing 15% SDS-PAGE.

[0539] The homogeneity of the Fab preparations was determined in the native state by size exclusion chromatography (HP-SEC) using calibration standards.

[0540] [Microscale production of IgG in mammalian cells] Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of IgG.

[0541] Cell culture supernatants were harvested 7 days after transduction and subjected to Protein A affinity chromatography (MabSelect SURE|GE Healthcare) using a liquid handling station.

[0542] Unless otherwise stated, samples were maintained in neutral elution buffer (NaPS: 137 mM Na phosphate, 81 mM NaCl, pH 7).

[0543] The samples were sterile filtered (0.2 μm pore size).

[0544] Protein concentration was measured by UV spectrophotometer, and IgG purity was analyzed under denaturing and reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA).

[0545] HP-SEC was performed to analyze the IgG preparations in the native state.

[0546] [Exploratory scale production of IgG] Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of IgG.

[0547] Cell culture supernatants were harvested 3 or 6 days after transduction and subjected to standard Protein A affinity chromatography (MabSelect SURE|GE Healthcare).

[0548] Unless otherwise stated, buffer exchange into 1x Dulbecco's PBS (pH 7.2 | Invitrogen) was performed and samples were sterile filtered (0.2 μm pore size).

[0549] Protein concentration was measured by UV spectrophotometry and IgG purity was analyzed using CE-SDS (LabChip GXII | Perkin Elmer | USA) under denaturing, reducing and non-reducing conditions.

[0550] HP-SEC was performed to analyze the IgG preparations in the native state.

[0551] [Exploratory-scale production of AviHis-tagged FabCys] Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of disulfide-bridged FabCysAviHis.

[0552] Cell culture supernatants were collected 3 or 7 days after transfection and subjected to metal ion affinity chromatography (Protino Ni-NTA|Macherey Nagel).

[0553] Unless otherwise stated, buffer exchange into 1x Dulbecco's PBS (pH 7.2 | Invitrogen) was performed and samples were sterile filtered (0.2 μm pore size).

[0554] Protein concentration was measured by UV spectrophotometry, and the purity of FabCysAviHis was analyzed using CE-SDS (LabChip GXII | Perkin Elmer | USA) under denaturing, reducing and non-reducing conditions.

[0555] HP-SEC was performed to analyze the FabCysAviHis preparation in the native state.

[0556] [IgG production for in vivo characterization] Material production was performed by establishing a stable HKB11 cell pool.

[0557] Eukaryotic HKB11 cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of IgG.

[0558] For the generation of stable expressing cell pools, each vector further contains a Zeocin resistance gene.

[0559] Three days after transduction, selection was initiated by adding 160 μg / mL Zeocin to the cell suspension.

[0560] During selection, cell number and viability initially declined.

[0561] 20-30 days after transduction, cells began to recover.

[0562] When viability reached approximately 80%, the stable pool was scaled up to the desired volume.

[0563] Six days after transduction, cell culture supernatants were collected and subjected to Protein A affinity chromatography (MabSelect SURE | GE Healthcare).

[0564] If necessary, a second purification step (preparative SEC | Superdex 200 | GE Healthcare) was performed to remove aggregates.

[0565] Buffer exchange into 1x Dulbecco's PBS (pH 7.2 | Invitrogen) was performed, and samples were sterile filtered (0.2 μm pore size).

[0566] Protein concentration was measured by UV spectrophotometry, and IgG purity was analyzed using CE-SDS (LabChip GXII | Perkin Elmer | USA) under denaturing, reducing and non-reducing conditions.

[0567] HP-SEC was performed to analyze the IgG preparations in the native state.

[0568] Endotoxin levels were measured by KQCL assay (Lonza).

[0569] Protein identity was confirmed using mass spectrometry (100MDL43).

[0570] Example 5 – Profiling of lead isolated clones [Cell-based IP-One Gαq assay for identification of functional primary hits in human PAR2] Clones were subjected to functional characterization after confirmation of PAR2 binding status by ELISA and full validation on IntelliCyt.

[0571] The exploratory scale purified IgG1f_AEASS clone was tested in replicates at two concentrations for its ability to inhibit activation of the PAR2 receptor by a synthetic agonist activating peptide (2-Furyol-LIGRO) using the Cisbio™ IP-One Gαq Assay.

[0572] HEK293F cells were infected in suspension culture format with 2% v / v human PAR2 BacMam virus in the presence of 0.5 mM sodium butyrate in growth medium (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin / streptomycin) for 24 h at 37°C in a humidified incubator with a 5% CO2 atmosphere.

[0573] On the day of the experiment, cells were harvested and resuspended to a density of 1x106 cells / mL in assay buffer (1 part stimulation buffer (Cisbio): 5 parts ddH2O) containing LiCl with 0.5% bovine serum albumin (BSA, Sigma).

[0574] Antibodies were tested at stock concentrations or initially diluted 1:2 in PBS.

[0575] 5 μL samples of either antibody concentration were added in duplicate to a half area white 96-well plate (Corning) followed by 25 μL of the PAR2 cell suspension.

[0576] Plates were incubated at 37° C. for 30 min before adding 5 μL / well of activating peptide, giving a final agonist elicitation concentration of 6.28 nM.

[0577] Final assay antibody screening was performed at 1:7 or 1:14 dilutions from Ab stocks.

[0578] The plates were reincubated at 37°C for an additional 30 min before the addition of 10 μL of IP-One Detection Kit (Cisbio) in lysis buffer.

[0579] The cell plate was placed on a plate shaker at room temperature (RT) for 1 hour, after which the plate was read on a PHERAstar FS microplate reader (BMG Labtech) using standard HTRF protocols, with excitation at 335 nm and emission read at both 620 nm and 665 nm.

[0580] The HTRF ratio was calculated as in Eq. 1.

[0581] Responses were normalized to % inhibition of the activating peptide IP-One response at a final assay concentration of 6.28 nM (EC80) (Figure 6).

[0582] Equation 1: Calculation of HTRF ratio

number

[0583] Table 2: Inhibition of peptide-induced agonist activity of human PAR2 Functional characterization of Y021171, Y022063, Y022054 and Y022065 hIgG1f_AEASS at highest replicate concentration in peptide inhibition assays using the Cisbio™ IP-One Gαq Assay. Also shown in FIG. [Table 2]

[0584] [Determination of affinity] [KD, ka and kD determination using antibody capture setup with SPR] For KD determination, monomeric fractions of antibody proteins (Fab fragments or IgG) were used (analyzed by analytical SEC to be >90% monomeric content).

[0585] Affinities were determined by kinetic characterization using either Biacore (Biacore T200) or Octet (QK384 or HTX) instruments as described below.

[0586] The antibodies were captured onto an anti-human IgG chip surface followed by injection of 100 nM PAR2.

[0587] Data were fitted to a 1:1 interaction model, except for the "Y021160" clone, where a heterogeneous ligand model was used with a slow dissociating component for comparison.

[0588] Table 3: Biacore of lead isolated clones [Table 3]

[0589] A suitable high-capacity capture surface was prepared, for example, by covalently immobilizing a suitable capture ligand onto a CM5 chip (Biacore, GE Healthcare) or an AR2G sensor (forteBIO, Pall Corp.) using EDC / NHS chemistry, or by loading an SA sensor with a biotinylated capture reagent.

[0590] Examples of suitable capture systems include anti-human Fc antibodies (Biacore, GE Healthcare), Protein A sensors (forteBIO, Pall Corp.), MabSelect Sure ligands (GE Healthcare), anti-His tag antibodies (Genscript), and the like.

[0591] Six to eight different analyte concentrations (2n-fold serial dilutions) were used for analysis during kinetic experiments.

[0592] After each cycle, the sensor surface was regenerated to remove the captured antibody / antigen complexes while maintaining the integrity of the capture surface.

[0593] A blank injection of running buffer was used for reference.

[0594] In all experiments, the assay buffer was adapted to the formulation of the PAR2 protein, ie buffer and detergent.

[0595] IgG was captured via the Fc fragment, and detergent (LMNG / CHS)-solubilized FL-N PAR2 StaR (PAR2-3) was used as the analyte in solution.

[0596] The assay buffer used was 50 mM HEPES pH 7.5, 150 mM NaCl, 0.02% LMNG.

[0597] A CM3 sensor chip was used as the substrate on which a high density of anti-hu-Fc capture antibody (BR-1008-39, GE Healthcare) was immobilized.

[0598] IgG was captured at a relatively high level, approximately 500 RU, to increase the ratio of specific to non-specific binding (while maintaining suitable conditions for kinetic characterization).

[0599] The sensorgrams were evaluated with the corresponding instrument evaluation software, i.e., Biacore T200 Evaluation Software 3.x (Biacore, GE Healthcare) or forteBIO Octet Data Analysis (forteBIO, Pall Corp.).

[0600] All sensorgrams were fitted to a 1:1 binding model and the kinetic constants ka and kD were determined and used to calculate the KD.

[0601] In Y021160, a heterogeneous ligand model was applied and ka, kD and KD were calculated using the slow dissociation component.

[0602] The affinity of three purified IgG1f_AEASS clones, Y021171, Y022054 and Y022063, to human PAR2-3 variant (P157:1-377, glycosylated, pBacMamHisStrepII, full-length) was determined by SPR by immobilizing IgG and probing with detergent (LMNG / CHS)-solubilized FL-N PAR2 StaR.

[0603] The affinities of Y022063 and Y021171 were 22 nM and 20 nM, respectively.

[0604] Y022054 showed an affinity of 2.5 nM, 10-fold higher than other candidates.

[0605] Y021171 showed fast association (ka) but also very fast dissociation (kD).

[0606] Extrapolating functional inhibition based on binding affinity, the affinity required for antagonistic anti-PAR2 antibodies was estimated to be in the range of <100 pM.

[0607] During the affinity maturation campaign, an “off-rate selection” strategy (Hawkins et al. 1992) was implemented to improve the KD properties, especially for Y021171.

[0608] The interaction between PAR2 and anti-PAR2 antibodies was assayed using a Biacore T200 instrument (GE Healthcare).

[0609] Anti-human IgG antibodies (Human Antibody Capture Kit, GE Healthcare) were immobilized on a sensor chip CM3 (GE Healthcare) by amine coupling at a surface density of 3500–4500 resonance units (RU).

[0610] Fixation was performed in HBS-EP+ buffer (GE Healthcare) at 25°C.

[0611] The buffer was then changed to 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% LMNG, 0.002% CHS.

[0612] Each cycle of the PAR2-antibody interaction assay consisted of antibody capture (100 nM antibody injected for 2 min at a flow rate of 5 μL / min), PAR2 injection (blank or 250 nM, contact and dissociation times of 2 and 3 min, respectively, at a flow rate of 20 μL / min), and regeneration of the chip surface (30 s at 20 μL / min using regeneration solution from the Human Antibody Capture Kit (GE Healthcare)).

[0613] The blank-subtracted data were fitted to a 1:1 interaction model to obtain the association and dissociation rate constants, ka (kon) and kd (koff), and the affinity constant, KD.

[0614] The measurements between each experimental data set were within the acceptable range of this methodology.

[0615] Table 4: Affinity determination of functional candidates Y021171, Y022063 and Y022054 by SPR The antibodies were captured onto an anti-human IgG chip surface followed by injection of 250 nM PAR2.

[0616] Data were fitted to a 1:1 interaction model. [Table 4]

[0617] Example 6 –Affinity maturation / lead optimization and phage selection Several IgG clones (including Y021171, Y022054, Y022063 and Y022065) partially inhibited the activation of PAR2-AP in the IP One assay.

[0618] Several other clones showed cross-reactivity with non-human primate (NHP) PAR2, but none of the four functional candidates showed any clear cross-reactivity to FlpIn CHO_maPAR2 (rhesus macaque, Macaca mulatta) and FlpIn CHO_moPAR2 (mouse) before affinity maturation, despite the high degree of identity between human and rhesus macaque PAR2 sequences in the extracellular domain.

[0619] [Mature Library] Construction of phage maturation libraries: Cloning of the maturation library was performed in a CysDisplay™ vector encoding the parental Fab fragments.

[0620] If not already present in the CysDisplay vector, the DNA sequences encoding the parental Fab fragments were transferred by restriction enzyme digestion and ligation into the respective vector prior to library cloning.

[0621] To increase the affinity and biological activity and reduce non-specificity of selected antibody candidates, the CDR-L3 and CDRH-1 / CDR-H2 regions were optimized in parallel.

[0622] Due to their modular structure, MorphoSys antibody libraries are designed for affinity optimization by CDR exchange, where single CDRs of an antibody are excised and replaced with individual members of a large CDR library (Prassler et al. 2009).

[0623] The Ylanthia™ Maturation Module (YMM) was based on the design of the Ylanthia library and was previously constructed with Slonomics™ technology (van den Brulle et al. 2008).

[0624] The generation of maturation libraries was performed separately for each maturation candidate.

[0625] To monitor the cloning efficiency, the parental CDR-L3 was replaced with Ylanthia Maturation Stuffer (YMS) before inserting the diversified LCDR-L3 YMM.

[0626] The digested vector fragment was ligated in a two-fold molar excess with the insert fragment containing the diversified CDR-L3.

[0627] The same procedure was applied to diversify CDRH-1 and CDR-H2.

[0628] The ligation mixture was electroporated into E. coli MC1061F' cells, yielding >108 independent colonies.

[0629] Library amplification was performed as described previously (Tiller et al. 2013).

[0630] For quality control, approximately 10 single clones per library were randomly picked and subjected to Sanger sequencing.

[0631] Maturation candidates were selected according to the following rationale and criteria: 1 Deselection of candidates with increased risk of off-target binding due to increased binding to: PAR2 negative antigen (ELISA): empty nanodisc, mouse IgG, anti-StrepII, bRIL ·PAR2 negative cell lines (FACS): Parental Flp-In CHO cells, Flp-In CHO_huPAR1 and Jurkat cells 2 Deselection of candidates that preferentially bind to the membrane-distal portion of the N-terminus (assessed by comparative ELISA with FL-StaR nanodiscs and truncated StaR nanodiscs)

[0632] Human PAR2-specific IgG1f_AEASS candidates were ranked as follows: 1 Functionally active candidates 2 As a prerequisite for functional evaluation, binding to cell surface expressed human PAR2 was performed primarily on a functionally active HEK293F cell line infected with WT FL-huPAR2 BacMam. 3 Binding competition with functionally active candidate Y021171 and / or functionally active R&D anti-PAR2 reagent mAb MAB3949, as the published PAR2 structure (Cheng et al., 2017) showed that this reagent antibody interacts with an extracellular loop. Table 5: Overview of maturation candidates [Table 5]

[0633] The main goal of affinity maturation is the improvement of existing properties and does not usually lead to the replacement of the target epitope.

[0634] In IgG characterization, it was observed that functional candidates did not cross-react with rhesus PAR2, whereas non-functional candidates bound to this species.

[0635] Due to the lack of recombinant rhesus PAR2 StaR antigenic material, a differential whole cell panning strategy (DWCP) was performed.

[0636] SPR data showed a fast off-rate of Y021171 (kD(1 / s) 0.01).

[0637] Therefore, during the third maturation panning round, a so-called "off-rate selection" procedure (Hawkins et al., 1992) was performed to enrich for candidates with improved off-rates.

[0638] Because SPR data were not available for all maturation candidates, the method was limited to a panning strategy that included only functional candidates.

[0639] During maturation, washing stringency was increased across panning rounds, for example by increasing the number and duration of washing steps (eg, overnight washes).

[0640] Extended or overnight washes were used in the off-rate selection strategy in combination with the addition of soluble antigen to the wash buffer to prevent rebinding of antibody phages to the immobilized antigen.

[0641] Nine parental clones were advanced into an affinity maturation campaign encompassing all available binding profiles and potential modes of action.

[0642] To increase the affinity and biological activity of previously selected antibody fragments, the CDR-L3 or CDR-H1 and CDR-H2 regions were exchanged in parallel with diversification modules (Prassler et al. 2009).

[0643] To avoid cross-cloning or the appearance of additional framework combinations, a specific Ylanthia™ Maturation Module (YMM) was used to conserve fixed VH / VL human germline framework pairs.

[0644] The parental Fab fragments were transferred from the corresponding expression vectors into the CysDisplay vector prior to library cloning for affinity maturation.

[0645] The CDR-L3 and CDR-H1 and CDR-H2 libraries were cloned separately for each maturation candidate and pooled.

[0646] Affinity maturation was designed separately for six individual candidates, namely, all functional candidates (Y021171, Y022054, Y022063, Y022065) as well as Y022059 and Y022069.

[0647] Weaker candidates were matured in pools (Y021160, Y022075 and Y022079).

[0648] Maturation libraries were generated for the separate diversification of CDR-L3 and CDR-H1 and CDR-H2.

[0649] Maturation panning was designed with a focus on high stringency solid-phase panning with full-length or N-terminally truncated Nanodisc-embedded PAR2 StaRs, as well as generating or improving rhesus PAR2 cross-reactivity.

[0650] After affinity maturation and screening, the project team succeeded in identifying a large set of highly specific Ylanthia™ antibody candidates.

[0651] Clones were progressed for IgG conversion and detailed characterization.

[0652] [Panning selection for affinity maturation] To increase the affinity and biological activity of previously selected antibody fragments, the CDR-L3 and CDR-H2 regions were exchanged in parallel with diversification modules (Prassler et al. 2009).

[0653] The parental Fab fragments were transferred from the corresponding expression vectors into the CysDisplay vector prior to library cloning for affinity maturation.

[0654] For selection of candidates with improved affinity, phages from the maturation library were subjected to three rounds of maturation panning.

[0655] Panning stringency was increased by decreasing the antigen concentration in each panning round (Low et al. 1996).

[0656] In addition to reducing the amount of antigen, off-rate selection was performed for the selected strategy (Hawkins et al. 1992).

[0657] These strategies were combined with longer wash steps in conjunction with the addition of soluble antigen to the wash buffer to prevent rebinding of antibody phages to the immobilized antigen.

[0658] After affinity maturation, the DNA fragments encoding the modified variable regions were directly substituted into the pYMex10_h_IgG1f_AEASS expression vector encoding the parent IgG.

[0659] Briefly, vector components encoding the parental VH or VL were removed with the appropriate restriction enzymes (NheI│XhoI for VH, NdeI│KpnI for VL) and fragments encoding the affinity matured variable regions were inserted and expressed and purified as described above to generate full-length IgG.

[0660] Example 7 – Lead optimization and clone profiling Profiling of purified Ylanthia™ IgG included assessment of binding specificity and cross-reactivity to human, mouse, rhesus and marmoset PAR2-expressing cells, as well as reconfirmation of binding to PAR2 by ELISA on Nanodisc-embedded StaRs and flow cytometry on PAR2-expressing cells.

[0661] After affinity maturation, several IgG clones, primarily derived from the functionally active parental IgG Y022065, were identified to have antagonistic activity due to their ability to completely inhibit activation of the human PAR2 receptor, highlighting their specificity for functionally relevant epitopes.

[0662] Primary screening of truncated or FL PAR2 StaR Nanodiscs in ELISA revealed significant improvement in binding compared to the parental antibody.

[0663] A kd-ranking ELISA was performed for all panning subcodes.

[0664] Plotting the data obtained in the classical and Koff-ranking ELISA setups showed a clear linear correlation of signals, indicating that the majority of maturation candidates have slow Koff values.

[0665] For the analysis, the reduction in binding signal was calculated as the ratio of signal to background (S / BG) ​​in the classical ELISA setting to S / BG in the Koff ranking ELISA: [truncated / FL PAR2 StaR nanodiscs S / BG] / [Koff ELISA truncated / FL PAR2 StaR nanodiscs S / BG].

[0666] Low Koff estimates by ELISA ranking correlated with low signals in a classical ELISA setting.

[0667] Candidates with greater than a two-fold decrease in signal were classified as potentially having fast off-rates.

[0668] 94% of the mature candidates showed no or low (<2-fold) signal change, of which 67% were derivatives of the parent candidate Y021171.

[0669] The parent Y021171 showed the greatest decrease in binding signal in the Koff ranking ELISA, whereas the other parent antibody candidates showed no or low change in signal. This result is consistent with previously generated SPR data for Y021171, which showed this candidate to exhibit a fast off-rate kd(1 / s)=0.01058. 6% of the matured candidates showed an elevated change in signal (2-4.5 fold), suggesting a fast off-rate.

[0670] Of these, 92% were mature derivatives of Y021171 (approximately 33% of the total number of Y021171 candidates) (Figure 7 ).

[0671] [Whole cell binding to PAR2 determined by Intellicyt] Screening of 2200 primary candidates in FlpIn CHO-V5His-huPAR2 and FlpIn CHO parental cell lines on Intellicyt revealed improved binding for most mature derivatives compared to their respective parental antibodies.

[0672] The best improvements were achieved with maturation panning of Y021171, Y022065, Y022069 and the pool-matured parents, with derivatives of Y022079 predicted to be the dominant family.

[0673] All candidates showed low binding (S / BG<1.5) to the FlpIn CHO parental cell line.

[0674] Over 800 candidates showed good or excellent binding to FlpIn CHO-V5His-huPAR2, with 2-5 fold, 5-10 fold or >10 fold (signal / parental signal) improved binding relative to FlpIn CHO parental cells (Figures 8-11).

[0675] [Protein panel profiling for PAR2 specificity (3P)] For protein panel profiling (Frese et al. 2013), 32 different proteins and controls were coated onto two 384-well MSD standard plates (Meso Scale Discovery, MTP 384-well MA6000, #L21XA) at a concentration of 1.0 μg / mL overnight at 4 °C.

[0676] The coating solution was discarded and the plates were blocked with 50 μL of PBS containing 3% (w / v) BSA or 3% (w / v) skim milk powder for 1 h at room temperature on a microtiter plate shaker (approximately 500 rpm), followed by three washing steps with 50 μL of wash buffer (PBS containing 0.05% (v / v) Tween 20).

[0677] Antibody samples (Fab fragments or IgG) were diluted to 100 nM and 10 nM in assay buffer (PBS containing 0.5% (w / v) BSA, 0.05% (v / v) Tween 20).

[0678] As controls, MOR reference mAb anti-lysozyme MOR03207 Fab or IgG (depending on sample format) and assay buffer were used.

[0679] Samples and controls were added (30 μL / well) and incubated for 3 hours at room temperature on a microtiter plate shaker.

[0680] Plates were washed three times and 30 μL of detection antibody (ECL-labeled anti-human Fab, diluted 1:2000) was added to each well and incubated for 1 hour on a microtiter plate shaker (approximately 500 rpm).

[0681] The MSD plate was washed and 35 μL / well of MSD Read Buffer T containing surfactant was added, after which the electrochemiluminescence signal was detected using a Sector Imager 6000 (Meso Scale Discovery | Gaithersburg | MD | USA).

[0682] For evaluation, the signal of the antibody sample on a particular protein was divided by the respective signal of the anti-lysozyme reference mAb MOR03207 to obtain the binding ratio.

[0683] Most clones, including all later mature clones, showed no or low propensity for non-specific binding.

[0684] Example 8 –Functional characterization of lead-optimized clones [Functional Assay: Cell-Based IP-One Gαq Assay] The assay method follows that of Example 5.

[0685] To determine IC50 values, antibody stocks were serially diluted 1:2 over a 10-point concentration curve.

[0686] Lead optimized antibody clones were tested in separate experiments upon challenge with the activating peptide (2-Furyol-LIGRO, 6.28 nM, Table 6), bovine trypsin (2 nM, Table 7) or the PAR1 activating peptide (SFLLR-NH2, 632 nM, Table 8).

[0687] IP-One HTRF results were normalized to the effect of 10 μM of MAB3949 in a challenge against the activating peptide during screening of the parental antibody clones (Figure 12). IP-One HTRF results were normalized to the effect of 1 μM of Benchmark 1 in a challenge against bovine trypsin during screening of parental antibody clones (Figure 13). IP-One HTRF results were normalized to the effect of 10 μM of MAB3949 during screening in a challenge against the PAR1 peptide SFFLR-NH2 trypsin (Table 8). The normalized data were fitted to a four-parameter sigmoidal dose-response curve (Equation 2) in GraphPad Prism version 7.04.

[0688] The data in Table 8 show that the Y022883 antibody can block the activation of PAR2 by the PAR1 activating peptide.

[0689] Equation 2: Fitting a four-parameter sigmoidal dose-response Y=Bottom+(Top-Bottom) / (1+((X^HillSlope) / (IC50^HillSlope))) where IC50 is the concentration (nM) that inhibits the AP / trypsin response by 50% and %max inhibition is the maximum inhibition of AP / trypsin (the value taken from the minimum asymptote or "Bottom" of the curve).

[0690] Table 6: Functional characterization of lead optimized clones that repeatedly show activity in the IP-One human PAR2 antagonist assay using the Cisbio™ IP-One Gαq kit against an activating peptide and share activity in the bovine trypsin challenge assay. [Table 6]

[0691] Table 7: Functional characterization of lead optimized clones that show activity in the IP-One human PAR2 antagonist assay using the Cisbio™ IP-One Gαq kit against bovine trypsin and share activity in the activating peptide challenge assay. If activity was observed in only one replicate, this is indicated in the "Replicate Number" column. [Table 7]

[0692] Affinity matured lead clones derived from the parental IgG Y022065 showed the highest percentage of active lead clones showing activity against both the activation peptide and trypsin.

[0693] Tables 6 and 7 list functional IgG actives that exhibit IC50 values ​​of n>1 against both the activation peptide and bovine trypsin.

[0694] Table 8: IP-One HTRF results normalized to the effect of 10 µM MAB3949 when screened with challenge against the PAR1 peptide SFFLR-NH2 trypsin. [Table 8]

[0695] [Functional assay: cell-based FLIPR calcium mobilization Gαq assay using HT-29 cell line] Confirmation of activity in an immortalized human colon adenocarcinoma cell line (HT-29, which endogenously expresses the PAR2 receptor) was performed following IC50 confirmation of lead clones in the IP-One recombinant human PAR2 in vitro assay.

[0696] HT-29 cells (ATCC HTB-38) were continuously cultured in a humidified incubator at 37°C with a 5% CO2 atmosphere in high glucose (25 mM), sodium pyruvate-free, GlutaMAX-containing DMEM medium (Gibco, Paisley, UK), 10% heat-inactivated fetal bovine serum, and penicillin / streptomycin (100 units / mL penicillin and 100 μg / mL streptomycin).

[0697] The culture medium was changed every 2 days from the second day after seeding, and the cells were harvested in the logarithmic growth phase after reaching 80-90% confluence with 0.05% trypsin / EDTA.

[0698] Cells were seeded in 384-well black wall plates (Corning) at a cell density of 5,000 cells / well in culture medium, 50 μL per well, and incubated for 24 h in a humidified incubator (37° C., 5% CO atmosphere).

[0699] On the day of the experiment, cell culture medium was removed and 50 μL of a 1:20 dilution of stock Calcium 5 dye (Molecular Devices) in assay buffer (HBSS 20 mM HEPES pH 7.4 buffer with 0.1% BSA) was added.

[0700] Plates were reincubated at 37°C for 45 min and then equilibrated at room temperature for an additional 15 min.

[0701] IgG lead clones were prepared in assay buffer and serially diluted to generate a 10-point curve.

[0702] IgG dose-response curves were added online using a FLIPR Tetra (Molecular Devices) pipettor (10 μL) and calcium responses were measured for 5 min.

[0703] Plates were reincubated at 37° C. for an additional 60 min, after which activating peptide (630 nM) or bovine trypsin (63 nM) was added at 10 μL / well online and calcium mobilization-activated human PAR2 was measured for an additional 5 min.

[0704] Data was extracted from the Max-Min raw data files and analyzed using Equation 2.

[0705] Data were normalized to MAB3949 or benchmark 1 for the activation peptide and bovine trypsin assays, respectively.

[0706] Table 9: Functional characterization of lead clones against bovine trypsin or activating peptide (AP) showing complete dose-dependent inhibition n>3 in the HT-29 FLIPR calcium mobilization assay normalized to the corresponding antibody control. [Table 9]

[0707] [Functional assay: Cell-based IP-One Gαq assay for cynomolgus monkey PAR2 and rhesus monkey PAR2] Functional characterization was performed using the Cisbio™ IP-One Gαq assay in replicates at two concentrations to test the cross-species antagonist activity of lead clones against cynomolgus and rhesus PAR2 receptors upon challenge with the synthetic agonist SLIGKV.

[0708] HEK293f cells were infected in suspension culture format with 2.5% v / v cynomolgus PAR2 BacMam virus or 2.5% v / v rhesus PAR2 BacMam virus in the presence of 0.5 mM sodium butyrate in growth medium (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin / streptomycin) for 24 hours at 37°C in a humidified incubator with a 5% CO2 atmosphere.

[0709] On the day of the experiment, cells were harvested and resuspended to a density of 1x106 cells / mL in assay buffer (1 part stimulation buffer (Cisbio): 5 parts ddH2O) containing LiCl with 0.5% bovine serum albumin (BSA, Sigma).

[0710] Antibody stocks were serially diluted 1:2 over a 10-point concentration curve and IC50 values ​​were determined.

[0711] Antibody lead clones were tested upon challenge against SLIGKV (1 μM).

[0712] 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 of the PAR2 cell suspension.

[0713] Plates were incubated at 37° C. for 30 min before adding 5 μL / well of SLIGKV for a final agonist elicitation concentration of 1 μM.

[0714] The plates were reincubated at 37°C for an additional 30 min before the addition of 10 μL of IP-One Detection Kit (Cisbio) in lysis buffer.

[0715] The cell plate was placed on a plate shaker at room temperature (RT) for 1 hour, after which the plate was read on a PHERAstar FS microplate reader (BMG Labtech) using standard HTRF protocols: Excitation was at 335 nm and emission was read at both 620 nm and 665 nm.

[0716] The HTRF ratio was calculated as in Eq. 1.

[0717] Responses were normalized to percent inhibition relative to SLIGKV alone at a final assay concentration of 1 μM, and results were fitted to a four-parameter sigmoidal dose-response curve (Equation 2) in GraphPad Prism version 7.04.

[0718] Table 10: Functional characterization of lead clones against SLIGKV that show antagonist activity in replicate Cisbio IP-One Cynomolgus PAR2 or Rhesus PAR2 receptor assays. [Table 10]

[0719] [Functional Assay: Cell-based IP-One Gαq Cell Assay for Human PAR1] Functional characterization was performed using the Cisbio™ IP-One Gαq assay in replicates at two concentrations to test the cross-species antagonist activity of lead clones against the human PAR1 receptor upon challenge with the synthetic agonist SFLLR.

[0720] HEK293f cells were infected in suspension culture format 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 for 24 h at 37°C in a humidified incubator with a 5% CO2 atmosphere.

[0721] On the day of the experiment, cells were harvested and resuspended to a density of 1x106 cells / mL in assay buffer (1 part stimulation buffer (Cisbio): 5 parts ddH2O) containing LiCl with 0.5% bovine serum albumin (BSA, Sigma).

[0722] Antibody stocks were serially diluted 1:2 over a 10-point concentration curve and IC50 values ​​were determined.

[0723] Antibody lead clones were tested upon challenge against SFLLR (100 nM).

[0724] 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 of the PAR2 cell suspension.

[0725] Plates were incubated at 37° C. for 30 min, after which 5 μL / well of SFLLR was added, resulting in a final agonist elicitation concentration of 100 nM.

[0726] The plates were reincubated at 37°C for an additional 30 min before the addition of 10 μL of IP-One Detection Kit (Cisbio) in lysis buffer.

[0727] The cell plate was placed on a plate shaker at room temperature (RT) for 1 hour, after which the plate was read on a PHERAstar FS microplate reader (BMG Labtech) using standard HTRF protocols: Excitation was at 335 nm and emission was read at both 620 nm and 665 nm.

[0728] The HTRF ratio was calculated as in Eq. 1.

[0729] Responses were normalized for percent inhibition relative to SFLLR alone at a final assay concentration of 100 nM, and results were fitted to a four-parameter sigmoidal dose-response curve (Equation 2) in GraphPad Prism version 7.04.

[0730] As a positive antagonist control for the PAR1 human in vitro assay, Vorapaxar (Axon Medchem Cat 1755), a small molecule PAR1 antagonist, was tested at a final assay concentration of 10 μM in a 3-fold dilution series for dose-response curves and incubations to determine IC50.

[0731] Table 11: Functional characterization of lead clones against SFLLR in the Cisbio IP-One human PAR1 selectivity assay [Table 11]

[0732] [Lead panel - Functional assay: Cell-based FLIPR calcium mobilization Gαq assay using HT-29 cell line] After scale-up, lead clones were characterized in HT-29 cells (following the methods in Example 8) and demonstrated antagonist activity against 0.1 nM bovine trypsin challenge against endogenously expressed PAR2 (FIG. 14).

[0733] Besides, it showed dual activity in proteolytic cleavage within the extracellular N-terminus by trypsin and in inhibiting PAR2-AP activation.

[0734] Analysis in the 3P specificity assay revealed several clones with increased propensity to bind non-specific antigens, thereby reducing the number of dually active IgGs for further consideration as therapeutic candidates.

[0735] To narrow down the target epitopes and for preliminary epitope evaluation, those IgG clones that passed the 3P assay QC were analyzed in an ELISA-based epitope binning experiment.

[0736] All candidates showed potential to compete with each other and likely target proximal binding regions distinct from the N-terminal epitope of Benchmark1.

[0737] Detailed characterization of the final candidate selection included affinity measurements by SPR.

[0738] The number of remaining clones was further reduced based on functional assays, sequence diversity and binding profiles.

[0739] These candidates were produced in a monovalent FabCys-AviH format and tested for cross-functionality against cynomolgus PAR2.

[0740] In addition, the optimized settings improved the affinity determination.

[0741] Y022870, Y022877 and Y022883 (three derivatives of Y022065) were selected as final candidates and defined as the "lead panel" or "lead set" primarily based on performance in functional assays, SPR affinity data, production QC and non-specific binding to 32 different proteins in protein panel profiling (3P) assays.

[0742] These three candidates were finally characterized against a set of six competing antibodies.

[0743] Detailed characterization demonstrated that the lead set binds purified cell surface-expressed PAR2 with affinities within the range of competing antibodies.

[0744] No off-target binding to the related proteins PAR1, PAR3 and PAR4 was detected.

[0745] In contrast to all competing (benchmark) antibodies, the antibodies of the lead set provided herein are able to inhibit not only protease activation but also peptide activation of PAR2, acting as dual inhibitors of PAR2 activation.

[0746] Table 12: In vitro cross-species summary of SLIGKV agonist challenge [Table 12]

[0747] Example 9 –Affinity and kinetics determination of affinity matured antibody clones [KD and koff determination using label-free methods for Biacore and Octet] For high throughput kinetic evaluation of IgG, dissociation rate constants (koff) were determined using either a Biacore T200 or Octet (QK384 or HTX) instrument.

[0748] The same basic assumptions, principles and considerations described for the full kinetic characterization were applied using the monomeric antigenic material (PAR2 StaR).

[0749] The koff for all 88 purified IgGs was determined by Octet and showed a net improvement over the parental antibody. When assessed with truncated huPAR2 StaR and FL-huPAR2 StaR, the kd[1 / s] ranged from 1.1E-4 to 1.0E-05.

[0750] Generally, only one analyte concentration was used for koff determination.

[0751] Samples assessed directly from IgG supernatants or bacterial lysates (Fab fragments) were subjected to koff determination using Octet in antibody capture format.

[0752] As a prerequisite, a monomeric antigen was used as the analyte.

[0753] IgG samples were captured at medium capture levels (0.4 nm) onto streptavidin sensors loaded with capture ligand (biotinylated MabSelect SuRe ligand) at high density.

[0754] Monomeric antigen proteins human PAR2-1 variant (P151 55-377, truncated) and human PAR2-2 variant (P166:1-377, full length) were used as analytes in solution.

[0755] Dissociation was monitored for up to 1500 seconds.

[0756] The recorded sensorgrams were fitted to a 1:1 binding model using the corresponding instrument-specific evaluation software (Biacore T200 Evaluation Software 2.x or 3.x|Octet Data Analysis 9.x or 10.x).

[0757] The affinity of all 11 IgG1f_AEASS to human PAR2 was determined by SPR.

[0758] IgG was captured via its Fc fragment, and detergent (LMNG / CHS)-solubilized FL-N PAR2 StaR was used as the analyte in solution.

[0759] The assay buffer used was 50 mM HEPES pH 7.5, 150 mM NaCl, 0.02% LMNG.

[0760] To minimize artifacts due to nonspecific interactions between FL-N PAR2 StaR and the sensor, a high density of anti-hu-Fc capture antibody (BR-1008-39, GE Healthcare) was immobilized on the CM3 sensor substrate.

[0761] IgG was captured at a relatively high level, approximately 500 RU, to increase the ratio of specific to nonspecific binding (while still maintaining suitable conditions for kinetic characterization).

[0762] The affinities ranged from 6.5 nM to 130 pM.

[0763] The koff values ​​of the best candidates were at the lower limit of the assay.

[0764] The smallest k reported was 2.00E-05 s-1 (corresponding to 3% dissociation in 1500 seconds).

[0765] Affinities were observed with K values ​​ranging from 6.5 nM to 130 pM, most of which showed improvement over the parent clone Y022065 (K = 5.3 nM) following the affinity maturation process, as expected.

[0766] Table 13: SPR kinetics of affinity matured clones determined using the Octet system and soluble PAR2 StaR™ [Table 13]

[0767] Table 14: SPR kinetics determined using the Biacore system with soluble PAR2 StaR™ and analysis of the top three affinity matured clones of interest. [Table 14]

[0768] Table 15: SPR kinetics determined using a Biacore system with human PAR2 StaR™-incorporated Nanodiscs - Comparison of three affinity matured clones with a non-affinity matured clone (Y021171). [Table 15]

[0769] [SPR affinity measurements using recombinant PAR2 StaR™ protein and evaluation of the effect of temperature on binding: Binding of human PAR2 to immobilized mAb at 25°C and 37°C] The interaction between PAR2 and anti-PAR2 antibodies was assayed using a Biacore T200 instrument (GE Healthcare).

[0770] Anti-human IgG antibodies (Human Antibody Capture Kit, GE Healthcare) were immobilized by amine coupling to the sensor chip CM3 (GE Healthcare) of the target and reference flow cells at a surface density of 2700 to 3000 resonance units (RU).

[0771] Fixation was performed in HBS-EP+ buffer (GE Healthcare) at 25°C.

[0772] The buffer was then changed to 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% LMNG, 0.002% CHS for the PAR2-antibody interaction assay, which was performed at either 25°C or 37°C.

[0773] Each cycle of the assay began with capturing antibody on the target flow cell to a level of 400-450 RU.

[0774] PAR2 was then injected over the target and reference flow cells at three concentrations for 5 min each, followed by 30 min of dissociation.

[0775] The surface was then regenerated with the solution provided by the kit (Human Antibody Capture Kit, GE Healthcare).

[0776] For each antibody tested, the first cycle was a blank (three injections of running buffer) and the second was a series of 50, 100 and 200 nM PAR2 injections.

[0777] The blank-subtracted data were fitted to a 1:1 interaction model to obtain the association and dissociation rate constants, ka and kd, and the affinity constant, KD.

[0778] Table 16: Summary of binding of lead clones after affinity maturation at 25°C and 37°C. [Table 16]

[0779] [Evaluation of lead clones to determine whether they exhibit pH-sensitive binding] Y22883 was captured to 280–320 RU and 100 nM PAR2 was injected for 2 min.

[0780] Approximately 400 seconds after the injection was stopped, a pH 6.0 buffer was injected for 5 minutes to see if it would cause faster dissociation.

[0781] The experiment was designed to prevent numerical evaluation of the dissociation rate constants.

[0782] Nevertheless, it is clear that dissociation is not accelerated under pH 6.0 conditions (Figure 15).

[0783] [Whole-cell apparent KD determination by KinExA] The KinExA method was applied to evaluate a subset of lead clones, namely Y022870 and Y02283, for determination of apparent KD with "native" PAR2 (ie, PAR2 expressed on the cell surface).

[0784] To determine the equilibrium KD for surface-expressed antigen, BacMam-infected HEK-293F cells expressing wild-type full-length human PAR2 were used in the titrations instead of soluble antigen.

[0785] Cells were titrated in incubation buffer (D-PBS from Gibco, 0.5% (w / v) BSA, 0.02% (v / v) sodium azide).

[0786] The experiment consisted of two titrations at two fixed IgG concentrations, low and high.

[0787] In both titration experiments (stoichiometry-controlled and affinity-controlled conditions), the maximum amount of cells must contain an effective ligand concentration high enough to completely saturate the IgG.

[0788] The mixture of IgG and cells was incubated overnight at room temperature under very gentle agitation to allow equilibrium formation.

[0789] Any cell-antibody complexes that had formed were then removed by centrifugation.

[0790] The amount of free IgG in the supernatant was measured by fluorescence detection using goat anti-human F(ab')2 fragment-specific antibody (Jackson ImmunoResearch, #109-005-097) and anti-human F(ab')2 fragment-specific Alexa Fluor 647 antibody (Jackson ImmunoResearch, #109-605-097) coated on PMMA (polymethylmethacrylate) beads.

[0791] Because IgG molecules were used in the KinExA experiments, we labelled the obtained values ​​as “apparent KD” and noted that bivalent binding on antigen-presenting cells cannot be excluded and that binding may be enhanced by the contribution of avidity.

[0792] The recorded titration curves were analyzed with KinExA Pro software 4.1.11 using the n-curve analysis tool applying the "equilibrium, whole cell" model.

[0793] Y022870 binds to wild-type human PAR2 expressed on whole cells with an affinity of approximately 370 pM, while Y022883 has an affinity of approximately 260 pM (Figure 16).

[0794] The values ​​are consistent with the SPR determined using PAR2 Nanodiscs (210 and 140 pM, respectively).

[0795] Example 10 –Epitope binning and analysis of affinity matured clones by HDX and flow cytometric analysis of receptor mutations [ELISA-based epitope binning by antibody competition] Epitope binning was performed by an ELISA-based assay.

[0796] Anti-human PAR2 IgG1f sample IgG was directly coated onto microtiter plates at a concentration of 100 nM.

[0797] In parallel, tagged antigen (e.g., nanodisc-embedded StaR) was incubated for 1 h at a constant concentration (12.5 nM–25 nM) with a 25-fold molar excess of a second anti-human PAR2 IgG1f to saturate all epitopes (maximum concentration 312.5 nM–625 nM, 7-point titration, 1:4) and allow complex formation.

[0798] An internal positive control (self-competition) was included for each sample IgG evaluated.

[0799] Antigen-IgG complexes were added to the IgG-coated plates for 30 min. Antigen-IgG complexes bound to the coated IgG were detected via tag-specific antibodies (e.g., anti-StrepII or anti-His) detected in combination with an appropriate alkaline phosphatase (AP)-conjugated secondary antibody and "AttoPhos" fluorescent substrate.

[0800] Multiple washing steps were performed between the individual assay steps.

[0801] If they compete for the same epitope, the antigen-IgG complex will not be able to bind to the IgG-coated plate, which will appear as a low signal.

[0802] By targeting different epitopes, the antigen-IgG complex can simultaneously bind to the coated IgG and to the IgG in solution, leading to a positive detection signal.

[0803] Using this competitive ELISA setup, available candidate target epitopes and epitope diversity were characterized by epitope binning.

[0804] Sixteen dual-function PAR2-specific candidates were tested for binding competition against each other and against reference antibodies such as MAB3949 (R&D Systems) and Benchmark 1.

[0805] This set of 16 antibodies included at least one derivative of each of the five parent families (i.e., clones derived by affinity maturation from parent clones Y022079, Y022069, Y022065, Y021171, Y022059).

[0806] All affinity matured families competed with each other and with anti-human PAR2 (MAB3949 R&D Systems).

[0807] This indicates that all of these antibodies target the proximal binding region (i.e., the extracellular domain other than the N-terminus).

[0808] None of the candidates competed with the benchmark mAb1.

[0809] Benchmark mAb1 is the only antibody in the panel tested that binds exclusively to full-length PAR2, i.e., to a linear N-terminal epitope. These results indicate that the affinity maturation candidates bind to distinct epitopes that are more closely related to or directly target the extracellular loop regions (Figure 17).

[0810] In addition, binding under StaR denaturing conditions was assessed, helping to classify epitopes as linear or conformational epitope recognition.

[0811] 97% of the IgG candidates tested showed reduced binding and were therefore determined to bind conformational epitopes.

[0812] [Hydrogen / Deuterium Exchange] Hydrogen / deuterium exchange (HDX) is one method that can be used to investigate and identify binding interfaces.

[0813] Protein-protein interaction sites are examined by cataloguing the hydrogen / deuterium exchange rates of amide hydrogens in the protein backbone.

[0814] How fast or slow this exchange occurs depends on the accessibility of these amide hydrogens to the solvent.

[0815] Therefore, amides in exposed regions should have faster exchange rates because they are more accessible to the solvent environment than amides buried within the protein-protein interface.

[0816] The following equipment and materials were used in this example:

[0817] Liquid chromatographic separation was performed on a Thermo Vanquish UHPLC (Thermo Scientific) equipped with two temperature-controlled column compartments.

[0818] Online digestion was performed on an enzymatic BEH pepsin column 2.1x50mm (Waters) at 15°C and the resulting peptides were separated on a Kinetex Evo C18 2.6μm 50x2.1mm (Phenomenex) at 4°C.

[0819] The mobile phases for the separation of peptides were acetonitrile / 0.1% formic acid (FA) and water / 0.1% FA (Fisher Chemical).

[0820] Separation was achieved using a 17.5 min gradient from 0% to 40% acetonitrile / 0.1% FA.

[0821] MS1 and MS / MS analyses of the resulting peptides were performed on an Orbitrap Fusion instrument (Thermo Scientific).

[0822] Peptide analysis was performed using Proteome Discoverer 2.4 with a Byonic node (Thermo Scientific), and HDX processing was performed using HDExaminer 2.4 (Sierra Analytics).

[0823] The genetically modified PAR2-2 1-377 (glycosylation site removed) PAR2 StaR protein was purified by LMNG / CHS.

[0824] The antigen-binding fragment (Fab) Y022883 was directly expressed (see Example 4) and the reference Fab was derived from a papain digest of R&D IgG MAB3949.

[0825] 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.

[0826] The PAR2-2 construct was diluted to 50 μM and incubated with equimolar concentrations of Fab Y022883 (or Fab MAB3949) for 30 min at 22°C.

[0827] HDX was initiated by diluting the protein mixture with deuterated buffer (10 mM phosphate buffer pH 7.5, 150 mM NaCl) at 22°C for 1 min, 10 min, 30 min, and 2 h (continuous labeling).

[0828] A 20-fold dilution was chosen to achieve 90-95% D-labeling of solvent-accessible hydrogens (typically, 2 µl of protein mixture mixed with 38 µl of deuteration buffer).

[0829] After the desired time, the exchange was quenched by adding 0.8% formic acid to lower the pH of the solution to approximately 2.5, and the solution was flash frozen on dry ice to reduce back exchange.

[0830] The samples were then thawed and subjected to enzymatic online digestion on a pepsin column followed by analysis by LC-MS.

[0831] The same protocol was then carried out for the PAR2-2 construct alone and for Fab Y022883 (or Fab MAB3949) alone to identify solvent / deuterium accessible hydrogens in the uncomplexed protein.

[0832] Comparing the deuteration level of the PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) complex with that of the homogeneous protein (Fab Y022883, Fab MAB3949 or PAR2-2) shows changes in the environment of solvent accessible hydrogens and such "mapping" can then be used to determine the PAR2-2 / Fab Y022883 (or PAR2-2 / Fab MAB3949) interaction site.

[0833] To validate this methodology, the HDX results obtained for PAR2 / MAB3949 Fab were mapped onto the published co-crystal structure of the complex of PAR2 and MAB3949 Fab (Cheng et al., Nature, 2017).

[0834] The interactions predicted from HDX were in good agreement with those observed in the crystal costructure.

[0835] Next, examining PAR2-2 / Fab Y022883 HDX, the data showed a different interaction than that observed for Fab MAB3949 (Cheng et al., Nature, 2017) (Figures 18-20).

[0836] The N-terminal region of PAR2 exhibits a characteristic deuterium-hydrogen exchange pattern, possibly suggesting that this region is involved in binding.

[0837] Table 17: List of PAR2-2 peptides identified by HDX, located in the interaction region, and therefore determined to contain the Y022883 antibody epitope.

[0838] [Table 17]

[0839] These HDX data suggest that it is highly unlikely that ECL1 or ECL2 of PAR2 is involved in a direct interaction with the Y022883 PAR2 antibody. The direct interactions observed using this method are located in segment 1, the helix 0 / 1 region (close to the transmembrane domain, in contrast to other PAR2-targeted antibodies that bind to more distal regions of the receptor) and ECL3.

[0840] [Mutation analysis using flow cytometry] The PAR2-binding epitope mutations were evaluated by flow cytometry on human PAR2-expressing HEK293F-cells as follows.

[0841] Expression levels were monitored using the Benchmark 1 antibody.

[0842] Mutations introduced into the WT PAR2 background targeted segment 1, helix 0 / 1 (single mutations F59A, S60W, D62G, D62F, E63K, E63A and double mutation D62G / E63G) or ECL3 (single mutations Q317A, G318F, Q319A, Q319I).

[0843] Various versions of PAR2 were introduced into HEK293F cells by BacMam infection.

[0844] 200 μL of HEK293F-cells at 4×106 cells / mL in FACS buffer (PBS (Sigma, #F9665), 1% BSA (Sigma, #A9647) and Roche Complete protease inhibitors (#11836145001)) were incubated with 20 nM Benchmark 1 or Y022883 for 1 h at 4°C.

[0845] Cells were pelleted and washed three times with 200 μL of FACS buffer.

[0846] The cells were then incubated with 200 μL of a secondary allophycocyanin (APC)-conjugated anti-human IgG antibody (20 nM) at 4° C. for 1 h and washed again three times with 200 μL of FACS buffer.

[0847] Mutant PAR2 and WT PAR2 showed very similar expression profiles when detected by Benchmark 1, except for D62F, which had partially reduced expression levels, whereas S60W, D62F, G318F and the double mutation D62G / E63G all had reduced expression profiles compared to WT when detected by Y022883, indicating that the regions identified by HDX analysis are involved in the binding of Y022883 to human PAR2.

[0848] The reduction in binding observed with selected mutations is stronger for segment 1 / helix 0 / 1 than for ECL3 (Figure 21).

[0849] The effect of these mutations is to result in structural changes that alter antibody epitopes and potentially lead to changes in the functional activity of the receptor.

[0850] Example 11 – Pharmacokinetics in rats The pharmacokinetics of Y022883 was studied in adult male Sprague Dawley rats.

[0851] Three rats were administered Y022883 intravenously at 10 mg / kg and blood was collected at various time points up to two weeks.

[0852] Blood samples were used to prepare serum, which were analyzed by a certified non-GLP ELISA method (generic PK kit) on the Gyrolab platform and PK parameters were calculated by non-compartmental analysis (NCA) using PhoenixTM WinNonlin.

[0853] Summary data is shown in FIG. 22 and Table 18.

[0854] These data are consistent with the expectation of a human mAb with no significant antigen sink, as predicted by the lack of binding of Y022883 to rat PAR2.

[0855] Table 18: Group PK parameters of Y022883 in rats (n=3).

[0856] Data are presented as mean values ​​with standard deviations in parentheses.

[0857] [Table 18]

[0858] Example 12 – Pharmacokinetics and Pharmacodynamics in Cynomolgus Monkeys The pharmacokinetics of Y022883 was investigated in adult male cynomolgus monkeys.

[0859] Three groups of three adult male cynomolgus monkeys were administered Y022883 intravenously at 10 mg / kg, 3 mg / kg, or 1 mg / kg, and blood was collected at various time points up to four weeks.

[0860] Blood samples were used to assess pharmacodynamics (including Figures 24-26) or serum was prepared for pharmacokinetics (see Figure 23).

[0861] Serum was analyzed by a certified non-GLP ELISA method (generic PK kit) on a Gyrolab platform and PK parameters were calculated by non-compartmental analysis (NCA) using PhoenixTM WinNonlin.

[0862] Summary data is shown in FIG. 23 and Table 19.

[0863] These data are consistent with the expectations of a human mAb that does not have a significant antigen sink.

[0864] Table 19: Group PK parameters of Y022883 in cynomolgus monkeys (n=3).

[0865] Data are presented as mean values ​​with standard deviations in parentheses.

[0866] [Table 19]

[0867] Pharmacodynamics were evaluated under sterile conditions at four time points (pre-dose, days 1, 13, and 29 post-dose).

[0868] One volume of PBS was added to each blood sample, and dextran T-500 was added at 6% for a final concentration of 2%.

[0869] After 25–40 min of incubation (the reaction was stopped when the leukocyte-rich and erythrocyte-rich phases were 50% each), the supernatant containing leukocytes was removed, washed with PBS, and resuspended in the original blood volume in culture medium (RPMI-1640, 2% FCS, 1% penicillin-streptomycin, 1% L-glutamine, 1% non-essential amino acids).

[0870] 1 mL cell samples were then challenged with PBS, 100 mcM 2f-LIGRLO, 100 nM trypsin or 100 pg / mL LPS and incubated at 37° C. for 6 hours.

[0871] Samples were then pelleted (306 g, 5 min, room temperature), the pellet resuspended, mixed with 0.48 mL of RNAlater, and then frozen at -70°C.

[0872] Sample RNA was then extracted using standard methods and the transcriptome was assessed using genome-based read mapping at a read depth of 50x.

[0873] The sequence reads (FASTQ files) were aligned to the reference genome and the number of reads mapped to each gene was counted, which generated a gene count table.

[0874] Next, analysis of gene expression differences between each stimulus at each time point for each dose group was performed (e.g., 1 mg / kg pre-PBS vs. 1 mg / kg pre-PAR2AP, 1 mg / kg pre-PBS vs. 1 mg / kg pre-trypsin, 1 mg / kg pre-PBS vs. 1 mg / kg pre-LPS).

[0875] Differential gene expression was then determined by creating a volcano plot, which is a scatter plot of fold change in gene expression versus statistical significance (P value).

[0876] Genes that showed a 2-fold or greater increase or decrease in expression with a significance level of 0.05 or less were considered as differentially expressed genes.

[0877] Thus, a differentially expressed gene set (from pre-treatment samples) was generated for each stimulus. The effect of mAb treatment was assessed by determining whether the pre-treatment differentially expressed gene set for each stimulus was still differentially expressed at each time point.

[0878] For example, 1816 genes were differentially regulated by LPS at pre-dose in the 1 mg / kg dose group (compared to pre-dose PBS). After 24 hours, 1379 of these genes were still differentially regulated by LPS (compared to 24 hour PBS). Thus, in the 1 mg / kg LPS dose group, 76% of the pre-dose LPS gene signature was present 24 hours after dosing.

[0879] Using this methodology, a single IV injection of 10 mg / kg Y022883 suppressed the PAR2AP-induced and trypsin-induced gene signatures at all time points, whereas the LPS gene signature was suppressed by 36% after 24 hours before recovering to 23% suppression by day 29 (Figure 24).

[0880] A similar set of observations occurred following administration of 3 mg / kg Y022883 (Figure 25), however with 1 mg / kg Y022883, the trypsin gene signature began to recover by day 29 (96%, 92%, and 91% inhibition at 24 hours, day 13, and day 29; Figure 26).

[0881] These data are consistent with Y022883 specifically antagonizing PAR2 (at least relative to TLR4) and blocking PAR2 on peripheral blood leukocytes for at least one month following a single injection of 1 mg / kg.

[0882] Partial suppression of the LPS-induced gene signature by PAR2 antagonism is consistent with literature reports linking TLR4 and PAR2 signaling cascades (Yamaguchi et al., 2016).

[0883] Example 13 Effects of SH-C and Benchmark II on trypsin and Par2-AP-induced p38 MAPK and pERK phosphorylation in T84 cell line T84 cells were treated with trypsin (1 μM) or PAR2-AP(2fu-LIGRLO) (10 μM) in growth medium.

[0884] Antibodies Y022883 (SH-C) and Benchmark 2 (SH-D) were tested at concentrations ranging from 10 nM to 3 μM.

[0885] Cells were lysed in lysis buffer (PBS + 1% Triton X-100, protease and phosphatase inhibitors) 5 or 30 min after treatment.

[0886] Total protein concentration was assessed by BCA followed by analysis of phosphorylated proteins.

[0887] Protein concentration was adjusted to 0.3 mg / mL and samples were loaded onto a 12-230 kDa Wes Separation Module 8 × 25 capillary cartridge (Protein Simple).

[0888] Rabbit antibodies were used to detect pERK and ERK (pERK, Cell Signaling, cat:9101S; ERK, Cell Signaling, cat:4695S) and phosphorylated p38 MAPK and p38 MAPK (Cell Signaling, cat:9211L; p38, Cell Signaling, cat:9212S).

[0889] Anti-rabbit detection module (Protein Simple, cat:DM-001) was used as the secondary antibody.

[0890] Data for phosphorylated ERK or phosphorylated p38 were normalized to data for total ERK or total p38. Biological duplicates were analyzed.

[0891] The percentage of maximum ratio was used to normalize between plates (Figures 27-29).

[0892] Treatment with trypsin or PAP2-AP increased pERK by more than eight-fold and phosphorylated p38-MAPK by more than three-fold.

[0893] Both SH-C and Benchmark II showed inhibition of ERK and p38-MAPK phosphorylation in trypsin-treated cells.

[0894] Only SH-C showed significant inhibition of phosphorylation in PAR2-AP-treated cells.

[0895] array Table 20-23: Antibody clone sequences for clones Y022065, Y022870, Y022877 and Y022883. CDRs were identified according to Kabat. [Table 20] [Table 21] [Table 22] [Table 23]

[0896] Table 24: Sequence Listing Keys [Table 24-1] [Table 24-2] [Table 24-3] [Prior art documents] [Non-patent literature]

[0897] [Non-Patent Document 1] Abbas , A. , Lichtman , A. and Pillai , S. , 2000 . 4th ed.

Outdoor Tool2

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[0898] The present invention is not limited in scope by the specific embodiments described herein. In addition to those actually described, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All references cited herein, including publications, patents, patent applications, etc., are incorporated herein by reference in their entirety.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to PAR2 and inhibits its activity, wherein the antibody or antigen-binding fragment contains the amino acid sequence of SEQ ID NOs. 41, 42, 43, and 44, or binds to an epitope consisting of the same sequence.

2. The antibody or antigen-binding fragment thereof according to Claim 1, (a) The amino acid sequence of SEQ ID NO: 5, 22, or 30; or an HCDR3 comprising or containing an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 5, 22, or 30. (b) HCDR3 having at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NOs. 5, 22, or 30, or (c) An HCDR3 amino acid sequence defined by Kabat or Chothia, derived from a VH domain selected from SEQ ID NOs: 2, 10, 13, 16, 19, or 27. An antibody or its antigen-binding fragment comprising a VH domain containing HCDR3 selected from the above.

3. The antibody or antigen-binding fragment thereof according to Claim 1, comprising a VH domain, wherein the VH domain is i) The amino acid sequence of SEQ ID NOs: 3, 11, 14, 17, 20, or 28; or an HCDR1 amino acid sequence selected from amino acid sequences having 3, 2, or 1 amino acid substitutions in SEQ ID NOs: 3, 11, 14, 17, 20, or 28, and / or ii) The amino acid sequence of SEQ ID NOs. 4, 12, 15, 18, 21, or 29; or an HCDR2 amino acid sequence selected from amino acid sequences having 3, 2, or 1 amino acid substitutions in SEQ ID NOs. 4, 12, 15, 18, 21, or 29. An antibody or its antigen-binding fragment, including the above.

4. An antibody or antigen-binding fragment according to Claim 1, comprising a V L domain, wherein the V L domain contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 6, 23, or 31.

5. The antibody or antigen-binding fragment thereof according to Claim 1, comprising LCDR3, a) The LCDR3 amino acid sequence is selected from the amino acid sequence of SEQ ID NO: 9, 26, or 33; or an amino acid sequence having 3, 2, or 1 amino acid substitutions in SEQ ID NO: 9, 26, or 33, or b) The LCDR3 amino acid sequence is defined by Chothia or Kabat and is derived from the VL domain by SEQ ID NO: 6, 23, or 31, or the LCDR3 sequence includes an amino acid sequence having 3, 2, or 1 amino acid substitutions in SEQ ID NO: 6, 23, or 31. An antibody or its antigen-binding fragment.

6. The antibody or antigen-binding fragment thereof according to claim 4, wherein the VL domain is a) i) The amino acid sequence of SEQ ID NO: 7, 24, or 32; or the LCDR1 amino acid sequence having 3, 2, or 1 amino acid substitutions in SEQ ID NO: 7, 24, or 32, or ii) An LCDR1 amino acid sequence defined by Chothia or Kabat and derived from the VL domain of SEQ ID NO: 6, 23, or 31; or an LCDR1 amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 6, 23, or 31, and / or b) i) The amino acid sequence of SEQ ID NO: 8 or 25; or the LCDR2 amino acid sequence having 3, 2, or 1 amino acid substitutions in SEQ ID NO: 8 or 25, or ii) An LCDR2 amino acid sequence defined by Chothia or Kabat and derived from the V L domain of SEQ ID NO: 6, 23, or 31; or an LCDR2 amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 6, 23, or 31. An antibody or its antigen-binding fragment, including the above.

7. The antibody or antigen-binding fragment thereof according to Claim 5, wherein the VL domain is a) i) The amino acid sequence of SEQ ID NO: 7, 24, or 32; or the LCDR1 amino acid sequence having 3, 2, or 1 amino acid substitutions in SEQ ID NO: 7, 24, or 32, or ii) An LCDR1 amino acid sequence defined by Chothia or Kabat and derived from the V L domain of SEQ ID NO: 6, 23, or 31, or an LCDR1 amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 6, 23, or 31, and / or b) i) The amino acid sequence of SEQ ID NO: 8 or 25; or the LCDR2 amino acid sequence having 3, 2, or 1 amino acid substitutions in SEQ ID NO: 8 or 25, or ii) An LCDR2 amino acid sequence defined by Chothia or Kabat, derived from the V L domain of SEQ ID NO: 6, 23, or 31, or an LCDR2 amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 6, 23, or 31. An antibody or its antigen-binding fragment, including the above.

8. The antibody or antigen-binding fragment thereof according to Claim 1, VH region selected from the amino acid sequences of SEQ ID NOs. 2, 10, 13, 16, 19, or 27, or at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% identical thereto, and VL region with an amino acid sequence identical to SEQ ID NOs. 6, 23, or 31, or at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% of these. An antibody or its antigen-binding fragment, including the above.

9. The antibody or antigen-binding fragment according to claim 8, wherein the VH region contains the same or at least 90% the same amino acid sequence as SEQ ID NO: 2, and the VL region contains the same or at least 90% the same amino acid sequence as SEQ ID NO:

6.

10. The antibody or antigen-binding fragment according to claim 8, wherein the VH region contains the same or at least 90% the same amino acid sequence as SEQ ID NO: 10, and the VL region contains the same or at least 90% the same amino acid sequence as SEQ ID NO:

6.

11. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VH region comprises the same or at least 90% the same amino acid sequence as SEQ ID NO: 13, and the VL region comprises the same or at least 90% the same amino acid sequence as SEQ ID NO:

6.

12. The antibody or antigen-binding fragment according to claim 8, wherein the VH region contains the same or at least 90% the same amino acid sequence as SEQ ID NO: 16, and the VL region contains the same or at least 90% the same amino acid sequence as SEQ ID NO:

6.

13. The antibody or antigen-binding fragment according to claim 8, wherein the VH region contains the same or at least 90% the same amino acid sequence as SEQ ID NO: 19, and the VL region contains the same or at least 90% the same amino acid sequence as SEQ ID NO:

23.

14. The antibody or antigen-binding fragment according to claim 8, wherein the VH region contains the same or at least 90% the same amino acid sequence as SEQ ID NO: 27, and the VL region contains the same or at least 90% the same amino acid sequence as SEQ ID NO:

31.

15. An antibody or antigen-binding fragment thereof according to Claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 3, (b) HCDR2 having the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 4, (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

5. The VL includes, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 7, (e) LCDR2 having the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 8, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

9. An antibody or its antigen-binding fragment, including the above.

16. An antibody or antigen-binding fragment thereof according to Claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 11, (b) HCDR2 having the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 12, and (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

5. The VL includes, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 7, (e) LCDR2 having the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 8, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

9. An antibody or its antigen-binding fragment, including the above.

17. An antibody or antigen-binding fragment thereof according to Claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 14, (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 15, (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

5. The VL includes, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 7, (e) LCDR2 having the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 8, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

9. An antibody or its antigen-binding fragment, including the above.

18. An antibody or antigen-binding fragment thereof according to Claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 17, (b) HCDR2 having the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 18, (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

5. The VL includes, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 7, (e) LCDR2 having the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 8, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

9. An antibody or its antigen-binding fragment, including the above.

19. An antibody or antigen-binding fragment thereof according to Claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 20, (b) HCDR2 having the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 21, (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

22. The VL includes, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 24, (e) LCDR2 having the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 25, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

26. An antibody or its antigen-binding fragment, including the above.

20. An antibody or antigen-binding fragment thereof according to Claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 28, (b) HCDR2 having the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 29, (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

30. The VL includes, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO: 32, (e) LCDR2 having the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having three, two, or one amino acid substitutions in SEQ ID NO: 25, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence containing three, two, or one amino acid substitutions in SEQ ID NO:

33. An antibody or its antigen-binding fragment, including the above.

21. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment inhibits the activation of the PAR2 peptide of PAR2.

22. The antibody or antigen-binding fragment thereof according to claim 1, wherein the inhibition of PAR2 activation includes inhibition of PAR2 tether ligand binding.

23. The antibody or antigen-binding fragment thereof according to claim 1, which inhibits the binding of a PAR2-activating peptide to PAR2.

24. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment inhibits the binding of a PAR2-activating peptide to PAR2, and the antibody or antigen-binding fragment inhibits the accumulation of inositol monophosphate (IP) via the PAR2-activating peptide with an IC50 of 1 to 100 nM.

25. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment inhibits the accumulation of trypsin-mediated IP with an IC50 of 1 to 300 nM.

26. An antibody or antigen-binding fragment thereof according to Claim 1, wherein the antibody or antigen-binding fragment inhibits the accumulation of inositol monophosphate (IP) via a PAR2-activated peptide with an IC50 of 1 to 100 nM.

27. ​​An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment inhibits calcium mobilization via a PAR2-activating peptide.

28. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment inhibits calcium mobilization via trypsin.

29. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment is not taken up into the cell when it binds to PAR2 on the cell surface.

30. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment does not inhibit the activation of PAR1 via ligand SFLLR, and the activation of PAR1 is determined using a ligand SFLLR-stimulated IP signaling assay.

31. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey PAR2 at an EC50 of 600 pM to 5 nM or less.

32. An antibody or antigen-binding fragment according to Claim 1, wherein the antibody or antigen-binding fragment binds to human PAR2 with a KD of 100 pM to 10 nM.

33. The antibody or antigen-binding fragment according to claim 1, wherein the binding of the antibody or its antigen-binding fragment to PAR2 is pH-independent between pH 7.5 and 6.

0.

34. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment does not bind to PAR1.

35. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment does not bind to PAR3.

36. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment does not bind to PAR4.

37. An antibody or antigen-binding fragment according to Claim 1, wherein 3 mg / kg of the antibody or antigen-binding fragment suppresses the PAR2 stimulation-induced response in leukocytes by more than 95% over a period of 30 days, and the suppression is measured by determining the stimulation-induced gene signature.

38. An antibody or antigen-binding fragment according to Claim 1, wherein 1 mg / kg of the antibody or antigen-binding fragment suppresses the PAR2 peptide-induced response in leukocytes by more than 90% over a period of 30 days, and the suppression is measured by determining the stimulus-induced gene signature.

39. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to PAR2 and inhibits cross-activation of PAR2 by a PAR1 tether ligand.

40. An antibody or antigen-binding fragment thereof according to claim 1, for use in therapeutic purposes.

41. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, for use in the treatment of PAR2-mediated diseases or conditions.

42. The antibody or antigen-binding fragment thereof according to claim 41, wherein the PAR2-mediated disease or condition is selected from atopic dermatitis, asthma, cancer (various cancers including breast cancer, melanoma, and head and neck cancer), pain (chronic, inflammatory, postoperative, neuropathic, fracture, gout, cancer, gastrointestinal tract 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 disease arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

43. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 39 in the manufacture of a pharmaceutical product for the treatment of PAR2-mediated diseases or conditions.

44. The use according to claim 43, wherein the PAR2-mediated disease or condition is selected from atopic dermatitis, asthma, cancer (various cancers such as breast cancer, melanoma, and head and neck cancer), pain (chronic, inflammatory, postoperative, neuropathic, fracture, gout, cancer, gastrointestinal tract 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 disease arthritis, Still's disease, dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

45. An antibody or antigen-binding fragment thereof according to claim 41, wherein the treatment further comprises administering an additional treatment, the additional treatment comprising 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, Bicozin, hydrocodone, morphine, oxycontin, roxicodone, Percocet, aspirin, celecoxib, pregabalin, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab, tocilizumab, and tofacitinib.

46. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 39 and a pharmaceutically acceptable excipient, diluent, or carrier.

47. Analgesics (e.g., anti-inflammatory agents (e.g., NSAIDs including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g., codeine, morphine, oxycodone, fentanyl, buprenorphine)), amitriptyline, gabapentin, anticancer agents (e.g., alkylating agents (e.g., nitrogen mustard, nitrourea), antimetabolites (e.g., folic acid analogs, pyrimidines and purine analogs), antibiotics and enzymes (e.g., dactinomycin, The present invention further includes one or more additional therapeutic agents independently selected from the group consisting of daunorubicin, doxorubicin, L-asparaginase), natural products (e.g., vinca alkaloids, taxen, tecan), hormones and antagonists (e.g., progestins, estrogens, GnRH, anti-estrogens), hydroxyureas, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecular targeted therapies (e.g., antibody conjugates), and platinum-based therapies (e.g., cisplatin, carboplatin, oxaliplatin), The pharmaceutical composition according to claim 46.

48. A pharmaceutical composition according to claim 46, wherein the pharmaceutical composition is for the treatment of PAR2-mediated diseases or conditions.

49. The pharmaceutical composition according to claim 48, wherein the PAR2-mediated disease or condition is selected from atopic dermatitis, asthma, cancer (various cancers such as breast cancer, melanoma, and head and neck cancer), pain (chronic, inflammatory, postoperative, neuropathic, fracture, gout, cancer, gastrointestinal tract 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.

50. A kit comprising the pharmaceutical composition described in claim 46, wherein the pharmaceutical composition is for use in the treatment of PAR2-mediated diseases or conditions.

51. A kit according to claim 50, wherein the PAR2-mediated disease or condition is selected from atopic dermatitis, asthma, cancer (various cancers such as breast cancer, melanoma, and head and neck cancer), pain (chronic, inflammatory, postoperative, neuropathic, fracture, gout, cancer, gastrointestinal tract 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.

52. The antibody or antigen-binding fragment according to claim 1, wherein the VH domain comprises the following: (a) the HCDR3 amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 including three, two, or one amino acid substitutions; and i) The HCDR1 amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 including three, two, or one amino acid substitutions; and / or ii) The HCDR2 amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4, including three, two, or one amino acid substitutions; (b) the HCDR3 amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 including three, two, or one amino acid substitutions; and i) The HCDR1 amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 11 including three, two, or one amino acid substitutions; and / or ii) The HCDR2 amino acid sequence of SEQ ID NO: 12 or containing three, two, or one amino acid substitutions; (c) the HCDR3 amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 including three, two, or one amino acid substitutions; and i) The HCDR1 amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 including three, two, or one amino acid substitutions; and / or ii) The HCDR2 amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 15, or containing three, two, or one amino acid substitutions; (d) the HCDR3 amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 including three, two, or one amino acid substitutions; and i) The HCDR1 amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 17 including three, two, or one amino acid substitutions; and / or, ii) The HCDR2 amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 18, including three, two, or one amino acid substitutions; (e) the HCDR3 amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 22 including three, two, or one amino acid substitutions; and i) The HCDR1 amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 20 including three, two, or one amino acid substitutions; and / or ii) The HCDR2 amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 21, including three, two, or one amino acid substitutions; (f) the HCDR3 amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 30 including three, two, or one amino acid substitutions; and i) The HCDR1 amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 28 including three, two, or one amino acid substitutions; and / or ii) The HCDR2 amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 29 containing three, two, or one amino acid substitutions; (g) an HCDR3 amino acid sequence derived from a VH domain selected from SEQ ID NO: 2, as defined by Chothia or Kabat; or an HCDR3 amino acid sequence derived from a VH domain selected from SEQ ID NO: 2, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain selected from SEQ ID NO: 2, as defined by Chothia or Kabat; or an HCDR1 amino acid sequence derived from a VH domain selected from SEQ ID NO: 2, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and / or ii) An HCDR2 amino acid sequence derived from a VH domain selected from SEQ ID NO: 2, as defined by Chothia or Kabat; or an HCDR2 amino acid sequence derived from a VH domain selected from SEQ ID NO: 2, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; (h) an HCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 10; or an HCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 10 containing three, two, or one amino acid substitutions; and i) an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 10; or an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 10 containing three, two, or one amino acid substitutions; and / or ii) An HCDR2 amino acid sequence derived from a VH domain selected from SEQ ID NO: 10, as defined by Chothia or Kabat; or an HCDR2 amino acid sequence derived from a VH domain selected from SEQ ID NO: 10, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; (i) an HCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 13; or an HDR3 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 13 containing three, two, or one amino acid substitutions; and i) an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 13; or an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 13 containing three, two, or one amino acid substitutions; and / or ii) An HCDR2 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 13; or an HCDR2 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 13 containing three, two, or one amino acid substitutions; (j) an HCDR3 amino acid sequence derived from a VH domain selected from SEQ ID NO: 16, as defined by Chothia or Kabat; or an HCDR3 amino acid sequence derived from a VH domain selected from SEQ ID NO: 16, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and i) an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 16; or an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 16 containing three, two, or one amino acid substitutions; and / or ii) An HCDR2 amino acid sequence derived from a VH domain selected from SEQ ID NO: 16, as defined by Chothia or Kabat; or an HCDR2 amino acid sequence derived from a VH domain selected from SEQ ID NO: 16, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; (k) an HCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 19; or an HCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 19 containing three, two, or one amino acid substitutions; and i) an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 19; or an HCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 19 containing three, two, or one amino acid substitutions; and / or ii) An HCDR2 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 19; or an HCDR2 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 19 containing three, two, or one amino acid substitutions; (l) an HCDR3 amino acid sequence derived from a VH domain selected from SEQ ID NO: 27, as defined by Chothia or Kabat; or an HCDR3 amino acid sequence derived from a VH domain selected from SEQ ID NO: 27, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and i) an HCDR1 amino acid sequence derived from a VH domain selected from SEQ ID NO: 27, as defined by Chothia or Kabat; or an HCDR1 amino acid sequence derived from a VH domain selected from SEQ ID NO: 27, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and / or ii) An HCDR2 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 27; or an HCDR2 amino acid sequence defined by Chothia or Kabat and derived from a VH domain selected from SEQ ID NO: 27 containing three, two, or one amino acid substitutions.

53. The antibody or antigen-binding fragment according to claim 1 or 52, wherein the VL domain comprises the following: (a) the LCDR3 amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 9 including three, two, or one amino acid substitutions; and i) The LCDR1 amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 including three, two, or one amino acid substitutions; and / or ii) The LCDR2 amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 including three, two, or one amino acid substitution; (b) The LCDR3 amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 26 including three, two, or one amino acid substitutions; and i) The LCDR1 amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 24, including three, two, or one amino acid substitutions; and / or ii) The LCDR2 amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 25, including three, two, or one amino acid substitutions; (c) The LCDR3 amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 33 including three, two, or one amino acid substitutions; and i) The LCDR1 amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 32 including three, two, or one amino acid substitutions; and / or ii) The LCDR2 amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 25, including three, two, or one amino acid substitutions; (d) an LCDR3 amino acid sequence derived from a VL domain selected from SEQ ID NO: 6, as defined by Chothia or Kabat; or an LCDR3 amino acid sequence derived from a VL domain selected from SEQ ID NO: 6, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and i) an LCDR1 amino acid sequence derived from a VL domain selected from SEQ ID NO: 6, as defined by Chothia or Kabat; or an LCDR1 amino acid sequence derived from a VL domain selected from SEQ ID NO: 6, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and / or ii) An LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 6, as defined by Chothia or Kabat; or an LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 6, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; (e) an LCDR3 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23, as defined by Chothia or Kabat; or an LCDR3 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and i) an LCDR1 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23, as defined by Chothia or Kabat; or an LCDR1 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; and / or ii) An LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23, as defined by Chothia or Kabat; or an LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 23, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions; (f) an LCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VL domain selected from SEQ ID NO: 31; or an LCDR3 amino acid sequence defined by Chothia or Kabat and derived from a VL domain selected from SEQ ID NO: 31 containing three, two, or one amino acid substitutions; and i) an LCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VL domain selected from SEQ ID NO: 31; or an LCDR1 amino acid sequence defined by Chothia or Kabat and derived from a VL domain selected from SEQ ID NO: 31 containing three, two, or one amino acid substitutions; and / or ii) An LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 31, as defined by Chothia or Kabat; or an LCDR2 amino acid sequence derived from a VL domain selected from SEQ ID NO: 31, as defined by Chothia or Kabat, containing three, two, or one amino acid substitutions.

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