Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• Anti-PAR2 antibodies Field of the Invention
• This invention relates to an antibody or antigen-binding fragment capable of binding to human PAR2.
• the invention further relates to antibodies, which specifically bind to an epitope of the human PAR2 receptor and block, antagonise, inhibit or prevent activation of human PAR2.
• the invention relates to methods for making, methods for using and pharmaceutical compositions comprising said antibodies.
• Background of the Invention Chronic pain and chronic inflammation are two of the biggest burdens on global health. Chronic pain alone affects approximately 50 million American adults or 20% of the population.
• Chronic pain is a debilitating condition which is defined as pain that persists and is experienced most days or every day for 6 months or more (https://uspainfoundation.org/wp-content/uploads/2018/03/Chronic- pain-facts-infographic.pdf).
• Chronic inflammation plays a central role in diseases that contribute to a high number of deaths including cancer, cardiovascular disease and diabetes. It has been predicted that chronic diseases will account for approximately three-quarters of all death worldwide by 2020 (Helamo, Delil and Dileba, 2017). Despite chronic pain being a global burden, patients only receive a 30% pain reduction from current available treatments (Rice, Smith and Blyth, 2016).
• Protease Activated Receptor 2 is a G protein-coupled receptor that belongs to a family of Protease-Activated Receptors (PAR).
• PAR2 is ascribed a critical role in inflammation, pain and other pathophysiological responses, where elevated levels of proteases are found.
• PAR2 is widely expressed with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression is detected in numerous epithelial and endothelial cells and organs, with limited evidence for expression in brain or skeletal muscle.
• PAR2 is also expressed on immune and inflammatory cells, such as T-cells, monocytes, macrophages, neutrophils, mast cells, and eosinophils.
• PAR2 antagonists are thus thought likely to provide benefit to a wide variety of patients and to have a potential to alleviate pain and/or inflammation-related conditions. Hence, PAR2 is regarded as a valuable therapeutic target for the treatment of several disease indications. There is a need to identify a therapeutic moiety that can specifically inhibit PAR2. Such an agent would be particularly desirable if it could inhibit all mechanisms of PAR2 activation.
• Summary of the Invention Provided herein are antibodies and antigen-binding fragments thereof that bind PAR2.
• the antibodies and antigen-binding fragments of the disclosure are useful, inter alia, for inhibiting PAR2- mediated signalling and for treating diseases and disorders caused by or related to PAR2 activity and/or signalling.
• the antibodies provided herein, or antigen-binding fragments thereof, specifically bind to and inhibit the activity of PAR2, wherein the antibody or fragment thereof binds to an epitope comprising an extracellular loop (ECL) and an N-terminal segment of PAR2 including helices 0 and 1. It is believed that binding to both of these regions can lead to comprehensive functional inhibition of PAR2 activity.
• the antibodies provided herein are dual-active in that they are able to inhibit both protease cleavage mediated activation of PAR2 (e.g. by trypsin) and peptide mediated activation of PAR2 (e.g.by PAR2-AP or PAR1-AP).
• the antibody or antigen-binding fragment thereof specifically bind to a discontinuous epitope of PAR2, wherein the epitope comprises one or more regions of non-helical Segment1 preceding Helix0/1, the Helix0/1 region and ECL3, optionally wherein the regions of Segment1, Helix0/1 and ECL3 are selected from V55-F77, L306-Y311 and F312-Y326 of PAR2 when numbered in accordance with the human PAR2 sequence of SEQ ID NO: 1.
• the antibody or antigen-binding fragment thereof specifically binds to and inhibits the activity of PAR2, and comprises a VH domain comprising a HCDR3, wherein: (a) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, 22 or 30; or SEQ ID NO: 5, 22 or 30 with 3, 2 or 1 amino acid substitutions thereto; (b) a HCDR3 comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, 22 or 30; or d) a HCDR3 amino acid sequence is as defined by Kabat or Chothia and is from a VH domain selected from SEQ ID NO: 2, 10, 13,16, 19 or 27.
• the antibody or antigen-binding fragment thereof comprises a VH domain, wherein the VH domain comprises: i. a HCDR1 amino acid sequence selected from SEQ ID NO: 3, 11, 14, 17, 20 or 28, optionally with 3, 2 or 1 amino acid substitution(s) thereto; and/or ii. a HCDR2 amino acid sequence selected from SEQ ID NO: 4, 12, 15, 18, 21 or 29, optionally with 3, 2 or 1 amino acid substitution(s) thereto.
• the antibody or antigen-binding fragment thereof comprises a VL domain, optionally a VL domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6, 23 or 31.
• the antibody or antigen-binding fragment thereof comprises a LCDR3, wherein a) the LCDR3 amino acid sequence is selected from: SEQ ID NO: 9, 26 or 33, optionally with 3, 2 or 1 amino acid substitution(s) thereto; or b) the LCDR3 amino acid sequence as defined by Chothia or Kabat and is from a VL domain according to SEQ ID NO: 6, 23 or 31, optionally wherein the LCDR3 sequence comprises 3, 2 or 1 amino acid substitution(s).
• the antibody or antigen-binding fragment thereof comprises a VL domain, wherein the VL domain comprises: a) i) a LCDR1 amino acid sequence of SEQ ID NO: 7, 24 or 32, optionally with 3, 2 or 1 amino acid substitution(s) thereto; or ii) a LCDR1 amino acid sequence, as defined by Chothia or Kabat, from a VL domain according to SEQ ID NO: 6, 23 or 31, optionally wherein the LCDR1 sequence comprises 3, 2 or 1 amino acid substitution(s); and/or b) i) a LCDR2 amino acid sequence of SEQ ID NO: 8 or 25, optionally with 3, 2 or 1 amino acid substitution(s) thereto; or ii) a LCDR2 amino acid sequence, as defined by Chothia or Kabat, from a VL domain according to SEQ ID NO: 6, 23 or 31, optionally wherein the LCDR2 sequence comprises 3, 2 or 1 amino acid substitution(s).
• an antibody or antigen-binding fragment thereof which 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 that is 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 that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical thereto.
• the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.
• the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13.
• the antibody or antigen- binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID 16.
• the antibody or antigen-binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID 19.
• the antibody or antigen- binding fragment thereof comprises a VH region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27.
• the antibody or antigen-binding fragment thereof comprises a V L region, wherein the V L region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23.
• the antibody or antigen-binding fragment thereof comprises a V L region, wherein the V L region comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the V H region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 2 and the V L region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 6.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the V H region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 10 and the VL region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 6.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the V H region comprises an amino acid sequence which is identical or at least 90% identical SEQ ID NO: 13 and the V L region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 6.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the V H region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 16 and the V L region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 6.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the V H region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 19 and the V L region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 23.
• the antibody or antigen-binding fragment thereof comprises a V H region, wherein the V H region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 27 and the V L region comprises an amino acid sequence which is identical or at least 90% identical to SEQ ID NO: 31.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H comprises: (a) the HCDR1 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 comprising 3, 2 or 1 amino acid substitution(s); (b) the HCDR2 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 comprising 3, 2 or 1 amino acid substitution(s); (c) the HCDR3 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 comprising 3, 2 or 1 amino acid substitution(s); and wherein the V L comprises: (d) the LCDR1 having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 comprising 3, 2 or 1 amino acid substitution(s); (e) the LCDR2 having the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 comprising 3, 2 or 1 amino acid substitution(s); and
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H comprises: (a) the HCDR1 having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 11 comprising 3, 2 or 1 amino acid substitution(s); (b) the HCDR2 having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 12 comprising 3, 2 or 1 amino acid substitution(s); and (c) the HCDR3 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 comprising 3, 2 or 1 amino acid substitution(s); and wherein the VL comprises: (d) the LCDR1 having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 comprising 3, 2 or 1 amino acid substitution(s), (e) the LCDR2 having the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 comprising 3, 2 or 1 amino acid substitution(s),
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H comprises: (a) the HCDR1 having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 comprising 3, 2 or 1 amino acid substitution(s); (b) the HCDR2 having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 15 comprising 3, 2 or 1 amino acid substitution(s); (c) the HCDR3 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 comprising 3, 2 or 1 amino acid substitution(s); and wherein the VL comprises: (d) the LCDR1 having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 comprising 3, 2 or 1 amino acid substitution(s), (e) the LCDR2 having the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 comprising 3, 2 or 1 amino acid substitution(s), and
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H comprises: (a) the HCDR1 having the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 17 comprising 3, 2 or 1 amino acid substitution(s); (b) the HCDR2 having the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 18 comprising 3, 2 or 1 amino acid substitution(s); (c) the HCDR3 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 comprising 3, 2 or 1 amino acid substitution(s); and wherein the VL comprises: (d) the LCDR1 having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 comprising 3, 2 or 1 amino acid substitution(s), (e) the LCDR2 having the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 comprising 3, 2 or 1 amino acid substitution(s), and
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H comprises: (a) the HCDR1 having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 20 comprising 3, 2 or 1 amino acid substitution(s); (b) the HCDR2 having the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 21 comprising 3, 2 or 1 amino acid substitution(s); (c) the HCDR3 having the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 22 comprising 3, 2 or 1 amino acid substitution(s); and wherein the VL comprises: (d) the LCDR1 having the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 24 comprising 3, 2 or 1 amino acid substitution(s), (e) the LCDR2 having the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 25 comprising 3, 2 or 1 amino acid substitution(s), and
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H comprises: (a) the HCDR1 having the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 28 comprising 3, 2 or 1 amino acid substitution(s); (b) the HCDR2 having the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 29 comprising 3, 2 or 1 amino acid substitution(s); (c) the HCDR3 having the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 30 comprising 3, 2 or 1 amino acid substitution(s); and wherein the VL comprises: (d) the LCDR1 having the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 32 comprising 3, 2 or 1 amino acid substitution(s), (e) the LCDR2 having the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 25 comprising 3, 2 or 1 amino acid substitution(s), and
• the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 77. In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 77. In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 78. In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 78. In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 79.
• the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 79. In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 80. In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 80. In embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 83. In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 83.
• the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 84. In embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 84. In embodiments, the antibody or antigen-binding fragment thereof comprises a V H domain wherein the V H domain comprises: a) the HCDR3 amino acid sequence of SEQ ID NO: 5, or SEQ ID NO: 5 which comprises 3, 2 or 1 amino acid substitution(s); and i. a HCDR1 amino acid sequence of SEQ ID NO: 3, or SEQ ID NO: 3 which comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 2
• the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 2 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 2, or wherein the HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 2 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 2, or wherein the HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 2 and comprises 3, 2 or 1 amino acid substitution(s), h) the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 10, or wherein the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 10 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 10, or wherein the HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 10 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 10, or wherein the HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 10 and comprises 3, 2 or 1 amino acid substitution(s),
• the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 13, or wherein the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 13 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 13, or wherein the HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 13 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 13, or wherein the HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 13 and comprises 3, 2 or 1 amino acid substitution(s), j) the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 16, or wherein the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 16 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 16, or wherein the HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 16 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 16, or wherein the HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 16 and comprises 3, 2 or 1 amino acid substitution(s), k) the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 19, or wherein the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 19 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 19, or wherein the HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 19 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 19, or wherein the HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 19 and comprises 3, 2 or 1 amino acid substitution(s), l) the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 27, or wherein the HCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 27 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 27, or wherein the HCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 27 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 27, or wherein the HCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V H domain selected from SEQ ID NO: 27 and comprises 3, 2 or 1 amino acid substitution(s).
• the antibody or antigen-binding fragment thereof comprises a V L domain wherein the V L domain comprises: a) the LCDR3 amino acid sequence of SEQ ID NO: 9, or SEQ ID NO: 9 which comprises 3, 2 or 1 amino acid substitution(s); and i. a LCDR1 amino acid sequence of SEQ ID NO: 7, or SEQ ID NO: 7 which comprises 3, 2 or 1 amino acid substitution(s); and/or ii. a LCDR2 amino acid sequence of SEQ ID NO: 8, or SEQ ID NO: 8 which comprises 3, 2 or 1 amino acid substitution(s); b) the LCDR3 amino acid sequence of SEQ ID NO: 26, or SEQ ID NO: 26 which comprises 3, 2 or 1 amino acid substitution(s); and i.
• a LCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 6, or wherein the LCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 6 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a LCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 6, or wherein the LCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 6 and comprises 3, 2 or 1 amino acid substitution(s), e) the LCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 23, or wherein the LCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 23 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a LCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 23, or wherein the LCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 23 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a LCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 23, or wherein the LCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 23 and comprises 3, 2 or 1 amino acid substitution(s), f) the LCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 31, or wherein the LCDR3 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 31 and comprises 3, 2 or 1 amino acid substitution(s); and i.
• a LCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 31, or wherein the LCDR1 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 31 and comprises 3, 2 or 1 amino acid substitution(s); and/or ii.
• a LCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 31, or wherein the LCDR2 amino acid sequence is as defined by Chothia or Kabat and is from a V L domain selected from SEQ ID NO: 31 and comprises 3, 2 or 1 amino acid substitution(s).
• antibodies of the invention are isolated or purified.
• the antibody or antigen-binding fragment thereof inhibits PAR2 peptide activation of PAR2. In embodiments, the antibody or antigen-binding fragment thereof specifically binds to and inhibits the activity of PAR2, wherein inhibiting PAR2 activity comprises binding to Segment1, Helix0/1, ECL3 of PAR2 receptor. In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits PAR2 activation, wherein inhibiting PAR2 activation comprises inhibiting PAR2 tethered ligand binding.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2 and inhibits PAR2 activation, wherein inhibiting PAR2 activation comprises inhibiting cross-activation by PAR1 tethered ligand in PAR1-PAR2 heterodimers.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 activating peptide to PAR2.
• the antibodies provided herein able to 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).
• the antibody or antigen-binding fragment thereof binds to an epitope that is identical to an epitope to which an antibody or fragment selected from clones Y022065, Y022870, Y022877, Y022883, Y022054 and/or Y021171 specifically bind.
• the antibody or antigen-binding fragment thereof binds to an epitope, wherein the epitope to which the antibody or fragment binds is identified by hydrogen deuterium exchange (HDX) and/or by site-directed mutagenesis and flow cytometry.
• HDX hydrogen deuterium exchange
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 activating peptide to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated accumulation of inositol monophosphate (IP) with an IC 50 of from 1 to 100 nM, optionally wherein PAR2 peptide mediated accumulation of IP is determined using a PAR2 peptide stimulated IP signalling assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated activation of PAR2.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated accumulation of IP with an IC 50 of from 1 to 300 nM; optionally wherein trypsin mediated accumulation of IP is determined using a trypsin stimulated IP signalling assay.
• the antibody or antigen-binding fragment thereof inhibits PAR2 activating peptide mediated accumulation of inositol monophosphate (IP) with an IC 50 of from 1 to 100 nM, optionally wherein PAR2 peptide inhibition is determined using an HTRF assay.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated calcium mobilisation, optionally with an IC 50 of from 1 to 100 nM, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay. In embodiments, the antibody or antigen-binding fragment thereof inhibits trypsin mediated calcium mobilisation, optionally with an IC 50 of from 1 to 200 nM, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay.
• the antibody or antigen-binding fragment thereof is not internalised into a cell upon binding to PAR2 on the surface of the cell, optionally wherein internalisation is determined by quantifying antibody or fragment binding using FACs.
• the antibody or antigen-binding fragment thereof does not inhibit ligand SFLLR mediated PAR1 activation, wherein PAR1 activation is determined by using a ligand SFLLR stimulated IP signalling assay.
• the antibody or antigen-binding fragment thereof binds to cynomolgus PAR2 with an EC 50 of from 600 pM to 5 nM or less, optionally wherein cynomolgus PAR2 binding is determined using flow cytometry.
• the antibody or antigen-binding fragment thereof binds to human PAR2 with a K D of 100 pM to 10 nM, optionally wherein binding affinity is determined using surface plasmon resonance (SPR) or KinExA.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 peptide to PAR2, wherein binding of the antibody or fragment to PAR2 is pH independent between pH 7.5 and 6.0.
• the antibody or antigen-binding fragment thereof does not bind to PAR1, optionally wherein PAR1 binding is determined using flow cytometry or ELISA.
• the antibody or antigen-binding fragment thereof does not bind to PAR3, optionally wherein PAR3 binding is determined using flow cytometry or ELISA. In embodiments, the antibody or antigen- binding fragment thereof does not bind to PAR4, optionally wherein PAR4 binding is determined using flow cytometry or ELISA. In embodiments, the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 peptide to PAR2, wherein 3mg/kg antibody or fragment supresses PAR2 stimulant induced response in leucocytes by >95% over a period of 30 days, wherein suppression is measured by determining stimulant induced gene signatures.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 peptide to PAR2, wherein 1mg/kg antibody or fragment supresses PAR2 peptide induced response in leucocytes by >90% over a period of 30 days, wherein suppression is measured by determining stimulant induced gene signatures.
• the antibody or antigen-binding fragment thereof competes with functional ligand AZ8838 in binding to PAR2.
• the antibody or antigen-binding fragment thereof directly competes with AZ8838 in binding to PAR2.
• the antibody or antigen-binding fragment thereof binds to PAR2 homodimers.
• the antibody or antigen-binding fragment thereof binds to PAR2-PAR1 heterodimers, optionally wherein binding inhibits cross activation of PAR2 by PAR1 tethered ligand.
• antibodies and antigen-binding fragments thereof that bind PAR2 for use in therapy are provided herein.
• the antibody or antigen-binding fragment thereof is for use in treating a PAR2- mediated disease or condition e.g.
• atopic dermatitis asthma, cancer (various including breast, melanoma, head and neck), pain (chronic, inflammatory, post-operative, neuropathic, fracture, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and associated 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.
• the antibody or antigen-binding fragment thereof is for use in the manufacture of a medicament for treating a PAR2-mediated disease or condition e.g. atopic dermatitis, asthma, cancer (various including breast, melanoma, head and neck), pain (chronic, inflammatory, post- operative, neuropathic, fracture, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and associated 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.
• a PAR2-mediated disease or condition e.g. atopic dermatitis, asthma, cancer (various including breast
• a method of treating a PAR2-mediated disease or condition e.g. pain, optionally wherein the pain is independently selected from chronic pain, inflammatory pain, post-operative pain, neuropathic pain, fracture associated pain, gout associated pain, cancer associated pain, gastrointestinal pain associated with inflammatory bowel disease etc.
• a PAR2-mediated disease or condition e.g. pain, optionally wherein the pain is independently selected from chronic pain, inflammatory pain, post-operative pain, neuropathic pain, fracture associated pain, gout associated pain, cancer associated pain, gastrointestinal pain associated with inflammatory bowel disease etc.
• the antibodies of the disclosure may be combined or administered alongside a further therapy, optionally wherein the further therapy comprises one or more further therapeutic agent(s) independently selected from the group consisting of analgesics including anti-inflammatory drugs (e.g.
• NSAIDS including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g. codeine, morphine, oxycodone, fentanyl, buprenorphine), amitriptyline, gabapentin; anticancer drugs including alkylating agents (e.g. nitrogen mustards, nitrourea), antimetabolites (e.g. folic acid analogues, pyrimidine and purine analogues), antibiotics and enzymes (e.g. dactinomycin, daunorubicin, doxorubicin, L-asparaginase), natural agents (e.g.
• vinca alkaloids e.g. vinca alkaloids, taxens, tecans
• hormones and antagonists e.g. progestins, estrogen, GnRH, anti-estrogens), hyroxyurea, immunomodulators, tyrosine kinase inhibitors, biological response modifiers, molecularly targeted therapies (e.g. antibody conjugated drugs), platinum based therapies (e.g. cisplatin, carboplatin, oxaliplatin); and/or optionally wherein the further therapy is selected from radiotherapy and/or surgical removal of tumours.
• hormones and antagonists e.g. progestins, estrogen, GnRH, anti-estrogens
• hyroxyurea e.g. immunomodulators, tyrosine kinase inhibitors, biological response modifiers
• molecularly targeted therapies e.g. antibody conjugated drugs
• platinum based therapies e.g. cisplatin, carbo
• a pharmaceutical composition comprising an antibody or fragment of the disclosure and a pharmaceutically acceptable excipient, diluent or carrier and optionally further comprising one or more further therapeutic agents independently selected from the group consisting of analgesics including anti-inflammatory drugs (e.g. NSAIDS including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g. codeine, morphine, oxycodone, fentanyl, buprenorphine), amitriptyline, gabapentin; anticancer drugs including alkylating agents (e.g. nitrogen mustards, nitrourea), antimetabolites (e.g.
• analgesics including anti-inflammatory drugs (e.g. NSAIDS including aspirin, ibuprofen, diclofenac, naproxen), paracetamol, opioids (e.g. codeine, morphine, oxycodone, fentanyl, buprenorphine),
• folic acid analogues pyrimidine and purine analogues
• antibiotics and enzymes e.g. dactinomycin, daunorubicin, doxorubicin, L-asparaginase
• natural agents e.g. vinca alkaloids, taxens, tecans
• hormones and antagonists e.g. progestins, estrogen, GnRH, anti-estrogens
• hyroxyurea e.g. progestins, estrogen, GnRH, anti-estrogens
• immunomodulators tyrosine kinase inhibitors
• biological response modifiers e.g. antibody conjugated drugs
• platinum based therapies e.g. cisplatin, carboplatin, oxaliplatin.
• compositions of the disclosure or a kit comprising said pharmaceutical composition, wherein the composition is for treating a PAR2 mediated disease or condition, e.g. selected from atopic dermatitis, asthma, cancer (various including breast, melanoma, head and neck), pain (chronic, inflammatory, post-operative, neuropathic, fracture, gout, cancer, gastrointestinal associated with inflammatory bowel disease), rheumatoid arthritis and associated 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.
• a PAR2 mediated disease or condition e.g. selected from atopic dermatitis, asthma,
• a pharmaceutical composition of the disclosure, or a kit of the disclosure in combination with a label or instructions for use to treat a disease or condition in a patient optionally wherein the label or instructions comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number); optionally wherein the kit comprises an IV or injection device that comprises said antibody or fragment.
• the amino acid substitution(s) comprise homologous substitution(s). These amino acid substitution(s) may be conservative substitution(s). Conservative amino acid substitution(s) refer to substitutions of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• 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
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• a predicted nonessential amino acid residue in an anti-PAR-2 antibody is replaced with another amino acid residue from the same side chain family.
• Methods of identifying amino acid conservative substitutions which 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)).
• the amino acid substitution(s) reduce the binding affinity of the antibody or antigen-binding fragment thereof for human PAR2 by no more than 1000, 800, 700, 500, 400, 300, 200, 100, 50 or 10-fold as compared to an antibody or antigen-binding fragment having a VH with an amino acid sequence of SEQ ID NO: 16 and VL with an amino acid sequence of SEQ ID NO: 6 when tested in a PAR2 binding assay, such as an SPR or Kinexa assay, at a pH of between 7.4 and 7.6.
• the antibodies provided herein may be human.
• the antibody is a monoclonal antibody.
• the antibody is an IgG antibody.
• the antibody or antigen-binding fragment thereof is an antigen-binding fragment.
• the antigen binding fragment is a scFv.
• the antigen-binding fragment is a Fab.
• the antibody or antigen-binding fragment thereof is humanized.
• the antibodies provided herein bind to human PAR2, and may also bind to Cynomolgus PAR2 and Rhesus PAR2 but not to mouse and/or rat PAR2.
• the antibody or antigen-binding fragment thereof prevents trypsin, tryptase and/or matriptase from interacting with PAR2.
• the antibody or antigen binding fragment thereof inhibits PAR2 activation by trypsin.
• the antibody or antigen-binding fragment thereof inhibits exposure of the tethered ligand. In embodiments, the antibody or antigen- binding fragment thereof prevents the tethered ligand from interacting with PAR2.
• nucleic acids capable of expressing the antibodies or antigen- binding fragments thereof. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 34, 70 and 72. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 34, 70 and 72.
• the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NO: 34, 70 and 72. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 35, 36, 37, 38, 71 and 73. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 35, 36, 37, 38, 71 and 73. In embodiments, the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NO: 35, 36, 37, 38, 71 and 73.
• the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 34. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 34. In embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 34. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 38. In embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 38. In embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 38.
• nucleic acids of the invention are isolated or purified.
• the disclosure provides for a vector comprising any of the nucleic acids disclosed herein.
• the disclosure provides for a set of vectors comprising any one or more of the nucleic acids disclosed herein.
• the disclosure provides for a host cell comprising any one or more of the vectors disclosed herein.
• the disclosure provides for a composition comprising a pharmaceutically acceptable carrier and any of the antibodies or antigen-binding fragments disclosed herein.
• the disclosure provides for a lyophilized composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein.
• the disclosure provides for a reconstituted lyophilized composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein.
• the composition is formulated for administration by lozenge, spray, oral administration, delayed release or sustained release, trans-mucosal administration, syrup, mucoadhesive, buccal formulation, mucoadhesive tablet, topical administration, parenteral administration, injection, subdermal administration, oral solution, rectal administration, buccal administration or transdermal administration.
• the disclosure provides for a kit comprising any of the antibodies or antigen- binding fragments disclosed herein or any of the compositions disclosed herein.
• the disclosure provides for a method for treating pain in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of any of the compositions disclosed herein.
• the pain is selected from the group consisting of: nociceptive, neuropathic, and mix-type pain.
• the pain is associated with a headache, chronic headache, a migraine headache, a cancer, a viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease, liver disease, multiple sclerosis, spinal cord injury, post herpetic neuralgia, diabetic neuropathy, lower back pain, inflammatory heart disease, kidney disease, gastritis, gingivitis, periodontal disease, asthma, chronic obstructive pulmonary disease, autoimmune disease, irritable bowel syndrome, fibromyalgia, leg pains, restless leg syndrome, diabetic neuropathy, an allergic condition, a surgical procedure, acute or chronic physical injury, bone fracture or a crush injury, spinal cord injury, an inflammatory disease, a non-inflammatory neuropathic or dysfunctional pain condition, or a combination thereof.
• the pain is osteoarthritis pain.
• the subject is a human.
• the disclosure provides for 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, purifying the antibody or antigen-binding fragment.
• the present invention provides antibodies or antigen-binding fragments that bind to human PAR2 receptor.
• the antibodies of the present invention are useful for inhibiting PAR2 and its downstream signalling cascade.
• PAR2 or GPR11 is a 44 kDa G protein-coupled receptor that belongs to a family of Protease-Activated Receptors (PAR). It is encoded by the gene F2RL1 (coagulation factor II receptor-like 1). PAR2 belongs to a family of Protease-Activated Receptors (PAR), which are activated by proteolytic cleavage within the extracellular N-terminus. This family comprises four members PAR1-PAR4 that are activated by different proteases.
• PAR2 is predominantly activated by the serine proteases tryptase and trypsin, while other PAR family members are mostly activated by thrombin, although proteinase 3, factor VIIa and factor Xa are also described to be involved in PAR activation.
• PAR2 is activated by three main mechanisms. One of the mechanisms involves the cleavage of the extracellular N-terminal domain by proteases. This results in the exposure of a tethered ligand, which binds to a conserved region on extracellular loop 2 on the receptor and triggers intracellular signalling.
• PAR2 can be activated by a synthetic short peptide known as activated peptide (PAR2-AP) that mimics the first six amino acids of the tethered N-terminal ligand.
• PAR2 can also be activated by cross-activation by PAR1 tethered ligand in PAR1-PAR2 hetero-dimerization.
• G ⁇ q and G i proteins are activated which in turn results in an influx of calcium, induction of MAPK signalling and downstream inflammatory signalling. This results in subsequent biological responses, such as proliferation or secretion of pro-inflammatory cytokines, e.g., IL-6, IL-8 (also known as CXCL8) and GM-CSF.
• pro-inflammatory cytokines e.g., IL-6, IL-8 (also known as CXCL8) and GM-CSF.
• PAR2 expression has been shown to be increased in synovial lining, chondrocytes, and tissues in human rheumatoid arthritis and animal models of arthritis (Amiable et al 2009). PAR2 also potentiates signalling via channels such as TRPV1 (Dai et al 2007), a ligand-gated ion channel involved in inflammatory pain. PAR2 signalling is also known to sensitize TRPV1 in vivo, resulting in thermal hyperalgesia (Amadesi et al., 2006). PAR2 activation has been shown to be responsible for various inflammatory signalling pathways. In mice lacking the PAR2 receptor, there is a delayed onset of inflammation in response to inflammatory mediators (Lindner et al, 2000).
• PAR2 plays an important role in pathophysiology of many disease conditions such as pain, musculoskeletal inflammation including osteoarthritis, neuro-inflammatory disorders, airway inflammation, itch, dermatitis, colitis and related conditions (Yau et al 2013).
• PAR2 receptor antagonists such as GB88 have also been shown to block inflammatory responses in vivo including the collagen-induced arthritis model in rats (Lohman et al 2012).
• Known small molecule PAR2 antagonists are not ideal therapeutics due to modest potency and broad specificity. Given the range of diseases in which PAR2 is considered pivotal there is a need to identify a potent, specific PAR2 antagonist.
• the antibodies of the present invention are potent and specific PAR2 antagonists that inhibit PAR2 activation mediated by cleavage of the N-terminal domain.
• 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
• the singular form "a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. It is convenient to point out here that "and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
• a and/or B is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
• 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.
• Nucleotides likewise, may be referred to by their commonly accepted single-letter codes. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations.
• Human protease activated receptor 2 or “human PAR2,” or “PAR2,” and the like, as used herein, refer to human PAR2 (wildtype or wt) with UniProt ID number: P55085, reproduced herein as SEQ ID NO: 1.
• Human PAR2 includes any sequence that is at least 99% or 100% identical to the amino acid sequence of any of SEQ ID NO: 1, or biologically active fragments thereof.
• tethered ligand 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 a proximal binding site on the PAR2 receptor to activate it.
• the tethered ligand at the N-terminus of the human PAR2 receptor comprises SEQ ID NO: 39.
• PAR2-AP “PAR2 activated peptide” or “PAR2 activating peptide,” it is meant a synthetic short peptide which mimics the cleaved tethered N-terminal ligand.
• the human peptide is SEQ ID NO: 39 and the murine peptide comprises SEQ ID NO: 40.
• a naturally occurring "antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The heavy chains comprise a variable region (VH) and a constant region, and the light chains comprise a variable region (VL) and a constant region.
• the VH and VL regions can be further divided into hypervariable (HV) and framework (FR) regions. Each VH and VL is composed of three complementarity determining regions (CDRs) and four FRs.
• antibody may refer, for example, to monoclonal antibodies, human antibodies, humanized antibodies, shark antibodies, camelid antibodies, or chimeric antibodies. Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the heavy and light chain determine antigen recognition and specificity.
• the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, trans-placental mobility, Fc receptor binding, complement binding, and the like.
• CL light chain
• CH2 or CH3 heavy chain
• the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody.
• the N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
• antibodies immunoglobulins
• immunoglobulins There are five major classes (isotypes) of immunoglobulins in humans: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses, e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
• the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al., (2000).
• CDRs are amino acid sequences with boundaries determined using any of a number of well-known schemes such as the “Kabat” and “Chothia” numbering scheme as shown in Table 1.
• the CDR amino acid residues in the heavy chain are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); whereas under Chothia the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3).
• Table 1 CDR definitions (numbering is according to the Kabat nomenclature).
• antigen-binding fragment or “epitope-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
• Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
• DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
• the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
• the two fragment variable (Fv) domains are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988; and Huston et al., 1988).
• single chain Fv single chain Fv
• Such single chain antibodies are also intended to be encompassed within the terms “fragment”, “epitope-binding fragment” or "antibody fragment”. These fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
• 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) minimal recognition units consisting of the amino acid residues that mimic the 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.
• CDR complementarity determining region
• 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 immune- pharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment,” as used herein.
• An antigen-binding fragment of an antibody will typically comprise at least one variable domain (e.g., at least one of a VH or VL).
• variable domain 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.
• the VH and VL domains may be situated relative to one another in any suitable arrangement.
• the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
• the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
• an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
• Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH-CH1 ; (ii) VH-CH2; (iii) VH- CH3; (iv) VH-CH1-CH2; (V) VH-CH1 -CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1 ; (ix) VL-CH2; (x) VL- CH3; (xi) VL-CH1-CH2; (xii) VL-CH1- CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
• variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
• a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
• the hinge region comprises a glycine-serine linker.
• an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
• antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).
• a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
• bispecific antibody refers to a bispecific antibody that binds to two different epitopes on a single PAR2 target.
• monovalent antibody refers to an antibody that comprises one epitope- binding moiety.
• bivalent antibody refers to an antibody that comprises two epitope-binding moieties.
• multivalent antibody refers to a single binding molecule with more than one valency, where "valency" is described as the number of antigen-binding moieties present per molecule of an antibody construct.
• the single binding molecule can bind to more than one binding site on a target molecule.
• multivalent antibodies include, but are not limited to bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, and the like, as well as multispecific antibodies and biparatopic antibodies.
• the multivalent antibody e.g., a PAR2 biparatopic antibody
• a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
• fragment crystallisable 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 a constant domain of an antibody.
• an Fc region may include a CH4 domain, present in some antibody classes.
• An Fc region may comprise the entire hinge region of a constant domain of an antibody.
• a constant region is modified compared to a wild-type constant region.
• the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant region domain (CL).
• Example modifications include additions, deletions or substitutions of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, etc.
• reference antibody refers to any antibody used in this disclosure during experimentation for reference against the anti-PAR2 antibodies of the present invention (e.g. for positive or negative controls and setting up assay conditions).
• 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 referred to as R001053 or PaB670129; Benchmark 3, a Regeneron Ab, also referred to as R001044 or H4H581P; Benchmark 4, an Amgen Ab, also referred to as R001048 or 1A1; MAB3949, a murine R&D systems mAb; and Benchmark 6 (Giblin et al 2011).
• epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
• a single antigen may have more than one epitope.
• different antibodies may bind to different areas on an antigen and may have different biological effects.
• Epitopes may be either conformational (e.g. discontinuous) or linear.
• a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
• a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
• an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
• binding site comprises an area on PAR2 target molecule to which an antibody or antigen-binding fragment selectively binds.
• antibodies specific for a particular target antigen will bind to an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
• affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, the stronger the affinity.
• the term "high affinity" for an IgG antibody or fragment thereof refers to an antibody having a KD of 10 -8 M or less, 10 -9 M or less, or 10 -10 M, or 10 -11 M or less, or 10 -12 M or less, or 10 -13 M or less for a target antigen.
• high affinity binding can vary for other antibody isotypes.
• high affinity binding for an IgM isotype refers to an antibody having a KD of 10 -7 M or less, or 10 -8 M or less.
• the term “avidity” refers to an informative measure of the overall stability or strength of the antibody-antigen complex.
• nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides can have any three-dimensional structure and can perform any function.
• polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), 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.
• a polynucleotide can be modified or substituted at one or more base, sugar and/or phosphate, with any of various modifications or substitutions described herein or known in the art.
• a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer.
• the sequence of nucleotides can be interrupted by non-nucleotide components.
• a polynucleotide can be further modified after polymerization, such as by conjugation with a labelling component.
• the term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
• 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 being transcribed and translated.
• ORF open reading frame
• a polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
• amino acid refers to natural and/or unnatural or synthetic amino acids, both the D and L optical isomers of any amino acid and amino acid analogues.
• a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short.
• biomarker is a nucleic acid, polypeptide or other organic or inorganic molecule expressed in humans and the presence or absence of a mutation or differential expression of the biomarker is used to determine sensitivity to any treatment comprising an anti-PAR2 antibody according to the invention.
• a protein is a biomarker for a cancer cell when it is deficient, mutated, deleted, or decreased in post-translational modification, production, expression, level, stability and/or activity, as compared to the same protein in a normal (non-cancerous) cell or control cell.
• polypeptide polypeptide
• the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
• the residues may be linked by peptide bonds or other bonds, e.g., ester, ether, etc.
• a polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence meaning that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
• This alignment and the percentage homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al., (1987).
• default parameters are used for alignment.
• Preferred alignment tools are provided on the European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI) webpage, using default parameters.
• expression product refers to the nucleic acids or amino acids (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
• 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 peptides, polypeptides or proteins.
• expression may include splicing of the mRNA in a eukaryotic cell.
• “Differentially expressed” as applied to a gene refers to the differential production of the mRNA transcribed from the gene or the protein product encoded by the gene.
• a differentially expressed gene may be overexpressed or underexpressed as compared to the expression level of a normal or control cell.
• overexpression is an increase in gene expression and generally is 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 expression over that detected in a normal or control counterpart cell or tissue.
• under expression is a reduction of gene expression and generally is 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 expression under that detected in a normal or control counterpart cell or tissue.
• the term “differentially expressed” also refers to where expression in a cancer cell or cancerous tissue is detected but expression in a control cell or normal tissue (e.g. non-cancerous cell or tissue) is undetectable.
• a high expression level of the gene can occur because of over expression of the gene or an increase in gene copy number.
• the gene can also be transcribed and translated into increased protein levels because of deregulation or absence of a negative regulator.
• the terms “inhibit” or “inhibiting” and the like refer to the interfering, antagonising or blocking of PAR2 receptor activation. Such terms denote quantitative differences between two states, e.g., refer to statistically significant differences between the two states. For example, "an amount effective to inhibit PAR2 activation” means that the activation of PAR2 due to agonist and PAR2 blocking antibody will be at least statistically significantly different from the cells treated with agonist alone.
• treatment generally mean obtaining a desired pharmacologic and/or physiologic effect, and may also be used to refer to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated.
• the effect may be prophylactic in terms of completely or partially delaying the onset or recurrence of a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition.
• Treatment covers any treatment of a disease or condition of a mammal, particularly a human, and includes any one or more of : (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).
• “treatment” of pain e.g., chronic or neuropathic pain
• the population of subjects treated by the method of the disease includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.
• therapeutically effective dose what is meant is a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., The Art, Science, and Technology of Pharmaceutical Compounding, 3rd Edition, 2008).
• nucleotide sequences encoding such antibodies have been determined, chimeric antibodies may be produced by recombinant methods. 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.
• 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. natural or recombinant).
• 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 substantially free of chemical precursors or other chemicals when chemically synthesized.
• 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.
• an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
• heterologous protein also referred to herein as a "contaminating protein”
• the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e. culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
• culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
• the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
• antibodies of the invention are isolated or purified.
• Provided herein are PAR2-directed antibodies and antigen-binding fragments thereof that bind PAR2.
• the antibody is an antagonising, neutralizing and/or blocking anti- PAR2 antibody or antigen-binding fragment.
• an “antagonising”, “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 the binding of PAR2 activating peptide to PAR2 or the activation of PAR2 by PAR2 activating peptide; and/or (ii) interferes with the interaction between PAR2 exposed tethered ligand and PAR2; and/or (iii) interferes with the interaction between PAR2 and a protease (e.g., trypsin, tryptase, matriptase, legumain); and/or (iv) inhibits PAR2 signalling (e.g.
• the antibodies or antigen-binding fragments of the disclosure inhibit activation of PAR2.
• the antibodies or antigen-binding fragments inhibit exposure of the tethered ligand.
• the antibodies or antigen- binding fragments inhibit activation of a PAR2 receptor by its exposed tethered ligand.
• the antibodies or antigen-binding fragments inhibit activation by the exposed tethered ligand of PAR2. In embodiments, the antibodies or antigen-binding fragments inhibit binding of the exposed tethered ligand to the second extracellular loop (ECL2) of PAR2. In embodiments, the antibodies or antigen binding fragments thereof bind to a discontinuous epitope, wherein binding to said epitope occludes binding of the exposed tethered ligand to ECL2 via steric hindrance.
• the inhibition caused by an anti- PAR2 neutralizing, blocking or antagonising antibody need not be complete so long as it is detectable using an appropriate assay.
• the antibody or antigen-binding fragment thereof inhibits PAR2 activity at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to uninhibited active PAR2.
• assays for detecting activity of a representative anti- PAR2 antibody or antigen-binding fragment are described in the exemplification section. The skilled worker is aware of additional anti-PAR2 antibody activity assays.
• provided herein are antibodies or antigen-binding fragments that interfere with the interaction between PAR2 and a protease.
• the protease is trypsin.
• the protease is neutrophil elastase.
• the protease is neutrophil proteinase 3.
• the protease is mast cell tryptase. In embodiments, the protease is tissue factor/factor Vila/factor Xa. In embodiments, the protease is a kallikrein-related peptidase. In embodiments, the protease is membrane-tethered serine proteinase- 1/matriptase 1. In embodiments, the protease is parasite cysteine proteinase. In embodiments, the antibodies or antigen-binding fragments inhibit/reduce inflammation-induced pain. Provided herein are PAR2-directed antibodies and antigen-binding fragments thereof that bind PAR2 molecules with high affinity at physiological, extracellular pH (i.e. pH 7.5).
• the antibodies or antigen-binding fragments of the present disclosure may possess one or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present disclosure will be evident to a person of ordinary skill in the art from a review of the present disclosure including the exemplification section provided herein.
• the anti-PAR2 antibodies of the disclosure are human antibodies.
• the term "human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
• 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), for example in the CDRs, and in embodiments, CDR3.
• human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• the antibodies of the disclosure may, in embodiments, be recombinant human antibodies.
• recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. 1992) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
• recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
• such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 activating peptide to PAR2 optionally wherein the antibody or fragment inhibits PAR2 activating peptide mediated accumulation of inositol monophosphate (IP) with an IC 50 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 wherein PAR2 peptide mediated accumulation of IP is determined using a PAR2 peptide stimulated IP signalling assay.
• IP inositol monophosphate
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 activating peptide to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated accumulation of inositol monophosphate (IP) with an IC 50 value of less than about 40 nM, 50 nM, 60 nM, 70 nM or 80 nM, optionally wherein PAR2 peptide mediated accumulation of IP is determined using a PAR2 peptide stimulated IP signalling assay.
• IP inositol monophosphate
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, and inhibits the binding of PAR2 activating peptide to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated accumulation of inositol monophosphate (IP) with an IC 50 of 40 nM or less, optionally wherein PAR2 peptide mediated accumulation of IP is determined using a PAR2 peptide stimulated IP signalling assay.
• IP inositol monophosphate
• the IP signalling assay is a Cisbio IP-One HTRF assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated accumulation of IP with an IC 50 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 wherein trypsin mediated accumulation of IP is determined using a trypsin stimulated IP signalling assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated accumulation of IP with an IC 50 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 wherein trypsin mediated accumulation of IP is determined using a trypsin stimulated IP signalling assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated accumulation of IP with an IC 50 of 70 nM or less; optionally wherein trypsin mediated accumulation of IP is determined using a trypsin stimulated IP signalling assay.
• the antibody or antigen-binding fragment thereof inhibits PAR2 activating peptide mediated accumulation of inositol monophosphate (IP) with an IC 50 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 wherein PAR2 peptide inhibition is determined using an HTRF assay.
• IP inositol monophosphate
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated calcium mobilisation, optionally with an IC 50 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, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated calcium mobilisation, optionally with an IC 50 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 or 160 nM, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay.
• the antibody or antigen-binding fragment thereof specifically binds to PAR2, wherein the antibody or fragment inhibits PAR2 activating peptide mediated calcium mobilisation, optionally with an IC 50 of 140 nM or less, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated calcium mobilisation, optionally with an IC 50 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, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated calcium mobilisation, optionally with an IC 50 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 250 nM, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay.
• the antibody or antigen-binding fragment thereof inhibits trypsin mediated calcium mobilisation, optionally with an IC 50 200 nM or less, and optionally wherein calcium mobilisation is determined using a PAR2 activating peptide stimulated calcium mobilisation assay
• the antibody or antigen-binding fragment thereof binds to cynomolgus PAR2 with an EC 50 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 wherein cynomolgus PAR2 binding is determined using flow cytometry.
• the antibody or antigen-binding fragment thereof binds to cynomolgus PAR2 with an EC 50 of less than about 400 pM, 800 pM, 1 nM, 5 nM or 10 nM, optionally wherein cynomolgus PAR2 binding is determined using flow cytometry.
• the antibody or antigen-binding fragment thereof binds to human PAR2 with a K D 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 wherein binding affinity is determined using surface plasmon resonance (SPR) or KinExA.
• SPR surface plasmon resonance
• KinExA KinExA
• the antibody or antigen-binding fragment thereof binds to human PAR2 with a K D of less than about 100 pM, 200 pM, 400 pM, 800 pM, 1 nM, 5 nM, 10 nM or 20 nM, optionally wherein binding affinity is determined using surface plasmon resonance (SPR) or KinExA. In embodiments, the antibody or antigen-binding fragment thereof binds to human PAR2 with a K D of 10 nM or less, optionally wherein binding affinity is determined using surface plasmon resonance (SPR) or KinExA.
• Figures Figure 1 Examples of specific PAR2 nanodisc (FL-StaR ND) versus empty nanodisc (empty ND) binding by positive control mAbs ‘Benchmark 1’ and a commercially available PAR2 mAb ‘R&D anti- huPAR2 mAb (MAB3949)’ and dose dependent binding observed for these and the three PAR2 phage-derived antibody clones shown. Antibodies were directly coated onto plates at 5 ⁇ g/mL. No significant background binding observed with the negative control, MOR03207, an anti-lysozyme antibody. Data are shown as signal divided by background (S/BG).
• Figure 2 Exemplification of degrees of competition (complete, partial and no competition) with the commercially available positive control mAb ‘MAB3949’ (R&D anti-huPAR2) on a panel of PAR2 mAbs (derived from the Ylanthia phage antibody library) using full length PAR2 StaR nanodiscs and therefore an indication of diversity. 50nM candidate mAb was titrated against a dose-response of R&D anti-huPAR2 (MAB3949) in competition for full length StaR nanodisc. Both complete competition and partial competition demonstrate a dose dependent binding effect.
• Figure 4 Understanding the ability of antibody clones to bind full length huPAR2 (FL-N, second row) and N-terminally truncated huPAR2 (truncate, first row) by flow cytometry.
• the resulting data demonstrates 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 (e.g. Y022065, Y022071) that bind to the truncated AND full length receptor, where the epitope also comprises part of the extracellular domain, and clones that bind the truncated but not full length receptor (e.g. Y022075).
• Figure 5 Multiplex profiling using IntelliCyt. The graphs show binding of purified IgG1f_AEASS on human PAR2-expressing cells. Data are exemplary for clone ‘Y022065’ (functional candidate).
• Figure 6 Functional characterization of Y021171, Y022063 and Y022054 & Y022065 hIgG1f_AEASS in the peptide inhibition assay using the Cisbio® IP-One G ⁇ q assay at two concentrations in replicate. Statistically significant inhibition of Activating Peptide induced agonism at human PAR2 was observed for four clones against the response of 6.28nM agonist alone ranging from 17-43% at the highest concentration tested. Y022065 identified as the most active candidate. Data are shown as mean response with standard deviation from 2 independent experiments run in singlicate.
• Figure 7 Koff-ranking ELISAs using nanodisc-embedded human PAR2 StaR® and purified soluble StaR® protein, where parental candidates represent the lineages of the 22 matured IgG candidates identified as functionally dual-active. Data are shown as signal divided by background (S/BG).
• Figure 8 Binding specificity of affinity matured clones shown by parental lineage on FlpIn CHO- V5His-huPar2 versus FlpIn CHO parental cells.
• Figure 9 Affinity matured clones, formatted as Fab fragments, bind to human PAR2 expressing CHO cells as assessed by flow cytometry. Data are shown as signal divided by background (S/BG).
• Figure 10 Affinity matured clones, formatted as IgGs or fAbs, bind to cynomolgus (IgGs)- and human-PAR2 (fAbs) BacMam-infected HEK293 cells as assessed by flow cytometry. Data are shown as signal divided by background (S/BG).
• Figure 11 Confirmation of binding to human and cynomolgus PAR2 expressing cells for the affinity matured lead mAb panel as Fab fragments and IgG in comparison to benchmark mAbs. Antibodies were incubated overnight with human PAR-2 and cyno PAR-2 over – expressing cells at ⁇ 5°C with sodium azide.
• Percentage inhibition ranges from 0 through to ⁇ 250% across the clones tested relative to the R&D Systems anti-human PAR2 MAB3949, suggesting that at comparable concentrations, a high number of lead clones show greater inhibition of Activating Peptide agonism of human PAR2 when compared to MAB3949.
• Lead clones derived from parent IgGs Y021171 and Y022065 by affinity maturation exhibit the largest proportion of actives.
• Figure 13 Functional characterization of lead optimized clones in inhibition assay against Bovine Trypsin. The IP-One assay is used to measure accumulation of IP as a function of G ⁇ q activation and human PAR2 activity in vitro.
• Each value represents % inhibition normalized against 1 ⁇ M Benchmark 1 at highest concentration tested in replicate for individual lead clones.
• Lead clones have been aligned with parental IgG. Inhibition of Bovine Trypsin was observed for lead clones matured from all parental IgGs. Percentage inhibition ranges from 0 through to ⁇ 130% across the clones tested relative to Benchmark 1.
• Lead clones identified from maturation of IgG Y022065 demonstrate the largest proportion of actives against Bovine Trypsin challenge. Two representatives derived from Y021171 demonstrate the highest % inhibition.
• Figure 14 Graphical representation of dose response inhibitor curves of parental clone (Y022065) and affinity matured lead representatives (Y022870, Y022877, Y022883) compared with Benchmark 1 and Benchmark 2 in calcium mobilisation assay measured in response to HT-29 challenge with Bovine Trypsin to activate endogenous PAR2. Data are shown as mean response with standard deviation from 3 independent experiments run as duplicate wells.
• Figure 15 SPR evaluation by Biacore to determine effect of pH on 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 by parental family derivation. Anti- huPAR2 is MAB3949.
• Figure 18 Back view of PAR2 (ECL3, Segment1 and Helix0/1). Regions of PAR2 that interact with Y022883, as derived by HDX, are shown as hatched areas.
• Figure 19 Top view of PAR2 (ECL3, Segment1 and Helix0/1). Regions of PAR2 that interact with Y022883, as derived by HDX, are shown as hatched areas.
• Figure 20 Side view of PAR2 (ECL3, Segment1 and Helix0/1). Regions of PAR2 that interact with Y022883, as derived by HDX, are shown as hatched areas.
• Figure 21 Expression profiles of WT and mutated (S60W, D62F, G318F, D62G & E63G) human PAR2 detected using flow cytometry of BacMam-infected HEK293F cells stained with Benchmark 1 (an N- terminal binder) or Y022883. 2 nd only: secondary FACS antibody only; n.i.: not infected.
• Figure 22 Rat serum mAb concentrations versus time following a single intravenous dose of 10mg/kg Y022883.
• Figure 25 Cynomolgus monkey ex vivo pharmacodynamics following a single 3mg/kg dose of Y022883. Data are shown as the percentage of pre-dose stimulant-induced (PAR2-AP as circles, trypsin as squares, LPS as triangles) gene signature.
• Figure 26 Cynomolgus monkey ex vivo pharmacodynamics following a single 1mg/kg dose of Y022883. Data are shown as the percentage of pre-dose stimulant-induced (PAR2-AP as circles, trypsin as squares, LPS filled triangles) gene signature.
• Figure 27 Inhibition of Trypsin induced phosphorylation of p38-MAPK and pERK in T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data show levels relative to treatment with vehicle and are normalised to total ERK or p38-MAPK.
• Figure 28 Inhibition of PAR2-AP induced phosphorylation of p38-MAPK and pERK in T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data show levels relative to treatment with vehicle and are normalised to total ERK or p38-MAPK.
• Figure 29 Inhibition of Trypsin and PAR2-AP induced phosphorylation of pERK (Left) and p38-MAPK (Right) in T84 cells by Y022883 (SH-C) and Benchmark II (SH-D). Data show levels as a percentage of the difference between the vehicle and the positive control. Examples Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.
• Example 1 Generation of full-length and N-terminally truncated human PAR2 constructs Designing PAR2 constructs
• the gene of the human Protease-Activated Receptor 2 (PAR2) was edited using standard site- directed mutagenesis strategies, based on Hutchison et al., 1978, to stabilize the receptor in the antagonist conformation by the introduction of nine point mutations (Cheng et al 2017).
• the stabilized receptor was also either genetically truncated at the N-terminus by 54 amino acids (first residue in encoded construct is V55) or kept full length (FL-N) using Polymerase Chain Reaction (PCR, based on Saiki et al., 1985), widely known in the Art.
• All four PAR2 proteins are also genetically truncated at the C-terminus by 20 amino acids (last residue K377) as part of the stabilized receptor process.
• the following constructs were generated: PAR2-1: P151: 55-377, glycosylated, pFastBacHisStrepII, truncated PAR2-2: P166: 1-377, de-glycosylated: 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 was sufficiently stable for use as antigen was to enable the discovery and identification of antibodies that bound to other regions of the receptor other than just the N terminus
• the potential glycosylation sites N30 and N222 were substituted by site-directed mutagenesis to glutamine residues in the FL-N protein PAR2-2.
• StrepII and deca-histidine tags were added by PCR and endonuclease cloning strategies, known to the skilled worker, to the C-terminus to allow affinity purification and specific selection in ELISA assays.
• PAR2-1 and PAR2-2 genes were cloned into a pFastBac1 vector (Thermofisher, #10360014). The multiple cloning site of the vector was modified to allow for insertion of genes using NheI as restriction endonuclease.
• the pFastBac1 vector is part of the commercial Bac-to-Bac expression system (Smith et al., 1983, Thermofisher, #10359016) for insect cells, which is known and widely used in the art. We followed the manufacturer’s instructions.
• the Bac-to-Bac expression system was used for PAR2-1 and -2 proteins.
• the pFastBac1 vector was modified to generate the pBacMam vector by introducing a human cytomegalovirus promotor 3’ following the polyhedron promotor, to allow for protein expression of the protein PAR2-3 and PAR2-4 in mammalian cells.
• the instructions of the Bac-to-Bac expression system were also applied to the pBacMam virus generation.
• Mammalian cell lines were used for expression of constructs in order to provide options to utilize antigen that contained mammalian-like glycosylation patterns.
• Proteins PAR2-1 and -2 were expressed in Spodoptera frugiperda Sf9 cells (Thermofisher, #89070101) in Expression Systems ES921 media (#96-001-01) with 10 % FBS (Sigma-Aldrich, #F9665). Cells were infected at a multiplicity of infection (MOI) of 2 at a cell density of 3.5 x 10 ⁇ 6/ml.
• MOI multiplicity of infection
• the membranes were collected by ultra- centrifugation for 1 hr at 135,000 g in a Beckman 45 Ti rotor, homogenized into PAR2/PI buffer and stored at -80 °C. All subsequent purification steps were performed at 4°C. Purification Thawed membranes were solubilized by the addition of 1% (w/v) LMNG (Anatrace, #NG310) /0.1% (w/v) cholesteryl hemisuccinate (CHS, Anatrace, #210) mixture for 1 hour. Insoluble material was removed by ultra-centrifugation for 30 minutes at 205,000 g in a Beckman 45 Ti rotor.
• Protein was batch bound for 2 hours to 8 ml NiNTA Superflow resin (Qiagen, #30430) in the presence of 8 mM imidazole.
• the resin was packed into a XK 16/20 column (GE Healthcare, #GE28-9889-37) and washed with 15 column volumes (CVs) 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 CVs of buffer A2 (50 mM HEPES pH 7.5, 250 mM NaCl, 0.02 % LMNG, 0.002% CHS, 75 mM imidazole).
• high-salt buffer A1 50 mM HEPES pH 7.5, 500 mM NaCl, 0.02 % LMNG, 0.002 % CHS, 75 mM imidazole
• buffer A2 50 mM HEPES pH 7.5, 250
• Protein was eluted with buffer A2 supplemented with 300 mM imidazole and concentrated to 0.5 ml using an Amicon Ultra-15 recycled cellulose concentrator with 100 kDa molecular weight cut-off (Millipore, #UFC900308). Aggregated material was removed by ultracentrifugation at 220,000 g for 10 min in a Beckman Coulter benchtop centrifuge using the TLA-100.2 rotor. The sample was then subjected to size exclusion chromatography in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.02 % LMNG, 0.002 % CHS, on a Superdex 20010/300 GL column (GE Healthcare, #17-5175-01).
• nanodisc-embedded human PAR2 StaR protein The preparation of nanodiscs is known in the art and based on Banerjee et al., 2008, using zebrafish apolipoprotein-1 (ZAP1) as the scaffold protein with a N-terminal hexa-histidine tag. Proteins PAR2-1 and -2 were re-constituted at 100 to 200 ⁇ M into nanodiscs and used as antigens for Fab-selection.
• ZAP1 zebrafish apolipoprotein-1
• the final molar ratios were 1:72:48:12:264 for PAR2:POPC:POPG:cholesterol:sodium-deoxycholate, respectively.
• the final sodium deoxycholate concentration was kept above 15 mM and adjusted with 10 mM HEPES 7.4, 150 mM NaCl buffer.
• zebrafish apolipoprotein 1 (ZAP1) was added in a molar ratio of 1:2 PAR2:ZAP1. The mix was incubated for 1 hour on ice.
• the detergent was removed by agitating the sample over night with Bio-beads SM-2 (Bio-Rad, #1528920) in a 1:1 ratio of protein solution to dry Bio-Bead weight (e.g.900 ⁇ l protein to 900 ⁇ g Bio-Beads).
• the nanodiscs were recovered as supernatant and the beads were washed with 50 mM HEPES pH 7.5, 150 mM NaCl for two volumes worth of weight of beads (e.g.1.8 mL for 900 ⁇ g beads).
• the protein solution was concentrated below 1 mL, centrifuged for 15 min in a table top centrifuge at 13,500 g and the supernatant was subjected to size exclusion chromatography on a Superdex 20010/300 GL column (GE Healthcare), as used above, in 50 mM HEPES pH 7.5, 150 mM NaCl. Fractions representing the nanodisc peak were pooled and concentrated to 1 – 2 mg/mL in an Amicon Ultra-4 recycled cellulose concentrator (100 kDa molecular weight cut-off, Millipore, #UFC810096) and frozen in aliquots at - 80°C.
• Example 2 Screening Antibodies or antigen-binding fragments of the disclosure were identified from phage display libraries.
• the phage display library used to identify the antibodies provided herein is the Ylanthia® phagemid based on the Ylanthia (Tiller et al. 2013) concept and employs CysDisplayTM technology to display Fab on the phage surface (Lohning et al.2000).
• Ten panning strategies, (including solid phase panning, capture panning and whole cell panning using techniques known in the art), were designed with an emphasis on solid phase pannings using full length or N-terminally truncated nanodisc-embedded human PAR2 StaR protein.
• Nanodisc-embedded human PAR2 StaR variants were used as panning antigen (i.e., a detergent free state).
• panning antigen i.e., a detergent free state
• nanodisc- embedded human PAR2 StaR protein was presented via tag-specific capture antibodies. Alternation of the capture mode was implemented to lower 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).
• differential whole cell pannings were conducted on human PAR2-expressing Flp-In CHO and BacMam-infected HEK293F cells. Alternation of panning on StaR followed by cell panning was employed to lower the risk of enrichment of candidates specific for other cellular targets (off-target binding).
• Ylanthia® candidates with mostly good specificity for the human PAR2 receptor expressed in a number of different cellular backgrounds.
• a pre-adsorption on irrelevant antigen components was performed prior to each round of solid phase pannings.
• Irrelevant antigen components included Ni-NTA, empty nanodisc as well as capture antibodies anti-StrepII mAb (StrepMAB-Immo) and anti-poly 6x Histidine-Ab IgG1 (R&D MAB050) or a nanodisc-embedded alternative GPCR (used exclusively in maturation pannings).
• Phage preparation Production of Fab-presenting phage particles New phage particles presenting Fab fragments on their surface were produced for each selection round. Thereby an E. coli TG1 culture was infected with phage derived from the previous selection round. Upon centrifugation, the bacterial pellets were re-suspended in fresh medium and plated on agar plates. After outgrowth, colonies were scraped off the plates and were used for phage rescue, polyclonal amplification of selected clones and phage production. With purified phage, the next panning round was started. Upon the last panning round single clones were picked from agar plates into the wells of a sterile microtiter plate pre-filled with medium.
• Example 3 Identification of hits by ELISA and whole cell binding by FACS and IntelliCyt screening ELISA ELISA techniques have been used for both screening of single Fab clones identified from panning outputs on target antigens as well as for characterization of purified antibodies. Optimal antigen and antibody concentrations as well as blocking conditions were determined according to state of the art methods. Direct coating of antigen Antigens were immobilized on microtiter plates. Plates were blocked and incubated with antibodies such as Fab containing crude E. coli lysates or purified Fab or IgG samples.
• Bound antibodies were detected using alkaline-phosphatase (AP) coupled secondary antibodies in combination with ‘AttoPhos’ fluorescence substrate. Multiple washing steps were performed in between individual assay steps.
• Antigen capture In other ELISA settings, antigens were captured to plates via a tag-specific antibody coated on microtiter plates (e.g. anti-StrepII, anti-His or benchmark 1 reference mAb). Bound antibodies were detected using respective alkaline-phosphatase (AP) coupled secondary antibodies in combination with ‘AttoPhos’ fluorescence substrate. Multiple washing steps were performed in between individual assay steps.
• Fab expression check by anti-Fd ELISA For verification of Fab expression in crude bacterial lysates, plates were coated with Fd-fragment specific antibodies.
• Bound Fabs were detected using respective alkaline-phosphatase (AP) coupled anti-Fab specific antibody in combination with ‘AttoPhos’ fluorescence substrate. Multiple washing steps were performed in between individual assay steps. K off ranking ELISA For a K off estimate, the ELISA plate was subjected to additional frequent and stringent washing upon the initial readout and a second detection was performed to identify candidates with a slow kd. Washing was performed using the following conditions: washing with 10 minute incubation 5 times, followed by overnight incubation, and then washing 5 times with 10 minute incubation, followed by 1h incubation, then washing 5 times with 5min incubation, before detection.
• AP alkaline-phosphatase
• Bound antibodies were detected using respective alkaline-phosphatase (AP) coupled secondary antibodies in combination with ‘AttoPhos’ fluorescence substrate.
• AP alkaline-phosphatase
• ELISA techniques were used for both screening of single Fab clones identified from panning outputs on target antigens as well as for characterization of purified antibodies. Optimal antigen and antibody concentrations as well as blocking conditions were evaluated and settings adjusted. Following panning selections, 368 clones from each third panning round output, were processed in primary screenings as bacterial lysates. Panning outputs from solid phase pannings were screened by FACS on StaR-coated magnetic beads or in ELISA using tagged-StaR variants or nanodisc-embedded StaRs.
• the IgGs Prior to functional characterization, the IgGs were re-screened by ELISA ( Figures 1 and 2) and for cell binding by IntelliCyt to reconfirm binding specificity. FACS and IntelliCyt Cell binding by flow cytometry and IntelliCyt to screen for positive identification of PAR2 binders Binding events to cell surface expressed antigen were identified by flow cytometry using either crude E. coli lysates from the panning output or purified antibodies.
• High throughput primary screens of panning outputs from differential whole cell pannings were mainly performed on human wild type PAR2-expressing Flp-In CHO cells (Flp-In CHO_huPAR2), GNTI BacMam wild type PAR2 cells and GNTI BacMam truncated PAR2 StaR versus parental Flp-In CHO cells (PAR2 negative) or non-infected HEK293GNTI- cells.
• Candidates showing an elevated background binding to parental Flp-In CHO cells were ascribed as either non-specific or off-target binders. All steps were performed in FACS buffer including FCS and sodium azide to prevent potential receptor internalization.
• PAR2 StaR nanodisc preparations presented high quality PAR2 receptors, in a detergent-free environment, comparable to the native receptor expressed on cells and led to the identification of highly promising, functionally active candidates.
• panning stringency is easily controlled using recombinant material, such a StaR nanodisc.
• Multiplexing by IntelliCyt To evaluate species cross-reactivity and/or unwanted binding to counter-targets simultaneously, screening in 384-well plate format was performed using the HTFC/ iQue screening platform from IntelliCyt. The HTFC/ iQue Screening System was also used for evaluation of binding to multiple target cell lines or evaluation of unwanted/non-specific binding in parallel, in other words multiplexing.
• Different cell populations could be distinguished by pre-labelling with distinct amounts of fluorescent dyes, such as Calcein or Cell-Tracker Green, establishing a unique signature of fluorescence intensity for each cell population, thereby producing a fluorescence coding system.
• the color-coded cell lines were then physically combined and mixed together with antibodies to be tested. Individual cell-lines could be identified via the fluorescence of the respective cell-line that had been pre-labelled. Crude bacterial cell lysates were combined with cells and incubated for 1 hour at room temperature in the dark, shaking gently. Fluorescence measurement was performed with the IntelliCyt HTFC/ iQue device. In between incubation steps, no washing was required. Raw data were evaluated with the help of the ‘ForeCyt’ software.
• the cell lines from each sample could be identified according to their fluorescence signature and individually evaluated for antibody binding. Staining-conditions for each cell line were optimized in order to find an assay set-up allowing the separation of distinct cell lines (Figure 5).
• Y022065 the parent of the final lead set, resulted from a panning strategy employing captured nanodisc-embedded deglycosylated full length hPAR2(PAR2-2, 1-377, N30Q, N222Q)_10xH_Sx2.
• the antigen was captured with anti-StrepII antibody.
• the antigen was captured with Benchmark 1 or coated on a Ni-NTA plate.
• Example 4 Expression and purification of Fab fragments and IgG Subcloning into Fab expression vector If required and to facilitate rapid expression of soluble Fab in E. coli, the Fab encoding inserts of the selected Ylanthia® phage were subcloned from pYPdis10 display vector into the Fab-expression vector (pYBex10_h_Fab-FH), comprising the features desired. Subcloning was performed by triple digest via XbaI ⁇ EcoRI-HF ⁇ PstI-HF. For full length FabCys expression in HKB11 cells, selected candidates were also cloned into the pYMex10_h_FabCys-AviH expression vector.
• Subcloning was performed by cutting out the antibody encoding fragments with the restriction enzymes NdeI ⁇ XhoI from the pYMex_h_IgG1f_AEASS source vector and ligation into the target vector pYMex10_h_FabCys-Avi-His.
• Subcloning into IgG expression vector For full length IgG expression in HKB11 cells, selected candidates were cloned into the pYMex10_h_IgG1f_AEASS expression vector. Subcloning was performed using a method for convenient and efficient conversion of a large amount of sequence-unique Fab clones into the IgG format.
• VH encoding fragment (flanked by restriction sites for NheI ⁇ XhoI)
• VL encoding fragment (flanked by restriction sites for NdeI ⁇ KpnI)
• a kappa or lambda specific eukaryotic pYMin expression cassette (flanked by restriction sites KpnI ⁇ NheI) were cloned into the acceptor vector for expression in mammalian cells (digested with NdeI ⁇ XhoI) in one-step or two- step cloning.
• E. coli XL-1 blue cells single clones were quality controlled via colony PCR and sequencing of the whole insert region.
• 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 using glycerol stocks from masterplates. Plates were incubated at 37°C for bacterial outgrowth and shaken overnight at 22°C for Fab expression. The next day expression cultures were lysed by addition of BEL buffer containing borate buffer, EDTA and lysozyme. Depending on the selected plate format and application, volumes were adjusted and the protocol for blocking was adapted accordingly. EDTA was omitted if lysates were used for sensitive cell screenings, e.g., functional assays.
• Buffer exchange to 1x Dulbecco ⁇ s PBS was performed using ‘PD10’ columns (GE Healthcare ⁇ Germany). Samples were sterile filtered (0.2 ⁇ m). Protein concentrations were determined by UV- spectrophotometry. The purity of the samples was analysed in denaturing, non-reducing 15% SDS- PAGE. The homogeneity of Fab preparations was determined in native state by size exclusion chromatography (HP-SEC) with calibration standards. Microscale production of IgG in mammalian cells Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both heavy and light chains of IgG.
• Protein concentrations were determined by UV-spectrophotometry and purity of IgG was analyzed under denaturing, reducing and non-reducing conditions using CE-SDS (LabChip GXII ⁇ Perkin Elmer ⁇ USA). HP-SEC was performed to analyse IgG preparations in native state. Exploratory scale production of AviHis tagged FabCys Eukaryotic HKB11 or HEK293-6E cells were transfected with mammalian expression vector DNA encoding both heavy and light chains of disulfide-bridged FabCysAviHis. Cell culture supernatants were harvested on day 3 or 7 post transfection and subjected to metal ion affinity chromatography (Protino Ni-NTA ⁇ Macherey Nagel).
• buffer exchange was performed to 1x Dulbecco’s PBS (pH 7.2 ⁇ Invitrogen) and samples were sterilefiltered (0.2 ⁇ m pore size). Protein concentrations were determined by UV-spectrophotometry and purity of FabCysAviHis was analysed under denaturing, reducing and non-reducing conditions using CE-SDS (LabChip GXII ⁇ Perkin Elmer ⁇ USA). HP-SEC was performed to analyse FabCysAviHis preparations in native state. IgG production for in vivo characterization Material production was undertaken by establishing stable HKB11 cell pools.
• Eukaryotic HKB11 cells were transfected with mammalian expression vector DNA encoding both heavy and light chains of IgG.
• respective vectors additionally contain a Zeocin resistance gene.
• Three days post-transfection selection was started by the addition of 160 ⁇ g/mL Zeocin to the cell suspension. During selection, cell count and viability initially decreased. 20 to 30 days after transfection cells started to recover. Reaching a viability of ⁇ 80 %, stable pools were scaled up to the desired volume. Cell culture supernatants were harvested on day 6 post transfection and subjected to Protein A affinity chromatography (MabSelect SURE ⁇ GE Healthcare).
• a second purification step (preparative SEC ⁇ Superdex 200 ⁇ GE Healthcare) was performed to remove aggregates. Buffer exchange was performed to 1x Dulbecco’s PBS (pH 7.2 ⁇ Invitrogen) and samples were sterile filtered (0.2 ⁇ m pore size). Protein concentrations were determined by UV-spectrophotometry and purities of IgG were analyzed under denaturing, reducing and non-reducing conditions using CE-SDS (LabChip GXII ⁇ Perkin Elmer ⁇ USA). HP-SEC was performed to analyze IgG preparations in native state. Endotoxin levels were determined by KQCL assay (Lonza). Protein identities were confirmed using Mass Spectrometry analysis (100MDL43).
• Example 5 Lead isolation clone profiling Cell-based IP-One G ⁇ q assay at human PAR2 for identification of functional primary hits Functional characterization was performed after full validation of clones in ELISA and IntelliCyt to confirm PAR2 binding status. Exploratory scale purified IgG1f_AEASS clones were tested for their ability to inhibit activation of the PAR2 receptor by the synthetic agonist, the Activating Peptide (2- Furyol-LIGRO) using the Cisbio® IP-One G ⁇ q assay at two concentrations in replicate.
• HEK293F cells were infected with a 2% v/v human PAR2 BacMam virus in the presence of 0.5mM sodium butyrate in growth media (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin/streptomycin) for 24h cultured in suspension format in a humidified incubator with 5 % CO2 atmosphere at 37°C.
• 0.5mM sodium butyrate in growth media Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin/streptomycin
• cells were harvested and re-suspended at a density of 1x10 ⁇ 6/ml in assay buffer at a density of 1x10 ⁇ 6/ml (1 part stimulation buffer (Cisbio): 5 parts ddH20) containing LiCl with 0.5% bovine serum albumin (BSA, Sigma).
• Antibodies were tested from stock concentration or following an initial 1:2 dilution in PBS.
• a 5 ⁇ l sample of either concentration of antibody in replicate was added to a half area white 96-well plates (Corning) followed by 25 ⁇ l PAR2 of cell suspension. Plates were incubated at 37°C for 30 minutes prior to addition of 5 ⁇ l/well Activating Peptide for a final agonist challenge concentration of 6.28nM.
• the final assay antibody screening was performed at a dilution of either 1:7 or 1:14 from Ab stock. Plates were re-incubated for a further 30 minutes at 37°C prior to addition of 10 ⁇ l IP-One detection kit (Cisbio) in lysis buffer.
• IP-One detection kit Cisbio
• HTRF ratios were calculated as in equation 1. Responses were normalised for % inhibition of Activating Peptide IP-One response at 6.28nM (EC80) final assay concentration ( Figure 6).
• Equation 1 Calculation of HTRF ratios
• Table 2 Inhibition of peptide-induced agonism of human PAR2 Functional characterization of Y021171, Y022063 and Y022054 & Y022065 hIgG1f_AEASS in the peptide inhibition assay using the Cisbio® IP-One G ⁇ q assay at the highest concentration in replicate. Also shown in Figure 6.
• Antibody clone ID % Max Inhibition AP ⁇ SD Number of repeats Y022054 17 ⁇ 10 2 Y021171 32 ⁇ 4 2 Y022063 27 ⁇ 5 2 Y022065 43 ⁇ 1 2 Affinity determination K D , ka and kd determination by SPR using antibody capture setup
• K D monomer fractions of antibody protein (Fab fragments or IgG) were used (at least 90% monomer content, as analysed by analytical SEC). Affinities were determined by kinetic characterization using either Biacore (Biacore T200) or Octet (QK384 or HTX) instruments as described below.
• the antibodies were captured on anti-human IgG chip surface followed by injection of 100 nM PAR2.
• CM5 chip Biacore, GE Healthcare
• AR2G Sensors fortéBIO, Pall Corp.
• SA sensors Biotinylated capture reagents.
• Appropriate capture systems were anti-hu-Fc antibody (Biacore, GE Healthcare), Protein A sensors (fortéBIO, Pall Corp.), MabSelect Sure Ligand (GE Healthcare), and anti-His-tag antibody (Genscript).
• the sensor surface was regenerated to remove captured antibody/antigen complexes, while maintaining the integrity of the capture surface.
• a blank injection of running buffer was used for referencing.
• the assay buffer was matched to the formulation of the PAR2 protein, i.e. base buffer and the detergent.
• the IgGs were captured via the Fc- fragment and detergent (LMNG/CHS) solubilised FL-N PAR2 StaR (PAR2-3) was used as analyte in solution. 50 mM HEPES pH 7.5, 150 mM NaCl, 0.02 % LMNG was used as assay buffer.
• CM3 sensor chips were used as basis to immobilize a high density of anti-hu-Fc capture antibody (BR-1008-39, GE Healthcare). IgGs were captured with relatively high levels of approx. 500 RU to achieve a higher ratio of specific to non-specific binding (while still maintaining conditions suitable for kinetic characterization). Sensorgrams were evaluated with the corresponding instrument’s evaluation software, i.e. Biacore T200 Evaluation Software 3.x (Biacore, GE Healthcare) or fortéBIO Octet Data Analysis (fortéBIO, Pall Corp.). All sensorgrams were fitted to a 1:1 binding model to determine ka and kd rate constants, which were used to calculate K D .
• Biacore T200 Evaluation Software 3.x (Biacore, GE Healthcare) or fortéBIO Octet Data Analysis (fortéBIO, Pall Corp.). All sensorgrams were fitted to a 1:1 binding model to determine ka and kd rate constants, which were used to calculate K D .
• Y022054 displayed a 10-fold higher affinity of 2.5 nM compared to the other candidates.
• Y021171 showed a fast association (ka) but also a very fast dissociation (kd).
• An “off rate selection” strategy (Hawkins et al. 1992) was implemented during the affinity maturation campaign to improve K D properties, notably for Y021171.
• the assay of PAR2 interaction with anti-PAR2 antibodies was carried out using a Biacore T200 instrument (GE Healthcare).
• Anti-human IgG antibody Human Antibody Capture Kit, GE Healthcare
• CM3 sensor chip
• RU resonance units
• Immobilisation was carried out at 25°C in HBS-EP+ buffer (GE Healthcare). The buffer was then changed to 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02 % LMNG, 0.002 % CHS.
• 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 ⁇ L/min), PAR2 injection (blank or 250 nM, with contact and dissociation times of 2 and 3 minutes, respectively, at a flow rate of 20 ⁇ L/minute), and chip surface regeneration (30 seconds at 20 ⁇ L/minute) using the regeneration solution from Human Antibody Capture Kit (GE Healthcare).
• the blank-subtracted data were fitted to 1:1 interaction model to obtain association and dissociation rate constants, ka (kon) and kd (koff), and the affinity constant K D . Measurements between each experimental data set were within the acceptable margins for this methodology.
• Table 4 Affinity determination of functional candidates Y021171, Y022063 and Y022054 by SPR
• the antibodies were captured on anti-human IgG chip surface followed by injection of 250 nM PAR2.
• the data were fitted to 1:1 interaction model.
• Example 6 Affinity maturation/lead optimisation and phage selections
• IgG clones including Y021171, Y022054, Y022063 and Y022065 partially inhibited PAR2-AP activation in the IP One assay.
• the DNA sequences encoding for the parental Fab fragments were transferred into the respective vector via restriction digest and ligation prior to library cloning.
• CDR-L3 and CDRH-1/ CDR-H2 regions were optimized in parallel.
• MorphoSys’ antibody libraries are designed for affinity optimization by CDR exchange, where single CDRs of antibodies are excised and replaced by individual members of large CDR libraries (Prassler et al. 2009).
• Ylanthia® maturation modules are based on the Ylanthia library design and were prebuilt with the Slonomics® technology (van den Brulle et al. 2008). The generation of the maturation libraries was performed for each maturation candidate individually.
• the parental CDR-L3 is replaced by an Ylanthia Maturation Stuffer (YMS), before the diversified LCDR-L3 YMM is inserted.
• YMS Ylanthia Maturation Stuffer
• Digested vector fragments were ligated with a 2-fold molar excess of the insert fragment carrying the diversified CDR-L3s. The same procedure was applied for diversification of CDRH-1 & CDR-H2. Ligation mixtures were electroporated in E.
• coli MC1061F cells yielding in >108 independent colonies. Amplification of the library was performed as described in the literature (Tiller et al.2013). For quality control, approximately 10 single clones per library were randomly picked and Sanger sequenced. Maturation candidates were selected according to the following scientific rationale and criteria: 1. Deselection of candidates with increased risk of off-target binding due to elevated binding on: • PAR2-negative antigens (ELISA): Empty nanodiscs, Mouse IgG, anti-StrepII, bRIL • PAR2-negative cell lines (FACS): parental Flp-In CHO cells, Flp-In CHO_huPAR1 and Jurkat cells 2.
• ELISA Empty nanodiscs
• Mouse IgG Mouse IgG, anti-StrepII, bRIL
• PAR2-negative cell lines FACS
• Human PAR2-specific IgG1f_AEASS candidates were ranked as follows: 1. Functionally active candidates; 2. Binding to cell surface expressed human PAR2 with a focus on the functionally active HEK293F cell line infected with WT FL-huPAR2 BacMam as a prerequisite for assessment of functionality; 3.
• Prolonged or overnight washing was used in off-rate selections strategies in combination with the addition of soluble antigen to the washing buffer to prevent any rebinding of antibody-phage to the immobilized antigen.
• Nine parental clones were progressed into an affinity maturation campaign covering all available binding profiles and potential modes of action.
• CDR-L3 or CDR-H1 & CDR-H2 regions were exchanged in parallel by diversified modules (Prassler et al.2009).
• Fixed VH/VL human germline framework pairs were preserved using specific Ylanthia® maturation modules (YMM) to avoid the emergence of cross-clones or additional framework combinations.
• YMM Ylanthia® maturation modules
• Fab fragments were transferred from the corresponding expression vector into the CysDisplayTM vector prior to library cloning for affinity maturation.
• CDR-L3 and CDR-H1 & CDR-H2 libraries were cloned separately for each maturation candidate and pooled.
• Affinity maturation was tailor made for 6 individual candidates, namely all functional candidates (Y021171, Y022054, Y022063, Y022065), as well as Y022059 and Y022069.
• Weaker candidates were matured in a pool (Y021160, Y022075 and Y022079).
• Maturation libraries were generated for individual diversification of CDR-L3 and CDR-H1 & CDR-H2.
• Maturation pannings were designed with an emphasis on high stringency solid phase pannings using full length or N- terminally truncated nanodisc-embedded PAR2 StaRs, as well as on the generation or improvement of rhesus PAR2 cross-reactivity.
• affinity maturation and screenings the project team was successful in identifying a large set of highly specific Ylanthia® antibody candidates. Clones were progressed into IgG conversion and in-depth characterization.
• Affinity maturation panning selections To increase affinity and biological activity of previously selected antibody fragments, CDR-L3 and CDR-H2 regions were exchanged in parallel by diversified modules (Prassler et al.2009).
• Fab fragments were transferred from the corresponding expression vector into the CysDisplayTM vector prior to library cloning for affinity maturation.
• phage derived from maturation libraries were subjected to three rounds of maturation panning. Panning stringency was increased by lowering the antigen concentration in each panning round (Low et al. 1996).
• off-rate selections were performed (Hawkins et al. 1992) for selected strategies. These strategies were combined with prolonged washing steps in combination with the addition of soluble antigen to the washing buffer to prevent any rebinding of antibody-phage to the immobilized antigen.
• the DNA fragment coding for the modified variable region was directly replaced in the pYMex10_h_IgG1f_AEASS expression vector, which codes for the parental IgG.
• the vector component coding for the parental VH or VL was removed with appropriate restriction enzymes (NheI ⁇ XhoI for VH, NdeI ⁇ KpnI for VL) and a fragment coding for the affinity matured variable region was inserted and expressed and purified as previously described to generate full length IgG.
• Example 7 Lead optimisation and clone profiling Profiling of purified Ylanthia® IgGs included binding specificity and cross-reactivity assessment to human, mouse, rhesus and marmoset PAR2-expressing cells, as well binding to PAR2 re-confirmed by ELISA to nanodisc-embedded StaRs and by flow cytometry to PAR2 expressing cells. Following affinity maturation, several IgG clones, mostly deriving from the functionally active parental IgG Y022065, were confirmed with antagonistic activity due to their ability to fully inhibit human PAR2 receptor activation and thus emphasizing their specificity for a functionally relevant epitope.
• IP-One HTRF results are normalised to effect of 10 ⁇ M MAB3949 from when screening parental antibody clones in challenge against Activating Peptide ( Figure 12) IP-One HTRF results are normalised to effect of 1 ⁇ M Benchmark 1 from when screening parental antibody clones in challenge against Bovine Trypsin ( Figure 13) IP-One HTRF results are normalised to effect of 10 ⁇ M MAB3949 when screening in challenge against PAR1 peptide SFFLR-NH2 Trypsin (Table 8) Normalised data was fitted in GraphPad Prism version 7.04 to a 4-parameter sigmoidal dose- response curve (Equation 2). The data in Table 8 shows that the Y022883 antibody is capable of blocking PAR2 activation by the PAR1 Activating Peptide.
• Table 6 Functional characterization of lead optimized clones against Activating Peptide that demonstrate activity in replicate in the IP-One human PAR2 antagonist assay using the Cisbio® IP- One G ⁇ q kit which also share activity in Bovine Trypsin challenge assay.
• Tables 6 and 7 list functional IgG actives which demonstrate IC 50 values n ⁇ 1 against both Activating Peptide and Bovine Trypsin.
• Table 8 IP-One HTRF results are normalised to effect of 10 ⁇ M MAB3949 when screening in challenge against PAR1 peptide SFFLR-NH2 Trypsin.
• HT-29 cells ATCC HTB-38 were kept in continuous culture using DMEM medium with high glucose (25 mM), without sodium pyruvate, but with GlutaMAX (Gibco, Paisley, UK), 10 % of heat-inactivated fetal bovine serum and penicillin/streptomycin (100 units/mL of penicillin and 100 ⁇ g/mL of streptomycin) in a humidified incubator with 5 % CO2 atmosphere at 37°C. Culture medium is changed every 2 days from the second day after seeding, and cells are harvested in the logarithmic phase of growth after reaching 80–90 % confluency by 0.05 % trypsin/EDTA.
• Cells were plated at 50 ⁇ L/well in culture media at a cell density of 5,000 cells per well in 384-well black wall plates (Corning) and incubated for 24 hours in a humidified incubator with 5% CO2 atmosphere at 37°C.
• cell media was removed and 50 ⁇ L assay buffer (HBSS 20mM HEPES pH7.4 buffer containing 0.1% BSA) containing Calcium 5 dye at a 1:20 dilution from stock (Molecular Devices). Plates were re-incubated at 37°C for 45 minutes prior to cells equilibrated for a further 15 minutes at room temperature.
• IgG lead clones were prepared in assay buffer and serially diluted to generate a 10-point curve.
• IgG dose response curves were added online using the FLIPR Tetra (Molecular Devices) pipettor (10 ⁇ L) and the calcium response measured over a 5-minute period. Plates were re-incubated again at 37°C for 60 minutes prior to a 10 ⁇ L /well online addition of either Activating Peptide (630nM) or Bovine Trypsin (63nM) and further measurement of human PAR2 activated by calcium mobilisation conducted over a 5 minute period. Data was analysed by extraction Max-Min raw data files and analysed by Equation 2. Data was normalised against MAB3949 or Benchmark 1 for Activating Peptide and Bovine Trypsin assays, respectively.
• Table 9 Functional characterization of lead clones against Bovine Trypsin or Activating Peptide (AP) that demonstrate full dose dependent inhibition n ⁇ 3 in the HT-29 FLIPR calcium mobilization assay normalized against corresponding antibody control.
• HEK293f cells were infected with a 2.5 % v/v Cynomolgus PAR2 BacMam virus, or a 2.5 % v/v Rhesus PAR2 BacMam virus, in the presence of 0.5mM sodium butyrate in growth media (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin/streptomycin) for 24h cultured in suspension format in a humidified incubator with 5 % CO2 atmosphere at 37 °C.
• 0.5mM sodium butyrate in growth media Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin/streptomycin
• HEK293f cells were infected with a 5 % v/v human PAR1 BacMam virus in the presence of 0.5mM sodium butyrate in growth media (Pro293, 5% FBS, 1% Glutamax, 0.4% penicillin/streptomycin) for 24 hours cultured in suspension format in a humidified incubator with 5 % CO2 atmosphere at 37°C.
• cells were harvested and re-suspended at a density of 1x10 ⁇ 6/mL in assay buffer at a density of 1x10 ⁇ 6/mL (1 part stimulation buffer (Cisbio): 5 parts ddH20) containing LiCl with 0.5% bovine serum albumin (BSA, Sigma).
• Antibody stocks are serially diluted 1:2 over a 10-point concentration curve in order to determine an IC 50 value.
• Antibody lead clones were tested in challenge against SFLLR (100nM). A 5 ⁇ L aliquot of either concentration of antibody in replicate was added to a half area white 96-well plates (Corning) followed by 25 ⁇ L PAR2 of cell suspension. Plates were incubated at 37°C for 30 minutes prior to addition of 5 ⁇ L/well SFLLR for a final agonist challenge concentration of 100nM. Plates were re- incubated for a further 30 minutes at 37°C prior to addition of 10 ⁇ L IP-One detection kit (Cisbio) in lysis buffer.
• IP-One detection kit Cisbio
• HTRF ratios were calculated as in equation 1. Responses were normalised for % fold inhibition over 100nM SFLLR alone final assay concentration and results fitted in GraphPad Prism version 7.04 to a 4-parameter sigmoidal dose-response curve (Equation 2).
• Table 11 Functional characterization of lead clones against SFLLR in Cisbio IP-One human PAR1 selectivity assay
• the lead set binds to purified and cell surface expressed PAR2 with affinities within the range of the competitor antibodies. No off-target binding to related proteins PAR1, PAR3 and PAR4 could be detected.
• the lead set of antibodies provided herein is able to inhibit not only protease activation, but also peptide activation of PAR2, thus acting as dual inhibitors of PAR2 activation.
• k off was determined by Octet and showed a net improvement over the parental antibodies.
• kd [1/s] range from 1.1E-4 to 1.0E-05 assessed on truncated huPAR2 StaR and FL-huPAR2 StaR. In general, only one analyte concentration was used for koff determination. Samples that were assessed directly from IgG supernatants or bacterial lysates (Fab fragments), were subjected to k off determination using Octet in an antibody capture format.
• a monomeric antigen was used as analyte.
• IgG samples were captured at a moderate capture level (0.4 nm) onto streptavidin sensors loaded with a high density of capture ligand (biotinylated MabSelect SuRe ligand).
• the 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. Dissociation was monitored for up to 1500 s. 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).
• affinities to human PAR2 were determined by SPR.
• the IgGs were captured via their Fc- fragment and detergent (LMNG/CHS) solubilised FL-N PAR2 StaR was used as analyte in solution.50 mM HEPES pH 7.5, 150 mM NaCl, 0.02 % LMNG was used as assay buffer.
• the CM3 sensors were used as basis to immobilize a high density of anti-hu-Fc capture antibody (BR- 1008-39, GE Healthcare).
• Table 13 SPR kinetics of affinity matured clones as determined using Octet system and soluble PAR2 StaR® Clone ka (1/Ms) Kd (1/s) KD (nM) Y022916 3.9E+4 2.5E-4 6.5 Y022856 1.2E+5 2.5E-4 2.5 Y022858 5.2E+4 7.3E-5 1.4 Y022870 9.6E+4 2.0E-5 0.21 Y022877 6.8E+4 2.0E-5 0.29 Y022879 5.9E+4 3.2E-5 0.5 Y022882 1.1E+5 2.0E-5 0.18 Y022883 1.4E+5 2.0E-5 0.14 Y022884 7.1E+4 8.4E-5 1.2 Y022885 1.5E+5 2.0E-5 0.14 Y022889 1.6E+5 2.0E-5 0.13 Benchmark1 1.2E+6 8.8E-4 0.76 Table 14: SPR kinetics as determined using Biacore system and soluble PAR2 StaR
• Anti-human IgG antibody Human Antibody Capture Kit, GE Healthcare
• CM3 Sensor Chip
• RU Reactive Radio Unit
• Immobilisation was carried out at 25 o C in HBS-EP+ buffer (GE Healthcare).
• 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 carried out at either 25 or 37 o C.
• Each cycle of the assay began with antibody capture in the target flow cell to a level of 400 to 450 RU.
• PAR2 was then injected over target and reference flow cells at three concentrations, 5 min each, followed by 30-min dissociation.
• the surface was then regenerated with a solution provided in the kit (Human Antibody Capture Kit, GE Healthcare). With every antibody tested the first cycle was a blank (three injections of the running buffer) and the second a series of 50, 100, and 200 nM PAR2 injections.
• the blank-subtracted data were fitted to 1:1 interaction model to obtain association and dissociation rate constants, k a and k d , and the affinity constant K D .
• Table 16 Summary of binding of lead clones after affinity maturation at 25°C and 37°C Clone Temp o C Dissociation ka (1/Ms) kd (1/s) KD (M) t1/2 (h) time (mins) Y22870 25 90 3.4E+04 7.0E-06 2.1E-10 27.6 Y22870 25 30 2.8E+04 1.2E-05 4.3E-10 16.1 Y22870 37 30 4.1E+04 1.9E-05 4.5E-10 10.3 Y22877 25 90 2.7E+04 8.0E-06 3.0E-10 24.1 Y22877 25 30 2.2E+04 5.1E-06 2.3E-10 37.9 Y22877 37 30 3.1E+04 1.7E-05 5.5E-10 11.5 Y22883 25 90 3.0E+04 1.2E-05 4.0E-10 16.2 Y22883 25 30 4.0E+04 8.1E-06 2.0E-10 23.7 Y22883 37 30 3.6E
• BacMam infected HEK-293F cells expressing the wild-type full-length human PAR2, were used for titration.
• the cells were titrated in incubation buffer (D-PBS from Gibco, 0.5% (w/v) BSA, 0.02% (v/v) Sodium azide).
• D-PBS incubation buffer
• the experiment consisted of two titrations with a low and a high fixed IgG concentration.
• the maximum amount of cells had to contain a high enough effective ligand concentration to completely saturate the IgG.
• the mixtures of IgG and cells were incubated at room temperature overnight under very gentle agitation to allow for equilibrium formation. Subsequently, the formed cell-antibody complex was removed by centrifugation. The free IgG amounts of the supernatants were determined with goat anti-human F(ab’) 2 fragment specific antibody (Jackson ImmunoResearch, #109-005-097) coated on PMMA (Polymethylmethacrylat) beads and fluorescence detection with anti-human F(ab’) 2 fragment specific Alexa Fluor 647 antibody (Jackson ImmunoResearch, #109-605-097).
• Example 10 Epitope binning and analysis of affinity matured clones by HDX and flow cytometry analysis of receptor mutations
• Epitope binning was performed by an ELISA-based assay.
• Anti-human PAR2 IgG1f sample IgGs were directly coated on a microtiter plate at a concentration of 100nM.
• a tagged antigen e.g.
• nanodisc-embedded StaR at a constant concentration (12.5 nM to 25 nM) was incubated with a 25- fold molar excess of a second anti-human PAR2 IgG1f to saturate all epitopes (maximum conc.312.5 nM to 625 nM, 7-point titration, 1:4) for 1 hour and complexes were allowed to form.
• An internal positive control self-competition
• Antigen-IgG complexes were added to IgG coated plates for 30 min. Antigen-IgG complexes bound to coated IgGs were detected via a tag-specific antibody (e.g.
• anti-StrepII or anti-His detected with a suitable alkaline-phosphatase (AP) coupled secondary antibody in combination with ‘AttoPhos’ fluorescence substrate. Multiple washing steps were performed in between individual assay steps.
• AP alkaline-phosphatase
• antigen-IgG complexes cannot bind to IgG coated plates which translates into low signals.
• Targeting of different epitopes allows antigen-IgG complexes to bind coated IgGs and IgGs in solution simultaneously, leading to positive detection signals.
• This competition ELISA set-up was employed to characterise the targeted epitope and epitope diversity of the available candidates by epitope binning.
• the 16 dual-functional PAR2-specific candidates were tested for binding competition against each other and against reference antibodies, including MAB3949 (R&D Systems) and Benchmark 1.
• This set of 16 antibodies included at least one derivative of each of the five parental families i.e., clones derived from the parental clones by affinity maturation (Y022079, Y022069, Y022065, Y021171, Y022059). All affinity matured families competed with each other, as well as anti-huPAR2 (MAB3949 R&D Systems). This indicates that all these antibodies target proximal binding regions (i.e., extracellular domains other than the N terminus). None of the candidates competed with Benchmark mAb 1.
• Benchmark mAb 1 is the only antibody in the panel tested that exclusively binds to full length PAR2, i.e., binds a linear N-terminal epitope; these results indicate that affinity matured candidates bind to a distinctly different epitope that is more closely related to or directly targeting the extracellular loop region (Figure 17).
• assessment of binding under StaR-denaturing conditions was implemented to aid categorization into recognition of linear or conformational epitope. 97% of tested IgG candidates displayed loss of binding and were therefore assigned as binding to a conformational epitope.
• Hydrogen Deuterium Exchange Hydrogen Deuterium Exchange (HDX) is one of the methods that can be used to interrogate and identify a binding interface.
• Protein–protein interaction sites are probed by cataloguing the hydrogen/deuterium exchange rate of the amide hydrogens in proteins backbone. How fast or slow this exchange takes place is determined by the accessibility of those amide hydrogens to the solvent. Therefore, amides in exposed regions should have a higher rate of exchange than those buried within a protein–protein interface since they have a higher accessibility to the solvent environment.
• the following equipment and materials were used in the example. Liquid chromatography separation was performed on Thermo Vanquish UHPLC (Thermo Scientific) equipped with two temperature-controlled column compartments.
• PAR2-2 construct was diluted to 50 ⁇ M and incubated with equimolar concentration of Fab Y022883 (or Fab MAB3949) for 30 minutes at 22 o C.
• HDX was initiated by dilution of protein mixture in a deuterated buffer (10mM phosphate buffer pH 7.5, 150mM NaCl) for each of several periods of time (continuous labelling): 1 min, 10 min, 30min, 2h at 22°C.
• the dilution of 20-fold was chosen to achieve up to 90-95% D labelling of solvent accessible hydrogens (usually 2 ⁇ l of protein mixture was mixed with 38 ⁇ l of deuterated buffer).
• Mutated PAR2 and WT PAR2 showed very similar expression profiles when detected with Benchmark 1, except for D62F, which shows a partially reduced expression level, whereas S60W, D62F, G318F and the double mutation D62G/E63G all show a reduced expression profile versus WT when detected with Y022883, indicating that the areas identified by HDX analysis are involved in Y022883 binding to human PAR2.
• the observed reductions in binding, with the chosen mutations are stronger for Segment1/Helix0/1 than ECL3 ( Figure 21).
• the impact of these mutations may confer a conformational change that alters the antibody epitope and may also cause a change in functional activity of the receptor.
• Example 11 Rat pharmacokinetics The pharmacokinetics of Y022883 were explored in adult male Sprague Dawley rats. 3 rats were intravenously injected with 10mg/kg Y022883 and blood samples taken at various timepoints out to 2 weeks. Blood samples were used to generate sera and these sera analysed by a qualified non-GLP ELISA method on the Gyrolab platform (generic PK kit) with PK parameters calculated by non- compartmental analysis (NCA) using PhoenixTM WinNonlin. Summary data are shown in Figure 22 and Table 18. These data are consistent with expectations of a human mAb that does not have a significant antigen sink which would be expected as Y022883 does not bind rat PAR2.
• RNA samples were then pelleted (306g, 5 minutes, room temperature), the pellet re-suspended and mixed with 0.48mL RNAlater before being frozen at -70°C.
• Sample RNA was then extracted using standard methods and the transcriptome assessed with genome-based read mapping at a read depth of 50x. Sequencing reads (FASTQ files) were aligned to a reference genome and the number of reads mapped to each gene counted which in turn generated a gene-count table. Differential gene expression analysis was then performed between each stimulus at each timepoint per dose group e.g.
• a set of differentially expressed genes was generated for each stimulus (from the pre-dose samples); the effect of treatment with mAb was evaluated by determining whether the set of pre-dose differentially expressed genes for each stimulus were still differentially expressed at each timepoint.
• 1816 genes were differentially regulated by LPS (vs predose PBS) at the predose timepoint in the 1mg/kg dose group; at 24h 1379 of these genes were still differentially regulated by LPS (vs 24h PBS) hence 76% of the predose LPS gene-signature was present at 24h post- dose in the 1mg/kg LPS dose group.
• Antibodies Y022883 (SH-C) and Benchmark 2 (SH-D) were tested at concentrations ranging from 10 nM to 3 ⁇ M.
• Cells were lysed at 5- or 30-min post treatment in lysis buffer (PBS + 1% Triton X-100, protease and phosphatase inhibitors).
• Total protein concentration was evaluated by BCA before phosphoprotein analysis. Protein concentration was adjusted to 0.3 mg/ml and samples were loaded onto a 12-230 kDa Wes Separation Module 8 x 25 capillary cartridges, Protein Simple.
• Rabbit Antibodies were used to detect pERK and ERK (pERK ,Cell signalling, cat: 9101S; ERK, Cell signalling, cat: 4695S) and phospho p38 MAPK and p38 MAPK (Cell signalling, cat: 9211L; p38, Cell signalling, cat: 9212S).
• Anti-rabbit detection module Protein Simple, cat: DM-001 was used as the secondary antibody. Data for phospho-ERK or phospho p38 was normalized to data for total ERK or total p38; biological duplicates were analyzed. Percentage of the maximum ratio was used to normalized between plates (Figs.27-29).
• Protease-activated receptor 2 sensitizes TRPV1 by protein kinase C ⁇ - and A-dependent mechanisms in rats and mice. J.Physiol, 575(2), pp.555–571. Amiable, N., Tat, S., Lajeunesse, D., Duval, N., Pelletier, J., Martel-Pelletier, J. and Boileau, C., 2009. Proteinase-activated receptor (PAR)-2 activation impacts bone resorptive properties of human osteoarthritic subchondral bone osteoblasts. Bone, 44(6), pp.1143-1150. Ausubel, F., 1987. Current protocols in molecular biology.

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