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
  • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/04—Antipruritics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • 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
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • 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
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• the present invention relates to antibodies to canine IL-31 receptor alpha that have a high binding affinity for canine IL-31 receptor alpha, and that can block the binding of canine IL-31 to the canine IL-31 receptor alpha.
• the present invention also relates to use of the antibodies of the present invention in the treatment of atopic dermatitis in dogs.
• the immune system comprises a network of resident and recirculating specialized cells that function collaboratively to protect the host against infectious diseases and cancer.
• the ability of the immune system to perform this function depends to a large extent on the biological activities of a group of proteins secreted by leukocytes and collectively referred to as interleukins.
• interleukins are four important molecules identified as interleukin-31 (IL-31), interleukin-4 (IL-4), interleukin- 13 (IL-13), and interleukin-22 (IL-22).
• IL-4, IL-13, IL-22, and IL-31 are critical cytokines for the development of immune responses that are required for protection against extracellular pathogens (e.g., tissue or lumen dwelling parasites), these cytokines also have been implicated in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.
• Atopic dermatitis is a relapsing pruritic and chronic inflammatory skin disease, that is characterized by immune system dysregulation and epidermal barrier abnormalities in humans.
• the pathological and immunological attributes of atopic dermatitis have been the subject of extensive investigations [reviewed in Rahman et al. Inflammation & Allergy-drug target 10:486- 496 (2011) and Harskamp et al., Seminar in Cutaneous Medicine and Surgery 32: 132-139 (2013)].
• Atopic dermatitis is also a common condition in companion animals, especially dogs, where its prevalence has been estimated to be approximately 10-15% of the canine population.
• atopic dermatitis in dogs and cats [reviewed in Nuttall et al., Veterinary Records 172(8):201-207 (2013)] shows significant similarities to that of atopic dermatitis in man including skin infiltration by a variety of immune cells and CD4 + Th2 polarized cytokine milieu including the preponderance of IL-31, IL-4, and IL-13.
• IL-22 has been implicated in the exaggerated epithelial proliferation leading to epidermal hyperplasia that is characteristic of atopic dermatitis.
• antibodies against canine IL-31 have been shown to have an effect on pruritus associated with atopic dermatitis in dogs [US 8,790,651 B2; US 10,093,731 B2],
• an antibody against human IL-31 receptor alpha (IL-3 IRA) has been tested and found to have an effect on pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New England Journal of Medicine, 376(9), 826-835 (2017)].
• JAK Janus kinase
• SYK spleen tyrosine kinase
• antagonists to a chemoattractant receptor-homologous molecule expressed on TH2 cells see e.g., U.S. 7,696,222, U.S. 8,546,422, U.S. 8,637,541, and U.S. 8,546,422]
• the present invention provides new mammalian antibodies, including caninized murine antibodies, to IL-31 receptor alpha (IL-3 IRA) from canines.
• the mammalian antibodies to canine IL-31 receptor alpha (cIL-3 IRA) are isolated antibodies.
• the mammalian antibodies or antigen binding fragments thereof bind canine IL-3 IRA.
• the mammalian antibodies or antigen binding fragments also block the binding of canine IL-3 IRA to canine interleukin-31.
• the mammalian antibodies are antibodies to canine IL-3 IRA.
• the mammalian antibodies are caninized antibodies.
• the caninized antibodies are caninized murine antibodies to canine IL-3 IRA.
• the present invention provides mammalian antibodies (including caninized antibodies) or antigen binding fragments thereof that bind canine IL-3 IRA, in which the antibody comprises a heavy chain and a light chain that together comprise a set of six complementary determining regions (CDRs), three of which are heavy chain CDRs: CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2) and CDR heavy 3 (HCDR3) and three of which are light chain CDRs: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3).
• CDRs complementary determining regions
• the mammalian antibody or antigen binding fragment thereof comprises an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 4, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 14, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 24.
• the mammalian antibody or antigen binding fragment thereof further comprises a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 33, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 37, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 46.
• the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 106. In other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by an amino acid sequence of SEQ ID NO: 108. In still other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds both to an epitope comprised by the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of SEQ ID NO: 108.
• the mammalian antibody or antigen binding fragment thereof comprises an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 5, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 15, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 25.
• the mammalian antibody or antigen binding fragment thereof further comprises a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 32, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 41, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 47.
• the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 98. In other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by an amino acid sequence of SEQ ID NO: 100. In still other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds both to an epitope comprised by the amino acid sequence of SEQ ID NO: 98 and the amino acid sequence of SEQ ID NO: 100.
• the mammalian antibody or antigen binding fragment thereof comprises an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 6, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 16, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 26.
• the mammalian antibody or antigen binding fragment thereof further comprises a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 32, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 42, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 48.
• the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 98. In other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by an amino acid sequence of SEQ ID NO: 101. In still other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds both to an epitope comprised by the amino acid sequence of SEQ ID NO: 98 and the amino acid sequence of SEQ ID NO: 101.
• the mammalian antibody or antigen binding fragment thereof comprises an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 7, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 17, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 27.
• the mammalian antibody or antigen binding fragment thereof further comprises a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 34, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 37, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 50.
• the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 106. In other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by an amino acid sequence of SEQ ID NO: 107. In still other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds both to an epitope comprised by the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of SEQ ID NO: 107.
• the mammalian antibody or antigen binding fragment thereof comprises an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 8, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 18, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 28.
• the mammalian antibody or antigen binding fragment thereof further comprises a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 35, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 43, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 51.
• the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 104. In other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by an amino acid sequence of SEQ ID NO: 105. In still other embodiments, the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds both to an epitope comprised by the amino acid sequence of SEQ ID NO: 104 and the amino acid sequence of SEQ ID NO: 105.
• the mammalian antibody or antigen binding fragment thereof comprises an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 9, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 19, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 29.
• the mammalian antibody or antigen binding fragment thereof further comprises a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 36, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 44, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 49.
• the mammalian antibody or antigen binding fragment thereof when bound to canine IL-3 IRA binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 109.
• the antibody and antigen binding fragment thereof bind canine IL-3 IRA and block the binding of canine IL-3 IRA to canine interleukin-31.
• the mammalian antibody to canine IL-3 IRA is a murine antibody.
• the mammalian antibody to canine IL-3 IRA is a caninized antibody.
• the caninized antibody to canine IL-3 IRA is a caninized murine antibody.
• the caninized antibodies of the present invention comprise a canine fragment crystallizable region (cFc).
• the caninized antibodies of the present invention also comprise a canine light chain constant region.
• the canine light chain constant region is a kappa canine light chain constant region.
• the kappa canine light chain constant region comprises the amino acid sequence of SEQ ID NO: 127.
• the caninized antibody or antigen binding fragment thereof can comprise a heavy chain that comprises a cFc region and a hinge region.
• the hinge region is preferably a canine hinge region.
• the canine hinge region can comprise a natural occurring: IgG-A hinge region, IgG-B hinge region, IgG-C hinge region, or IgG-D hinge region.
• the hinge region is a corresponding modified canine hinge region.
• the hinge region is the IgG-A hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 112.
• the hinge region is the IgG-B hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 113.
• the hinge region is the IgG-C hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 114.
• the hinge region is a modified IgG-D hinge region comprising the amino acid sequence of SEQ ID NO: 115.
• the canine Fc region can be an IgG-A, IgG-B, IgG-C, an IgG-D or modifications thereof.
• a caninized antibody or antigen binding fragment thereof comprises an IgG-Bm.
• a caninized antibody or antigen binding fragment thereof comprises an IgG-A that comprises an amino acid sequence that has at least 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 116.
• a caninized antibody or antigen binding fragment thereof comprises an IgG-B that comprises an amino acid sequence that has at least 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 110.
• a caninized antibody or antigen binding fragment thereof comprises an IgG-C that comprises an amino acid sequence that has at least 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 117.
• a caninized antibody or antigen binding fragment thereof comprises an IgG-D that comprises an amino acid sequence that has at least 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 118.
• a caninized antibody or antigen binding fragment thereof comprises an IgG-Bm that comprises an amino acid sequence that has at least 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 111, wherein both the aspartic acid residue (D) at position 31 of SEQ ID NO: 110 and the asparagine residue (N) at position 63 of SEQ ID NO: 110, remain substituted by an alanine residue (A) in the sequence of IgG-Bm.
• the caninized antibody or antigen binding fragment thereof comprises the canine IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 112.
• the caninized antibody comprises a heavy chain that comprises an IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 113.
• the caninized antibody comprises a heavy chain that comprises an IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising an amino acid sequence comprising at least 90%, 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 114.
• the caninized antibody comprises a heavy chain that comprises an IgG-D, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 115.
• the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93. In other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94. In yet other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95. In still other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96. All of these heavy chain variable regions can further comprise a canine hinge region and/or a cFc that have been aforementioned above.
• the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128. In other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 129. In still other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 129. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 129. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 129. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 129. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130.
• the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 90. In other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 91. In still other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In still other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86. In other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87. In yet other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88. In still other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 89.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence of SEQ ID NO: 91. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.
• the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72. In other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 73. In still other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 74. In yet other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75.
• the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 76. In other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 77. In yet other embodiments, the caninized antibody against canine IL-3 IRA or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 78.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 76. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 77. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 78.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 76. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 77. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 78.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 74 and a light chain comprising the amino acid sequence of SEQ ID NO: 76. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 74 and a light chain comprising the amino acid sequence of SEQ ID NO: 77. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 74 and a light chain comprising the amino acid sequence of SEQ ID NO: 78.
• the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 76. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 77. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 78.
• the present invention further provides canine or caninized antibodies or antigen binding fragment thereof that bind to canine interleukin-31 receptor alpha (canine IL-3 IRA) and block the binding of canine IL-3 IRA to canine IL-31 and that when bound to canine IL-3 IRA the antibody binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 or any combination thereof.
• canine or caninized antibodies or antigen binding fragment thereof that bind to canine interleukin-31 receptor alpha (canine IL-3 IRA) and block the binding of canine IL-3 IRA to canine IL-31
• the mammalian antibody when bound to canine IL-3 IRA, binds to an epitope comprised by the amino acid sequence of SEQ ID NO: 104 or SEQ ID NO: 105, or to both SEQ ID NO: 104 and SEQ ID NO: 105; or SEQ ID NO: 98 or SEQ ID NO: 100, or to both SEQ ID NO: 98 and SEQ ID NO: 100; or alternatively to SEQ ID NO: 106 or SEQ ID NO: 108, or to both SEQ ID NO: 106 and SEQ ID NO: 108 .
• the identification of the epitopes can be based on chemical crosslinking and mass spectrometry detection.
• the mammalian antibody when bound to canine IL-3 IRA, binds at least one amino acid residue, preferably one to three amino acid residues, more preferably two to five amino acid residues, and/or more preferably three to eight amino acid residues or more within the amino acid sequence of SEQ ID NO: 104 or within SEQ ID NO: 105, or that are within the amino acid sequences of both SEQ ID NO: 104 and SEQ ID NO: 105; or SEQ ID NO: 98 or SEQ ID NO: 100, or to both SEQ ID NO: 98 and SEQ ID NO: 100; or alternatively, to SEQ ID NO: 106 or SEQ ID NO: 108, or to both SEQ ID NO: 106 and SEQ ID NO: 108.
• the mammalian antibody that binds to SEQ ID NO: 104 binds to the arginine residue at position 225 of SEQ ID NO. 2, z.e., R225.
• the mammalian antibody that binds to SEQ ID NO: 104 binds to the threonine residue at position 236 of SEQ ID NO. 2, z.e., T236.
• the mammalian antibody that binds to SEQ ID NO: 105 binds to the arginine residue at position 298 of SEQ ID NO. 2, z.e., R298.
• the mammalian antibody that binds to SEQ ID NO: 105 binds to the lysine residue at position 305 of SEQ ID NO. 2, z.e., K305. In still other embodiments, the mammalian antibody that binds to SEQ ID NO: 105 binds to the threonine residue at position 306 of SEQ ID NO. 2, z.e., T306. In yet other embodiments, the mammalian antibody binds to at least two amino acid residues from the group consisting of: R225 and/or T236 and/or R298 and/or K305 and/or T306 of SEQ ID NO: 102. In a specific embodiment of this type, the mammalian antibody binds to R225, T236, R298, K305, and T306 of SEQ ID NO. 2. The present invention further provides antigen binding fragments of these mammalian antibodies.
• the mammalian antibody that binds to SEQ ID NO: 98 binds to the lysine residue at position 102 of SEQ ID NO. 2, z.e., K102.
• the mammalian antibody that binds to SEQ ID NO: 98 binds to the serine residue at position 110 of SEQ ID NO. 2, z.e., Suo.
• the mammalian antibody that binds to SEQ ID NO: 98 binds to the lysine residue at position 112 of SEQ ID NO. 2, z.e., K112.
• the mammalian antibody that binds to SEQ ID NO: 98 binds to the lysine residue at position 118 of SEQ ID NO. 2, z.e., Kus.
• the mammalian antibody that binds to SEQ ID NO: 98 binds to the arginine residue at position 119 of SEQ ID NO. 2, z.e., R119.
• the mammalian antibody that binds to SEQ ID NO: 100 binds to the threonine residue at position 176 of SEQ ID NO. 2, z.e., T176.
• the mammalian antibody that binds to SEQ ID NO: 100 binds to the tyrosine residue at position 178 of SEQ ID NO. 2, i.e., Yrzs. In still other embodiments, the mammalian antibody that binds to SEQ ID NO: 100 binds to the serine residue at position 189 of SEQ ID NO. 2, i.e., Si89. In yet other embodiments, the mammalian antibody binds to at least two amino acid residues from the group consisting of: K102 and/or Suo and/or K and/or Kus and/or R119 and/or T176 and/or Yns and/or Si89 of SEQ ID NO: 102.
• the mammalian antibody binds to K102, Suo, Km, Kus, Rii9, T176, Y178, and Si89 of SEQ ID NO. 2.
• the present invention further provides antigen binding fragments of these mammalian antibodies.
• the mammalian antibody that binds to SEQ ID NO: 106 binds to the arginine residue at position 430 of SEQ ID NO. 2, z.e., R430. In other embodiments, the mammalian antibody that binds to SEQ ID NO: 106, binds to the lysine residue at position 435 of SEQ ID NO. 2, z.e., K435. In yet other embodiments, the mammalian antibody that binds to SEQ ID NO: 106, binds to the threonine residue at position 438 of SEQ ID NO. 2, z.e., T438.
• the mammalian antibody that binds to SEQ ID NO: 106 binds to the tyrosine residue at position 441 of SEQ ID NO. 2, z.e., Y441.
• the mammalian antibody that binds to SEQ ID NO: 108 binds to the serine residue at position 472 of SEQ ID NO. 2, z.e., S472.
• the mammalian antibody that binds to SEQ ID NO: 108 binds to the threonine residue at position 479 of SEQ ID NO. 2, z.e., T479.
• the mammalian antibody that binds to SEQ ID NO: 108 binds to the threonine residue at position 487 of SEQ ID NO. 2, z.e., T487.
• the mammalian antibody binds to at least two amino acid residues from the group consisting of: R430 and/or K435 and/or T438 and/or Y441 and/or S472 and/or T479 and/or T487 of SEQ ID NO: 102.
• the mammalian antibody binds to R430, K435, T438, Y441, S472, T479, and T487 of SEQ ID NO. 2.
• the present invention further provides antigen binding fragments of these mammalian antibodies.
• the present invention also provides nucleic acids, including isolated nucleic acids, that encode any of: the sets of 3 HCDRs or 3 LCDRs; the heavy chain variable regions of the caninized antibodies or antigen binding fragments thereof; the heavy chains of the caninized antibodies or antigen binding fragments thereof, the light chain variable regions of the caninized antibodies or antigen binding fragments thereof, and/or the light chains of the caninized antibodies or antigen binding fragments thereof.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain of a specific caninized antibody of any one of the antibodies of the present invention and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain of that (said) specific caninized antibody.
• the present invention further provides expression vectors that comprise such pairs of nucleic acids, or alternatively individual nucleic acids of the present invention.
• the present invention provides pairs of expression vectors, wherein one of the pair of expression vectors comprises a nucleic acid comprising a nucleotide sequence that encodes the light chain of a specific caninized antibody of any one of the antibodies of the present invention, and the other of the pair of expression vectors comprises a nucleic acid comprising a nucleotide sequence that encodes the heavy chain of that (said) specific caninized antibody.
• the present invention provides nucleic acids that encode a set of the three heavy chain complementary determining regions (CDRs), a CDR heavy 1 (HCDR1), a CDR heavy 2 (HCDR2), and a CDR heavy 3 (HCDR3) of a mammalian antibody (including a caninized antibody) of the present invention, nucleic acids that encode a set of the three light chain complementary determining regions (CDRs), a CDR light 1 (LCDR1), a CDR light 2 (LCDR2), and a CDR light 3 (LCDR3) of a mammalian antibody (including of a caninized antibody) or an antigen binding fragment thereof of the present invention, or pairs of such light chains and heavy chain CDRs.
• CDRs three heavy chain complementary determining regions
• HCDR1 CDR heavy 1
• HCDR2 CDR heavy 2
• HCDR3 CDR heavy 3
• the nucleic acid encodes an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 4, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 14, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 24.
• the nucleic acid encodes a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 33, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 37, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 46.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the nucleic acid encodes an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 5, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 15, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 25.
• the nucleic acid encodes a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 32, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 41, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 47.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the nucleic acid encodes an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 6, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 16, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 26.
• the nucleic acid encodes a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 32, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 42, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 48.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the nucleic acid encodes an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 7, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 17, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 27.
• the nucleic acid encodes the amino acid sequence of SEQ ID NO: 34, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 37, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 50.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the nucleic acid encodes an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 8, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 18, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 28.
• the nucleic acid encodes a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 35, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 43, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 51.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the nucleic acid encodes an HCDR1 that comprises the amino acid sequence of SEQ ID NO: 9, an HCDR2 that comprises the amino acid sequence of SEQ ID NO: 19, and an HCDR3 that comprises the amino acid sequence of SEQ ID NO: 29.
• the nucleic acid encodes a LCDR1 that comprises the amino acid sequence of SEQ ID NO: 36, a LCDR2 that comprises the amino acid sequence of SEQ ID NO: 44, and the LCDR3 that comprises the amino acid sequence of SEQ ID NO: 49.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the present invention also provides nucleic acids that encode the heavy chain variable region of a mammalian antibody (including a caninized antibody) or an antigen binding fragment thereof of the present invention.
• the present invention further provides nucleic acids that encode the heavy chain of a mammalian antibody (including a caninized antibody) or an antigen binding fragment thereof of the present invention.
• the present invention also provides nucleic acids that encode the light chain of a mammalian antibody (including a caninized antibody) or an antigen binding fragment thereof of the present invention.
• the present invention further provides as a pair, a nucleic acid encoding this set of the three heavy chain CDRs and a nucleic acid that encodes this set of the three light chain CDRs.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• a nucleic acid of the present invention encodes a heavy chain variable region of a caninized antibody or antigen binding fragment thereof in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 95.
• a nucleic acid encodes the light chain variable region of the caninized antibody or antigen binding fragment thereof in which the light chain variable region comprises the amino acid sequence of SEQ ID NO: 129.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 95 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain variable region that comprises the amino acid sequence of SEQ ID NO: 129.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain variable region of a caninized antibody or antigen binding fragment thereof in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 95.
• a nucleic acid encodes the light chain variable region of the caninized antibody or antigen binding fragment thereof in which the light chain variable region comprises the amino acid sequence of SEQ ID NO: 130.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 95 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain variable region that comprises the amino acid sequence of SEQ ID NO: 130.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain variable region of a caninized antibody or antigen binding fragment thereof in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 96.
• a nucleic acid encodes the light chain variable region of the caninized antibody or antigen binding fragment thereof in which the light chain variable region comprises the amino acid sequence of SEQ ID NO: 129.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 96 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain variable region that comprises the amino acid sequence of SEQ ID NO: 129.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain variable region of a caninized antibody or antigen binding fragment thereof in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 96.
• a nucleic acid encodes the light chain variable region of the caninized antibody or antigen binding fragment thereof in which the light chain variable region comprises the amino acid sequence of SEQ ID NO: 130.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 96 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain variable region that comprises the amino acid sequence of SEQ ID NO: 130.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain of a caninized antibody or antigen binding fragment thereof in which the heavy chain comprises the amino acid sequence of SEQ ID NO: 88.
• a nucleic acid encodes the light chain of the caninized antibody or antigen binding fragment thereof in which the light chain comprises the amino acid sequence of SEQ ID NO: 91.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain that comprises the amino acid sequence of SEQ ID NO: 88 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain that comprises the amino acid sequence of SEQ ID NO: 91.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain of a caninized antibody or antigen binding fragment thereof in which the heavy chain comprises the amino acid sequence of SEQ ID NO: 88.
• a nucleic acid encodes the light chain of the caninized antibody or antigen binding fragment thereof in which the light chain comprises the amino acid sequence of SEQ ID NO: 92.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain that comprises the amino acid sequence of SEQ ID NO: 88 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain that comprises the amino acid sequence of SEQ ID NO: 92.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain of a caninized antibody or antigen binding fragment thereof in which the heavy chain comprises the amino acid sequence of SEQ ID NO: 89.
• a nucleic acid encodes the light chain of the caninized antibody or antigen binding fragment thereof in which the light chain comprises the amino acid sequence of SEQ ID NO: 91.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain that comprises the amino acid sequence of SEQ ID NO: 89 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain that comprises the amino acid sequence of SEQ ID NO: 91.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid of the present invention encodes a heavy chain of a caninized antibody or antigen binding fragment thereof in which the heavy chain comprises the amino acid sequence of SEQ ID NO: 89.
• a nucleic acid encodes the light chain of the caninized antibody or antigen binding fragment thereof in which the light chain comprises the amino acid sequence of SEQ ID NO: 92.
• the present invention further provides a pair of nucleic acids, wherein one of the pair of nucleic acids comprises a nucleotide sequence that encodes the heavy chain that comprises the amino acid sequence of SEQ ID NO: 89 and the other of the pair of nucleic acids comprises a nucleotide sequence that encodes the light chain that comprises the amino acid sequence of SEQ ID NO: 92.
• the present invention also provides a kit containing this pair of two nucleic acids.
• the present invention also provides a kit containing this pair of two nucleic acids.
• a nucleic acid encoding the set of the three heavy chain CDRs encodes the heavy chain of a caninized antibody and the corresponding nucleic acid encoding the set of the three light chain CDRs encodes the light chain of that caninized antibody.
• the present invention provides expression vectors that comprise one or more of the nucleic acids of the present invention that express one or more of the nucleic acids of the present invention, and pairs of those expression vectors.
• one of the pair of expression vectors expresses a heavy chain of a caninized antibody of the present invention and the other expresses a light chain of that caninized antibody.
• Host cells that comprise the expression vectors of the present invention, including such pairs of such expression vectors are also provided.
• the present invention further provides pharmaceutical compositions that comprise the caninized antibodies and antigen binding fragments thereof of the present invention along with a pharmaceutically acceptable carrier and/or diluent.
• the present invention further provides pharmaceutical compositions that comprise a nucleic acid of the present invention, along with a pharmaceutically acceptable carrier and/or diluent, and/or an expression vector that comprise one or more of the nucleic acids of the present invention, along with a pharmaceutically acceptable carrier and/or diluent.
• the present invention also provides methods of treating atopic dermatitis comprising administering one of the aforesaid pharmaceutical compositions to an animal subject that has atopic dermatitis.
• the animal subject is a canine.
• the present invention also provides methods of aiding in blocking pruritus associated with atopic dermatitis in an animal subject, comprising administering to an animal subject in need thereof of a therapeutically effective amount of a pharmaceutical composition of the present invention.
• the animal subject is a canine.
• the present invention provides methods of producing a caninized antibody or antigen binding fragment thereof that binds canine IL-3 IRA.
• the method includes culturing a host cell comprising one or more expression vectors that encode and express the light chain of a caninized antibody of the present invention and the heavy chain of that caninized antibody in a culture medium under conditions in which the nucleic acid is expressed, thereby producing a polypeptide comprising the light chain of a caninized antibody of the present invention, and/or the heavy chain of that caninized antibody.
• the polypeptides are then recovered from the host cell or culture medium.
• the polypeptides comprising the light chain of a caninized antibody of the present invention and the polypeptides comprising the heavy chain of that caninized antibody are combined with each under conditions that are conducive to form a caninized antibody.
• Figure 1 shows the binding of IL-31 to IL-3 IRA.
• the extracellular domain (ECD) of canine IL-3 IRA was tested for its ability to bind to canine IL-31.
• the results indicate that IL- 3 IRA ECD binds in a dose-dependent manner to biotinylated canine IL-31 with an EC50 of 0.55 ng/ml.
• Figures 2A-2B show the binding of xIL-3 IRA monoclonal antibodies (mABS) to IL- 3 IRA.
• the selected mouse mAbs were tested for their reactivity to canine IL-3 IRA.
• the results indicate that the selected mouse mAbs bind to canine IL-3 IRA in a dose-dependent manner.
• All of the 10 mouse monoclonal antibodies have strong binding reactivity to canine IL-3 IRA.
• Figure 2A depicts mouse mAbs: 51F8 (•), 74H10 (®), 100H8(A), 209G5 ( ⁇ ), 224G3( ), and Iso control (o).
• Figure 2B depicts mouse mAbs: 55B3 (•), 65G9 (®), 85C10 (A), 218D9 ( ⁇ ), 227E7( ), and Iso control (o).
• Figure 3 shows the blocking of the binding of IL-31 to IL-3 IRA by monoclonal antibodies (mABS) to IL-3 IRA.
• the selected mouse mAbs (anti-canine IL-3 IRA) were tested for their ability to block the binding of IL-31 with IL-31RA/OSMR by Flow Cytometry.
• the FACS result indicates that the ten mouse mAbs can block the binding of IL-31 with the IL- 31RA/OSMR complex presented on CHO- IL-31RA/OSMR cells.
• Antibodies 51F8, 74H10, 100H8, 209G5, and 218D9 exhibited superior blocking activity.
• FIG. 4 shows the induction of STAT-3 phosphorylation by IL-31.
• Ba/f3-OI cells expressing the IL-31 receptor complex were tested for IL-31 -induced STAT-3 phosphorylation.
• the results indicate that STAT-3 phosphorylation was induced by IL-31 in the Baf3-OI cells (®) in a dose-dependent manner, implying that: (i) the canine IL-31 receptor complex is successfully expressed on the cell surface; (ii) that the binding of canine IL-31 to the IL-31 receptor can stimulate the endogenous STAT3 phosphorylation; and (iii) then initiate its downstream signaling pathway.
• Ba/f3 cells (o) were used as the control.
• Figures 5A-5B show the inhibition of IL-31 -mediated STAT-3 phosphorylation in Ba/f3- 01 cells by the selected xIL-3 IRA antibodies. The results indicate that the selected mAbs inhibit IL-31 mediated STAT-3 phosphorylation in a dose-dependent manner in Ba/f3-OI cells.
• Figure 5A depicts mouse mAbs 209G5 (®), 218D9 (A), 85C10 ( ⁇ ), and IL-31 protein ( ⁇ ).
• Figure 5B depicts mouse mAbs 100H8 (•), 74H10 (®), 85C10 (A), 51F8 ( ⁇ ), and cIL-31 protein ( ⁇ ).
• Figures 6A-6E provides the epitopes on canine IL-3 IRA for the antibodies 100H8, 51F8, 218D9, 85C10, and 224G3, respectively.
• Figure 6A depicts the epitope for 100H8; the epitope comprises the amino acid sequences of SEQ ID NO: 97 (within SEQ ID NO: 119) and SEQ ID NO: 103 (within SEQ ID NO: 120), respectively.
• Figure 6B depicts the epitope for 51F8; the epitope comprises comprises the amino acid sequences of SEQ ID NO: 98 (within SEQ ID NO: 121) and SEQ ID NO: 100 (within SEQ ID NO: 122).
• Figure 6C depicts the epitope for 218D9; the epitope comprises the amino acid sequences of SEQ ID NO: 104 (within SEQ ID NO: 123) and SEQ ID NO: 105 (within SEQ ID NO: 124), respectively.
• Figure 6D depicts the epitope for 85C10; the epitope comprises the amino acid sequences of SEQ ID NO: 106 (within SEQ ID NO: 125) and SEQ ID NO: 108 (within SEQ ID NO: 126), respectively.
• Figure 6E depicts the epitope for 224G3; the epitope comprises the amino acid sequence of SEQ ID NO: 109 (also within SEQ ID NO: 126). The position of binding residues of the amino acid sequence of SEQ ID NO: 2 for the respective epitopes on the cIL-31R ECD antigen are also denoted.
• Figures 7A-7E provides plots for the binding activity of the identified murine-canine chimeric or caninized antibodies to canine IL-3 IRA. The results indicate that the caninized antibodies have similar binding affinity as their corresponding parental antibodies (as represented by the murine-canine chimeric antibodies).
• Figure 7A depicts the binding plots for monoclonal 51F8 antibodies: Chimeric 51F8 (•) c51F8VH3VL6 (®), c51F8VH3VL7 (A), c51F8VH4VL6 ( ⁇ ), and c51F8VH4VL7 ( ⁇ ); and the iso control (o).
• Figure 7B depicts the binding plots for monoclonal 100H8 antibodies: Chimeric 100H8 (•), C100H8VH5VL4 (®), and C100H8VH7VL4 (A).
• Figure 7C depicts the binding plots for monoclonal 85C10 antibodies: Chimeric 85C10 (•), c85C10VH3VL2 (®), and C85C10VH1VL2(A).
• Figure 7D depicts the binding plots for monoclonal 218D9 antibodies: Chimeric 218D9 (•), c218D9VH3VL2 (®), c218D9VH3VL3 (A), c218D9VH4VL2 ( ⁇ ), and c218D9VH4VL3 ( ⁇ ); and the iso control (o).
• Figure 7E depicts the binding plots for monoclonal 224G3 antibodies: m224G3 Chim (•), c224G3VH2VL2 (®), and c224G3VH2VL3(A).
• the term “Chimeric” before the antibody number signifies that the antibody is a murinecanine chimeric antibody, e.g., Chimeric 218D9 or Chimeric 51F8.
• an “m” before the antibody number followed by a “Chim” signifies that the antibody is a murine-canine chimeric antibody, e.g., m224G3 Chim.
• the lower case “c” before the antibody number signifies that it is a caninized antibody, e.g., c218D9VH4VL2.
• Figure 8 shows the blocking of the binding of IL-31 to IL-3 IRA by the inhibition of the IL-31 -mediated STAT-3 phosphorylation in Ba/f3-OI cells.
• the results indicate that the caninized 218D9 antibodies can inhibit IL-31 mediated STAT-3 phosphorylation in a dosedependent manner in Ba/f3-OI cells, and that the constructs c218D9VH3VL3 and c218D9VH4VL3 have the same inhibitory activity as the parental mouse-canine chimeric 218D9 antibody: Chimeric 218D9 (•), c218D9VH3VL2 (®), c218D9VH3VL3 (A), c218D9VH4VL2 ( ⁇ ), and c218D9VH4VL3 ( ⁇ ); and the IL-31 only control (o).
• the present invention provides formulations and methodology that can achieve a significant effect on the skin inflammation associated with atopic dermatitis.
• FR Antibody framework region the immunoglobulin variable regions excluding the
• V region The segment of IgG chains which is variable in sequence between different antibodies.
• administering refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal e.g., a canine subject, cell, tissue, organ, or biological fluid.
• Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
• administering and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
• subject includes any organism, preferably an animal, more preferably a mammal (e.g., canine or feline) and most preferably a canine.
• Treating means to administer a therapeutic agent, such as a composition containing any of the antibodies of the present invention, internally or externally to e.g., a canine subject or patient having one or more symptoms, or being suspected of having a condition, for which the agent has therapeutic activity.
• the agent is administered in an amount effective to alleviate and/or ameliorate one or more disease/condition symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree.
• the amount of a therapeutic agent that is effective to alleviate any particular disease/condition symptom may vary according to factors such as the disease/condition state, age, and weight of the patient (e.g., canine), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease/condition symptom has been alleviated or ameliorated can be assessed by any clinical measurement typically used by veterinarians or other skilled healthcare providers to assess the severity or progression status of that symptom.
• an embodiment of the present invention may not be effective in alleviating the target disease/condition symptom(s) in every subject, it should alleviate the target disease/condition symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student’s t-test, the chi 2 -test, the U-test according to Mann and Whitney, the Kruskal -Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.
• any statistical test known in the art such as the Student’s t-test, the chi 2 -test, the U-test according to Mann and Whitney, the Kruskal -Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.
• Treatment refers to therapeutic treatment, as well as research and diagnostic applications.
• Treatment as it applies to a human, veterinary (e.g., canine), or research subject, or cell, tissue, or organ, encompasses contact of the antibodies of the present invention to e.g., a canine or other animal subject, a cell, tissue, physiological compartment, or physiological fluid.
• the term "canine” includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.
• the term “feline” refers to any member of the Felidae family. Members of this family include wild, zoo, and domestic members, including domestic cats, pure-bred and/or mongrel companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.
• canine frame refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues defined herein as CDR residues.
• CDR residues the amino acid sequences of the native canine CDRs are replaced with the corresponding foreign CDRs (e.g., those from a mouse or rat antibody) in both chains.
• the heavy and/or light chains of the canine antibody may contain some foreign non-CDR residues, e.g., so as to preserve the conformation of the foreign CDRs within the caninized antibody, and/or to modify the Fc function, as exemplified below and/or disclosed in U.S. 10,106,607 B2, hereby incorporated by reference herein in its entirety.
• Fc fragment crystallizable region
• Fc receptors cell surface receptors
• the canine fragment crystallizable region (cFc) of each of the four canine IgGs were first described by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001); see also, Bergeron et al., Vet. Immunol. Immunopathol. 157: 31-41 (2014) and U.S. 10,106,607 B2],
• canine Fc (cFc) “IgG-Bm” is canine IgG-B Fc comprising two (2) amino acid residue substitutions, D31 A and N63 A, as in the amino acid sequence of SEQ ID NO: 111 of IgG-B (see below) and without the c-terminal lysine (‘K”). Both the aspartic acid residue (D) at position 31 of SEQ ID NO: 110 and the asparagine residue (N) at position 63 of SEQ ID NO: 110, are substituted by an alanine residue (A) in IgG-Bm.
• amino acid sequence of IgG-Bm SEQ ID NO: 111, is provided below.
• substitution of an amino acid residue” with another amino acid residue in an amino acid sequence of an antibody for example is equivalent to “replacing an amino acid residue” with another amino acid residue and denotes that a particular amino acid residue at a specific position in the amino acid sequence has been replaced by (or substituted for) by a different amino acid residue.
• substitutions can be particularly designed /. ⁇ ., purposefully replacing an alanine with a serine at a specific position in the amino acid sequence by e.g., recombinant DNA technology.
• a particular amino acid residue or string of amino acid residues of an antibody can be replaced by one or more amino acid residues through more natural selection processes e.g., based on the ability of the antibody produced by a cell to bind to a given region on that antigen, e.g., one containing an epitope or a portion thereof, and/or for the antibody to comprise a particular CDR that retains the same canonical structure as the CDR it is replacing.
• substitutions/replacements can lead to “variant” CDRs and/or variant antibodies.
• antibody refers to any form of antibody that exhibits the desired biological activity.
• An antibody can be a monomer, dimer, or larger multimer. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies and camelized single domain antibodies.
• Parental antibodies are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as caninization of an antibody for use as a canine therapeutic antibody.
• antibodies of the present invention that "block” or is “blocking” or is “blocking the binding” of e.g., a canine receptor to its binding partner (ligand), is an antibody that blocks (partially or fully) the binding of the canine receptor to its canine ligand and vice versa, as determined in standard binding assays (e.g., BIACore®, ELISA, or flow cytometry).
• standard binding assays e.g., BIACore®, ELISA, or flow cytometry
• an antibody or antigen binding fragment of the invention retains at least 10% of its canine antigen binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis.
• an antibody or antigen binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine antigen binding affinity as the parental antibody.
• an antibody or antigen binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.
• isolated antibody refers to the purification status and in such context means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with experimental or therapeutic use of the binding compound as described herein.
• an antibody is said to bind specifically to a polypeptide comprising a given antigen sequence (in this case a portion of the amino acid sequence of canine IL-3 IRA) if it binds to polypeptides comprising the portion of the amino acid sequence of canine IL-3 IRA, but does not bind to other canine proteins lacking that portion of the sequence of canine IL-3 IRA.
• a polypeptide comprising canine IL-3 IRA may bind to a FLAG®-tagged form of canine IL-3 IRA, but will not bind to other FLAG®-tagged canine proteins.
• antibody fragment or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen (e.g., canine IL-3 IRA) bound by the full-length antibody, e.g. fragments that retain one or more CDR regions.
• antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
• An antibody, or binding compound derived from the antigen-binding site of an antibody binds to its canine antigen, or a variant or mutein thereof, “with specificity” when it has an affinity for that canine antigen or a variant or mutein thereof which is at least ten-times greater, more preferably at least 20-times greater, and even more preferably at least 100-times greater than its affinity for any other canine antigen tested.
• a "chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species.
• variable domains are obtained from an antibody from an experimental animal (the "parental antibody”), such as a rodent, and the constant domain sequences are obtained from the animal subject antibodies, e.g., human or canine so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human or canine subject respectively, than the parental (e.g., rodent) antibody.
• parental antibody an antibody from an experimental animal
• the constant domain sequences are obtained from the animal subject antibodies, e.g., human or canine so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human or canine subject respectively, than the parental (e.g., rodent) antibody.
• the term "caninized antibody” refers to forms of antibodies that contain sequences from both canine and non-canine (e.g., mouse) antibodies.
• the caninized antibody will comprise substantially all of at least one or more typically, two variable domains in which all or substantially all of the hypervariable loops correspond to those of a noncanine immunoglobulin (e.g., comprising 6 CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) are those of a canine immunoglobulin sequence.
• a caninized antibody comprises both the three heavy chain CDRs and the three light chain CDRS from a murine (mouse) anti-canine antigen antibody together with a canine frame or a modified canine frame.
• a modified canine frame comprises one or more amino acids changes as exemplified herein that further optimize the effectiveness of the caninized antibody, e.g., to increase its binding to its canine antigen and/or its ability to block the binding of that canine antigen to the canine antigen’s natural binding partner.
• variable regions of each light/heavy chain pair form the antibody binding site.
• an intact antibody has two binding sites.
• the two binding sites are, in general, the same.
• the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR).
• CDRs complementarity determining regions
• FR framework regions
• the CDRs are usually aligned by the framework regions, enabling binding to a specific epitope.
• both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
• hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen-binding.
• the hypervariable region comprises amino acid residues from a "complementarity determining region” or "CDR" (i.e. LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain).
• CDR complementarity determining region
• framework or "FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.
• IgG heavy chain subtypes of dog IgG There are four known IgG heavy chain subtypes of dog IgG and they are referred to as IgG- A, IgG-B, IgG-C, and IgG-D.
• the two known light chain subtypes are referred to as lambda and kappa.
• a canine or caninized antibody against its antigen of the present invention optimally has two attributes:
• ADCC antibody-dependent cytotoxicity
• CDC complement-dependent cytotoxicity
• IgG-B can be purified using protein A, but has high level of ADCC activity.
• IgG-A binds weakly to protein A, but also displays ADCC activity.
• neither IgG-C nor IgG-D can be purified on protein A columns, although IgG-D displays no ADCC activity. (IgG-C has considerable ADCC activity).
• an “antipruritic agent” is a compound, macromolecule, and/or formulation that tends to inhibit, relieve, and/or prevent itching. Antipruritic agents are colloquially referred to as anti -itch drugs.
• an “antipruritic antibody” is an antibody that can act as an antipruritic agent in an animal, including a mammal such as a human, a canine, and/or a feline, particularly with respect to atopic dermatitis.
• the antipruritic antibody binds to specific proteins in the IL-31 signaling pathway, such as IL-31 or its receptor IL-3 IRA.
• the binding of the antipruritic antibody to its corresponding antigen inhibits the binding of e.g., IL-31 with IL-3 IRA, and interferes with and/or prevents the successful signaling of this pathway, and thereby inhibits, relieves, and/or prevents the itching that is otherwise caused by the IL-31 signaling pathway.
• corresponding antigen e.g., IL-31 or IL-3 IRA
• Homology refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are optimally aligned.
• a position in both of the two compared sequences is occupied by the same base or amino acid residue, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
• the percent of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous when the sequences are optimally aligned then the two sequences are 60% homologous.
• the comparison is made when two sequences are aligned to give maximum percent homology.
• Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.
• one amino acid sequence is 100% "identical” to a second amino acid sequence when the amino acid residues of both sequences are identical.
• an amino acid sequence is 50% "identical” to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical.
• the sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In particular embodiments, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.
• Sequence similarity includes identical residues and nonidentical, biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable.
• Consatively modified variants or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity /hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein.
• Those of skill in this art recognize that, in general, single amino acid substitutions in non- essential regions of a polypeptide do not substantially alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.;
• “Function-conservative variants,” as used herein, refers to antibodies or fragments in which one or more amino acid residues have been changed without altering a desired property, such an antigen affinity and/or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table A above.
• isolated nucleic acid molecule means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.
• a nucleic acid molecule comprising a particular nucleotide sequence does not encompass intact chromosomes.
• Isolated nucleic acid molecules "comprising" specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.
• the present invention provides isolated caninized antibodies of the present invention, methods of use of the antibodies in the treatment of a condition e.g., the treatment of atopic dermatitis in canines.
• IgG heavy chains there are four IgG heavy chains referred to as A, B, C, and D. These heavy chains represent four different subclasses of dog IgG, which are referred to as IgG- A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC) and IgG-D (or IgGD).
• Each of the two heavy chains consists of one variable domain (VH) and three constant domains referred to as CH-1, CH-2, and CH-3.
• the CH-1 domain is connected to the CH-2 domain via an amino acid sequence referred to as the “hinge” or alternatively as the “hinge region”.
• nucleic acid and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. The amino acid and nucleic sequences for these heavy chains are also available from the GenBank data bases.
• the amino acid sequence of IgGA heavy chain has accession number AAL35301.1
• IgGB has accession number AAL35302.1
• IgGC has accession number AAL35303.1
• IgGD has accession number (AAL35304.1).
• Canine antibodies also contain two types of light chains, kappa and lambda.
• the DNA and amino acid sequence of these light chains can be obtained from GenBank Databases.
• the kappa light chain amino acid sequence has accession number ABY 57289.1 and the lambda light chain has accession number ABY 55569.1.
• the known amino acid sequences of the four unmodified canine IgGs are: clgG-A [SEQ ID NO: 116] LGGPSVLI FPPKPKDILRITRTPEVTCWLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGT YRWSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTV S ITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCA VMHETLQNHYTDLSLSHSPGK clgG-B [SEQ ID NO: 110]
• amino acid sequence of the kappa canine light chain constant region is: [SEQ ID NO: 127]
• Caninized mouse anti -canine antibodies that bind canine IL-3 IRA include, but are not limited to: antibodies of the present invention that comprise canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and/or canine kappa or lambda light chains together with mouse anticanine IL-3 IRA CDRs.
• the present invention provides caninized mouse anticanine antibodies of the present invention, including isolated caninized mouse anti-canine antibodies, that bind to canine IL-3 IRA and that preferably also block the binding of that canine IL-3 IRA to canine IL-31.
• the present invention further provides caninized mouse antibodies and methods of use of the antibodies of the present invention in the treatment of a condition e.g., the treatment of atopic dermatitis in canines.
• the present invention further provides full length caninized heavy chains that can be matched with corresponding light chains to make a caninized antibody. Accordingly, the present invention further provides caninized mouse anti-canine antigen antibodies (including isolated caninized mouse anti-canine antibodies) of the present invention and methods of use of the antibodies of the present invention in the treatment of a condition e.g., the treatment of atopic dermatitis in canines.
• the present invention also provides antibodies of the present invention that comprise a canine fragment crystallizable region (cFc region) in which the cFc has been genetically modified to augment, decrease, or eliminate one or more effector functions.
• the genetically modified cFc decreases or eliminates one or more effector functions.
• the genetically modified cFc augments one or more effector function.
• the genetically modified cFc region is a genetically modified canine IgGB Fc region.
• the genetically modified cFc region is a genetically modified canine IgGC Fc region.
• the effector function is antibody-dependent cytotoxicity (ADCC) that is augmented, decreased, or eliminated.
• ADCC antibody-dependent cytotoxicity
• the effector function is complement-dependent cytotoxicity (CDC) that is augmented, decreased, or eliminated.
• CDC complement-dependent cytotoxicity
• the cFc region has been genetically modified to augment, decrease, or eliminate both the ADCC and the CDC.
• mutant canine IgGB heavy chains were generated. These variants may include one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A.
• Variant heavy chains i.e., containing such amino acid substitutions
• Intact antibodies are expressed and purified from HEK 293 cells and then can be evaluated for binding to Fc Y RI and Clq to assess their potential for mediation of immune effector functions. [See, U.S. 10,106,607 B2, the contents of which are hereby incorporated by reference in its entirety.]
• the present invention also provides modified canine IgG-Ds which in place of its natural
• IgG-D hinge region they comprise a hinge region from:
• IgG-A FNECRCTDTPPCPVPEP SEQ ID NO: 112
• IgG-B PKRENGRVPRPPDCPKCPAPEM SEQ ID NO: 113; or
• IgG-C AKECECKCNCNNCPCPGCGL SEQ ID NO: 114.
• the IgG-D hinge region can be genetically modified by replacing a serine residue with a proline residue, i.e., PKESTCKCIPPCPVPES, SEQ ID NO: 115 (with the proline residue (P) underlined and in bold substituting for the naturally occurring serine residue).
• PKESTCKCIPPCPVPES a proline residue
• SEQ ID NO: 115 with the proline residue (P) underlined and in bold substituting for the naturally occurring serine residue.
• the modified canine IgG-Ds can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis el al. , Molecular Cloning, A Laboratory Manual (1982)].
• nucleic acids encoding the amino acid sequence of canine IgG-D can be modified so that it encodes the modified IgG-Ds.
• the modified nucleic acid sequences are then cloned into expression plasmids for protein expression.
• the six complementary determining regions (CDRs) of a caninized mouse anti-canine antibody can comprise a canine antibody kappa (k) or lambda (/) light chain comprising a mouse light chain LCDR1, LCDR2, and LCDR3 and a canine antibody heavy chain IgG comprising a mouse heavy chain HCDR1, HCDR2, and HCDR3.
• k canine antibody kappa
• / lambda
• IgG comprising a mouse heavy chain HCDR1, HCDR2, and HCDR3.
• the present invention further comprises the nucleic acids encoding the antibodies of the present invention (see e.g., Examples below).
• nucleic acids that encode immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of the caninized antibodies, with the exception of the CDRs which do not change, provided herein when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.
• the present invention further provides nucleic acids that encode immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when the comparison is performed with a BLAST algorithm, wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.
• nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program using the default parameters.
• GCG Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin
• BLAST ALGORITHMS Altschul, S.F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W ., et al., Nature Genet. 3:266-272 (1993); Madden, T.L., et al., Meth. EnzymoL 266: 131-141(1996); Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J.C., et al., Comput. Chem.
• the canine heavy chain constant region can be from IgGA, IgG-B, IgGC, IgGD, or a modified cFc, such as the IgG-Bm used herein [see, U.S. 10,106,607 B2, hereby incorporated by reference in its entirety] and the canine light chain constant region can be from kappa or lambda.
• the antibodies can be engineered to include modifications to the canine framework and/or the canine frame residues within the variable domains of a parental (i.e., mouse) monoclonal antibody, e.g. to improve the properties of the antibody.
• caninized anti-canine IL-31 receptor alpha monoclonal antibodies can be performed by determining a DNA sequence that encodes the heavy and light chains of canine IgG were determined.
• the DNA and protein sequence of the canine heavy and light chains are known in the art and can be obtained by searching of the NCBI gene and protein databases.
• IgG subtypes IgG-A, IgG-B, IgG-C, and IgG-D, and two types of light chains, i.e., kappa and lambda.
• a caninized mouse anti-canine IL-3 IRA antibody can be produced recombinantly by methods that are known in the field.
• Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines.
• ATCC American Type Culture Collection
• Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
• insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
• Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
• the antibody or antigen binding fragment comprises a heavy chain constant region, e.g., a canine constant region, such as IgG-A, IgG-B, IgG-C and IgG-D canine heavy chain constant region or a variant thereof.
• the antibody or antigen binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, such as lambda or kappa canine light chain region or variant thereof.
• the canine heavy chain constant region can be from IgG-B and the canine light chain constant region can be from kappa.
• antibodies with their cognate protein antigens is mediated through the binding of specific amino acids of the antibodies (paratopes) with specific amino acids (epitopes) of target antigens.
• An epitope is an antigenic determinant that causes a specific reaction by an immunoglobulin.
• An epitope consists of a group of amino acids on the surface of the antigen.
• a protein of interest may contain several epitopes that are recognized by different antibodies. The epitopes recognized by antibodies are classified as linear or conformational epitopes.
• Linear epitopes are formed by a stretch of a continuous sequence of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence, but are brought together upon three-dimensional protein folding.
• Epitope mapping refers to the process of identifying the amino acid sequences (i.e., epitopes) that are recognized by antibodies on their target antigens. Identification of epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it can aid in the development of new therapeutics, diagnostics, and vaccines. Epitope mapping can also aid in the selection of optimized therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on IL-31 receptor alpha can also elucidate unique epitopes and define the protective or pathogenic effects of vaccines. Epitope identification also can lead to development of subunit vaccines based on chemical or genetic coupling of the identified peptide epitope to a carrier protein or other immunostimulating agents.
• Epitope mapping can be carried out using polyclonal or monoclonal antibodies and several methods are employed for epitope identification depending on the suspected nature of the epitope (i.e., linear versus conformational). Mapping linear epitopes is more straightforward and relatively, easier to perform. For this purpose, commercial services for linear epitope mapping often employ peptide scanning. In this case, an overlapping set of short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind antibodies of interest. The strategy is rapid, high-throughput, and relatively inexpensive to perform.
• mapping of a discontinuous epitope is more technically challenging and requires more specialized techniques such as x-ray co-crystallography of a monoclonal antibody together with its target protein, Hydrogen-Deuterium (H/D) exchange, Mass Spectrometry coupled with enzymatic digestion as well as several other methods known to those skilled in the art.
• H/D Hydrogen-Deuterium
• An anti-canine IL-3 IRA antibody or antigen-binding fragment thereof of the present invention includes any antibody or antigen-binding fragment thereof that binds to the same epitope in canine IL-3 IRA as the one of the antibodies, disclosed herein, bind, e.g., such as the 218D9 antibody which binds to the epitope comprising the amino acid sequence either SEQ ID NO: 104, SEQ ID NO: 105, or both SEQ ID NO: 104 and SEQ ID NO: 105, or the 51F8 antibody which binds to the epitope comprising the amino acid sequence either SEQ ID NO: 98, SEQ ID NO: 100, or both SEQ ID NO: 98 and SEQ ID NO: 100, including caninized antibodies, and any antibody or antigen-binding fragment that cross-blocks (partially or fully) or is crossblocked (partially or fully) by an antibody or fragment discussed herein for canine IL-3 IRA binding; as well as any variant thereof.
• the cross-blocking antibodies and antigen-binding fragments can be identified based on their ability to cross-compete with e.g., the 218D9 or 51F8 antibody in standard binding assays (e.g., BIACore®, ELISA, as exemplified below, or flow cytometry).
• standard ELISA assays can be used in which a recombinant canine IL-3 IRA protein is immobilized on the plate, one of the antibodies is fluorescently labeled and the ability of non-labeled antibodies to compete off the binding of the labeled antibody is evaluated.
• BIAcore® analysis can be used to assess the ability of the antibodies to cross-compete.
• test antibody to inhibit the binding of the e.g., 51F8 or 218D9 antibody, to canine IL-3 IRA demonstrates that the test antibody can compete with the 51F8 or 218D9 antibody for binding to canine IL-3 IRA and thus, may, in some cases, bind to the same epitope on canine IL-3 IRA as the 51F8 and/or 218D9 antibody binds.
• Antibodies and fragments thereof that bind to the same epitope as any of the anti -canine IL-3 IRA antibodies or fragments of the present invention also form part of the present invention.
• compositions comprising the antibodies of the present invention
• these antibodies can be admixed with a pharmaceutically acceptable carrier or excipient.
• a pharmaceutically acceptable carrier or excipient See, e.g., Remington ’s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].
• Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions [see, e.g., Hardman, el al. (2001) Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, el al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al.
• the antibodies of the present invention are diluted to an appropriate concentration in a sodium acetate solution pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.
• Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index (LD50/ ED50).
• antibodies exhibiting high therapeutic indices are desirable.
• the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in canines.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration.
• the mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial.
• the antibodies of the present invention can be administered by an invasive route such as by injection.
• the antibodies of the present invention, or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery.
• Administration by non-invasive routes e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.
• compositions can be administered with medical devices known in the art.
• a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.
• the pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos.: 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.
• compositions disclosed herein may also be administered by infusion.
• implants and modules form administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
• the administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibodies, the level of symptoms, the immunogenicity of the therapeutic antibodies and the accessibility of the target cells in the biological matrix.
• the administration regimen delivers sufficient therapeutic antibodies to effect improvement in the target disease/condition state, while simultaneously minimizing undesired side effects.
• the amount of biologic delivered depends in part on the particular therapeutic antibodies and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available [see, e.g., W awrzynczak Antibody Therapy, Bios Scientific Pub.
• Determination of the appropriate dose is made by the veterinarian, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of the symptoms.
• Antibodies provided herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc.
• Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation.
• a total weekly dose is generally at least 0.05 pg/kg body weight, more generally at least 0.2 pg/kg, 0.5 pg/kg, 1 pg/kg, 10 pg/kg, 100 pg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more [see, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346: 1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych.
• Doses may also be provided to achieve a pre-determined target concentration of antibodies of the present invention in the canine’s serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 pg/ml or more.
• antibodies of the present invention is administered subcutaneously or intravenously, on a weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.
• inhibit or “treat” or “treatment” includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder.
• the terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.
• a beneficial result has been conferred on a vertebrate subject (e.g., a canine) with a disorder, condition and/or symptom, or with the potential to develop such a disorder, disease or symptom.
• the terms “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to an amount of antibodies of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, e.g., canine, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition.
• a therapeutically effective dose further refers to that amount of the antibodies sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
• a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.
• An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%.
• An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess severity of the condition.
• the nucleotide sequence of SEQ ID NO: 1 encodes the extracellular domain of the canine IL-31 receptor alpha (cIL-3 IRA) fused to a HIS tag.
• Canine IL-3 IRA ECD HIS-tagged protein comprises the amino acid sequence of SEQ ID NO: 2.
• the nucleotide sequence was prepared by chemical synthesis and then cloned into expression plasmids that are suitable for production of the corresponding proteins in eukaryotic cells, either HEK-293 or CHO cells.
• Canine IL-3 IRA ECD-lOHis [SEQ ID NO: 1] gtgctgcccgccaagcccgagaacatcagctgcatcttctactacgaggagaacttcacctgcacctggag cccgagaaggaggccagctacacctggtacaaggtgaagagaacctacagctacggctacaagagcgaca tctgcagcaccgacaacagcaccagaggcaaccacgccagctgcagcttcctgcccccaccatcaccaac ccgacaactacaccatccaggtggaggcccagaacgccgacggcatcatgaagagcgacatcacctactg gaacctggacgccatcatgaagatcgagccccccgagatcttcagcgtg
• Plasmids comprising the nucleotide sequence of SEQ ID NO: 1 were transfected into HEK-293 or CHO cells using electroporation via the MaxCyte instrument as per the manufacturer’s recommendation. Several days following transfection, the supernatants of transfected cells and un-transfected controls were harvested and spun down to remove cellular debris. IL-3 IRA with the HIS tag was purified from cell culture fluids by passing the clarified harvested fluid from transfected cells over nickel columns as per the manufacturer’s recommendation. Purified proteins were quantified by measuring their absorbance of ultraviolet light at 280 nm.
• HRP-Streptavidin Dilute horse raddish peroxidase-Streptavidin (HRP-Streptavidin) to a final dilution of 1 : 1000 in 1% NFDM in PBST.
• TMP 3,3',5,5'-tetramethylbenzidine
• the extracellular domain (ECD) of canine IL-3 IRA was tested for its ability to bind to canine IL-31 (see, Figure 1). The results indicate that IL-3 IRA ECD binds in a dose-dependent manner to biotinylated canine IL-31 with an EC50 of 0.55 ng/ml.
• MONOCLONAL ANTIBODIES AGAINST CANINE IL-31 RECEPTOR alpha Monoclonal antibodies (mAbs) against canine IL-3 IRA were produced by the immunization of mice multiple times with canine IL-3 IRA ECD. Mice were immunized via the Intraperitoneal route with IL-3 IRA ECD in GS proprietary adjuvant 3 times on days 0, 14, and 28 using 50 pg per mouse for first immunization and 25 pg per mouse for the subsequent boosts. On day 48 mice were immunized once more with 25 pg of antigen and 4 days later their spleen cells were fused with the myeloma SP2/0 cell line to produce hybridomas secreting antibodies.
• mice were collected from mice and tested against canine IL-3 IRA by ELISA.
• the spleen cells from the mouse with highest IL-3 IRA ECD reactivity were fused with the myeloma SP2/0 cell line to produce hybridomas.
• supernatants from growing hybridomas were screened by flow cytometry using cells expressing the IL-3 IRA protein.
• the reactivities of hybridoma were confirmed by ELISA as follows: Procedure for the ELISA :
• the selected mouse mAbs were tested for their reactivity to canine IL-3 IRA.
• the results indicate that the selected mouse mAbs bind to canine IL-3 IRA in a dose-dependent manner.
• Ten (10) mouse monoclonal antibodies were obtained that have strong binding reactivity to canine IL-3 IRA, as shown in Figures 2A-2B.
• amino acid sequences of the heavy and light chain variable regions of these ten mouse monoclonal antibody are provided below.
• DIKMTQSPSS I FASLGERVTITCKASQDINSYLNWFQQKPGKSPKTLIYRADRLVDGVPSRFSGS GSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELN
• KSRLTISKDTSKNQVFLKIANVDTADTATYYCARIAGGLRRAPYAMDSWGQGTSVTVSS 218D9VL [SEQ ID NO: 69] DIQMTQSPASLSVSVGETVTITCRASENIYSSLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGS GSGTQYSLKINSLQSEDSGNYYCQHFRDTPPTFGGGTKLEIK
• Table 2 below, provides the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD), as analyzed by Octet Kinetics (see also, Example 9 below). These constants reflect the binding affinity of the individual monoclonal antibodies for canine IL-31 RA.
• the results show that the selected mAbs have low nanomolar to sub-picomolar binding affinities ranging from about 0.85 nM to about 1 pM.
• HCDRl all of the HCDRl’s in this group of four antibodies have the identical amino acid sequence (SEQ ID NO: 3). Whereas the four HCDR2’s differ, they do not differ much.
• the HCDR2 of 100H8 differs from the three other HCDR2’s by having an isoleucine residue at the ninth position rather than a threonine residue.
• 74H10 further differs from 100H8 by possessing an aspartic acid residue at its C-terminus rather than a glycine residue.
• the other three HCDR2’s have a glycine residue at their C-terminus.
• both HCDR’s have three additional amino acid residues (FDV) at their C-terminus.
• FDV amino acid residues
• Both 74H10 and 55B3 have a leucine at their fourth position, rather than a valine residue like 100H8, whereas 222E7 has a glutamine residue.
• both 100H8 and 74H10 have a proline residue at the third position
• both 222E7 and 55B3 have a glutamine residue at the third position.
• LCDRl Three of the four LCDRl ’s in this group of four antibodies have the identical amino acid sequence (SEQ ID NO: 30), but whereas the LCDR1 of 55B3 shares their first four amino acid residues, its LCDR1 differs from the other three LCDRls in its remaining seven amino acid residues.
• the LCDR2 of 100H8 and 74H10 have identical amino acid sequences (SEQ ID NO: 40), but the LCDR2 of 55B3 and 222E7 both differ from the other two LCDR2s by having an asparagine residue at position three instead of the aspartic acid residue of 100H8 and 74H10.
• the LCDR2 of 222E7 has a valine residue at position two rather than an alanine residue found in the LCDR2 of the three other antibodies.
• all four LCDR3’s in this group of four antibodies have the identical amino acid sequence (SEQ ID NO: 45).
• the remaining six (6) antibodies to canine IL-3 IRA detailed above, i.e., 65G9, 85C10, 224G3, 51F8, 209G5, and 218D9, can be further broken down into two pairs of antibodies that have noticeable identity in their respective CDR amino acid sequences, and two antibodies that are relative outliers. Accordingly, there is appreciable amino acid sequence identity between the sets of 6CDRs of antibody 65G9 with that of antibody 85C10 (see, Table 3).
• antibodies 65G9 and 85C10 both bind two linear sequences in the C-terminal region of IL-31RA- ECD, one of which is the same (SEQ ID NO: 106; see, Table 6 and Figure 6D) and the other one has substantial overlap (compare SEQ ID NO: 107 with SEQ ID NO: 108; see, Table 6).
• one of the outliers, antibody 244G3 binds to an epitope comprising a single linear sequence in the C-terminal region of IL-31RA-ECD (see, Figure 6E), which is contained with the second linear sequence of antibody 85C10 (compare SEQ ID NO: 109 with SEQ ID NO: 108; see, Table 6).
• the second group contains antibodies 51F8 and 209G5, which also have appreciable amino acid sequence identity between their respective sets of 6CDRs (see, Table 3). Consistently, antibodies 51F8 and 209G5 both bind two linear sequences in the N-terminal region of IL-31 RA-ECD, one of which is the same (SEQ ID NO: 98; see, Table 6 and Figure 6B), whereas the second linear sequence of the IL-31RA-ECD that antibody 209G5 binds (SEQ ID NO: 101) is within the amino sequence of the second linear sequence of IL-31RA-ECD that antibody 51F8 binds (SEQ ID NO: 100; see, Table 6).
• the other outlier, antibody 218D9 binds to two linear amino acid sequences located in the middle portion of the amino acid sequence of IL-31RA-ECD (SEQ ID NOs: 104 and 105; see, Figure 6C and Table 6). This antibody proved to be both a strong binder of IL-3 IRA and a good blocker of the binding of IL-3 IRA with IL-31.
• nucleotide sequences of the canine IL-3 IRA with c-terminal Flag tag and OSMR with c-terminal HA tag were prepared by chemical synthesis and then cloned into lentivirus vector Lenti-puro and Lenti-Hygro, respectively.
• the lentivirus Lenti- puro-IL3 IRA-Flag and Lenti-Hygro-OSMR-HA prepared from the Lenti-X 293T cells were co-transfected into CHO-kl cells.
• the CHO stable cell pool co-expressing canine IL-3 IRA and OSMR was selected by FACS with anti-flag and anti-HA antibodies. Single cell clones were isolated from the stable pool.
• the developed CHO-IL-31RA/OSMR table cell line is applied to screen anti-canine IL-3 IRA monoclonal antibodies for blocking of IL-31 with its receptor complex IL-31RA/OSMR.
• Cell line CHO-IL-31RA/OSMR stable cell line
• Cell growth medium F-12K Medium with 10% FBS, 8pg/ml Puromycin and 200pg/ml hygromycin
• CHO-IL-31RA/OSMR cells were grown in the growth medium in T75 flask.
• the FACS result indicate that nine of the ten mouse anti -canine IL-3 IRA mAbs can significantly block the binding of IL-31 with the IL-31RA/OSMR complex presented on the CHO-IL-31RA/OSMR cells.
• the lead antibodies are 51F8, 74H10, 100H8, 209G5 and 218D9.
• Stat-3 is known to be activated by IL-31 in cells comprising the the heterodimeric receptor for IL-31.
• the nucleotide sequences encoding IL-3 IRA and OSMR, respectively were prepared by chemical synthesis and then cloned into expression vectors pcDNA3.1.
• the vectors containing the IL-3 IRA and OSMR nucleotide sequences, respectively, were co-transfected into Ba/f3 cells and the transfected cells, denoted as “Ba/f3-OI”, were grown as a pool under antibiotic selection.
• the ability of canine IL-31 to induce STAT-3 activation was tested as follows.
• Starvation medium the growth medium without mIL-3 and cIL-31 P-STAT3 (Tyr705) Assay Kit: PerkinElmer, ALSU-PST3-A-HV
• Figure 4 shows the induction of STAT-3 phosphorylation by canine IL-31, which stimulates activation of STAT-3 in Ba/f3-OI cells in a dose dependent manner.
• Ba/f3 cells were used as the control.
• Ba/f3-OI cells expressing the IL-31 receptor complex were tested for IL-31- induced STAT-3 phosphorylation.
• the IC50 for the various 218D9 antibody constructs was calculated to be approximately: 2.2 nM for the chimeric antibody; 230 nM for C218D9VH3VL2; 8.3 nM for c218D9VH4VL2; 2.9 nM for c218D9VH4VL3; and 2.5 nM for C218D9VH3VL3.
• Binding affinity measurement results indicate that all the tested monoclonal antibodies have low nanomolar to sub-picomolar binding affinities ranging from about 1.5 nM to about 1 pM (see, Table 2 above).
• the DNA and protein sequence of the canine heavy and light chains are known and can be obtained by searching of the NCBI gene and protein databases.
• canine antibodies there are four known IgG subtypes: IgG-A, IgG-B, IgG-C, and IgG-D, and two types of light chains, i.e., kappa and lambda.
• the process of producing caninized heavy and light chains that can be mixed in different combinations to produce caninized anti-canine IL-31 receptor alpha mAbs involves the following scheme: i) Identify the DNA sequence of VH and VL domains comprising the CDRs of desired anti- IL-31 receptor alpha mAbs ii) Identify the H and L chain CDRs of desired anti-IL-3 IRA mAbs iii) Identify a suitable sequence for H and L chain of canine IgG iv) Identify the DNA sequence encoding the endogenous CDRs of canine IgG H and L chains of the above sequence.
• step (v) Replace the DNA sequence encoding endogenous canine H and L chain CDRs with DNA sequences encoding the desired anti-IL-3 IRA CDRs. In addition, optionally replace some canine framework residues with selected residues from the desired anti -IL-31 receptor alpha mAb framework regions.
• step (v) Synthesize the DNA from step (v), clone it into a suitable expression plasmid, and transfect the plasmids containing desired caninized H and L chains into HEK 293 cells.
• vii) Purify expressed caninized antibody from HEK 293 supernatant.
• the caninized antibodies were tested for reactivity with canine IL-3 IRA as follows:
• TMB 3,3 ',5,5'-tetramethylbenzidine
• the caninized antibodies were tested for their reactivity to canine IL-3 IRA.
• the results indicate that the caninized antibodies have similar binding affinity as their corresponding parental antibodies (represented by their chimeric antibodies).
• Table 5A provides the relative binding affinities of the different caninized antibodies (EC50), relative to their corresponding mouse-canine chimera antibody (see, Figures 7A-7E). Although these are just relative numbers, caninized antibody 218D9 stands out as an antibody that has essentially the same binding affinity as its parental chimeric murine antibody.
• the term “Chimeric” before the antibody number signifies that the antibody is a murinecanine chimeric antibody, e.g., Chimeric 218D9 or Chimeric 51F8.
• an “m” before the antibody number followed by a “Chim” signifies that the antibody is a murine-canine chimeric antibody, e.g., m224G3 Chim.
• the lower case “c” before the antibody number signifies that it is a caninized antibody, e.g., c218D9VH4VL2.
• Table 5B shows the binding constants of the caninized antibodies of 51F8 and 218D9. The results again indicate that caninized 218D9 antibodies have essentially the same binding affinity as their parental chimeric murine antibody, whereas caninized 51F8 antibodies have slightly weaker binding affinity than their parental chimeric murine antibody.
• Antibodies 100H8, 51F8, 209G5, and 55B3 share epitopes towards the N-terminus, while antibodies 65G9, 85C10 and 224G3 share epitopes towards the C-terminus of the IL-31RA-ECD.
• Antibody 218D9 has two unique epitopes located at middle portion of IL-31RA-ECD.
• Lys Thr Phe Gin Cys lie Glu Ala Met Gin Ala Cys Leu Thr Gin Asp

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