WO2020086886A1 - Il4/il13 receptor molecule for veterinary use - Google Patents

Il4/il13 receptor molecule for veterinary use Download PDF

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Publication number
WO2020086886A1
WO2020086886A1 PCT/US2019/057922 US2019057922W WO2020086886A1 WO 2020086886 A1 WO2020086886 A1 WO 2020086886A1 US 2019057922 W US2019057922 W US 2019057922W WO 2020086886 A1 WO2020086886 A1 WO 2020086886A1
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Prior art keywords
seq
polypeptide
heterodimeric protein
amino acid
il13r
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PCT/US2019/057922
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French (fr)
Inventor
Hangjun Zhan
Lam Nguyen
Fawn Qian
Shyr Jiann Li
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Kindred Biosciences, Inc.
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Publication date
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Priority to EP19875949.0A priority Critical patent/EP3870601A4/en
Priority to US17/287,756 priority patent/US20210395340A1/en
Publication of WO2020086886A1 publication Critical patent/WO2020086886A1/en

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    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5406—IL-4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5437—IL-13

Definitions

  • This present disclosure relates to heterodimeric proteins comprising interleukin 4 receptor and interleukin 13 receptor fragments derived from companion animal species that bind to IL4 and/or IL13 of a companion animal species, for example, canine IL4 and canine IL13.
  • the present disclosure also relates to methods of using the heterodimeric proteins, for example, for treating IL4 and/or ILl3-induced conditions or reducing IL4 and/or IL13 signaling activity in cells, for instance in companion animals, such as canines, felines, and equines.
  • Sequence Listing is provided as a file entitled “20l9-l0-24_0l l57-0027-00PCT_ST25.txt” created on October 25, 2019, which is 334 kilobytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
  • Interleukin 4 is a cytokine promoting differentiation of naive helper T cells to Th2 cells.
  • Interleukin 13 has similar effects on immune cells. Both IL4 and IL13 play important roles in T cell-mediated immune responses that are directly associated with allergy, for example, atopic dermatitis and asthma. It is generally understood that IL4 can form a signaling complex either with heterodimeric receptors IL4 receptor subunit alpha (IL4R) and yc or IL4R and IL13 receptor subunit alpha-l (IL13R). IL13 can form a signaling complex with heterodimeric receptors IL4Ra and ILl3Ral . Extracellular domains of IL4Ra or ILl3Ral may bind to IL4 and/or IL13 and reduce the free concentrations of the cytokines, thus diminishing the clinical signs and symptoms associated with dermatitis, asthma and other disorders.
  • Companion species animals such as cats, dogs, and horses, suffer from many allergic diseases similar to human allergic diseases, including atopic dermatitis and asthma.
  • methods and compounds that can be used specifically to bind companion animal IL4 and/or IL13 for treating IL4/IL 13 -induced conditions and for reducing IL4/IL13 signaling activity.
  • a heterodimeric protein comprising:
  • IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
  • Embodiment 2 The heterodimeric protein of embodiment 1, wherein the first contiguous polypeptide and/or the second contiguous polypeptide comprises one, two, three, or four IL4R ECDs and/or one, two, three, or four IL13R ECDs.
  • Embodiment 3 The heterodimeric protein of any one of the preceding
  • first contiguous polypeptide and/or the second contiguous polypeptide further comprises at least one binding partner other than IL4R ECD or IL13R ECD.
  • Embodiment 4 The heterodimeric protein of embodiment 3, wherein the at least one binding partner comprises IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
  • Embodiment 5 The heterodimeric protein of any one of the preceding
  • the heterodimeric protein binds to IL13 and/or IL4 with a dissociation constant (Kd) of less than 5 x 10 6 M, less than 1 x 10 6 M, less than 5 x 10 7 M, less than 1 x 10 7 M, less than 5 x 10 8 M, less than 1 x 10 8 M, less than 5 x 10 9 M, less than 1 x 10 9 M, less than 5 x 10 10 M, less than 1 x 10 10 M, less than 5 x 10 11 M, less than 1 x 10 11 M, less than 5 x 10 12 M, or less than 1 x 10 12 M, as measured by biolayer interferometry.
  • Kd dissociation constant
  • Embodiment 6 The heterodimeric protein of any one of the preceding
  • heterodimeric protein reduces IL13 and/or IL4 signaling in a companion animal species.
  • Embodiment 7 The heterodimeric protein of any one of the preceding
  • companion animal species is canine, feline, or equine.
  • Embodiment 8 The heterodimeric protein of any one of the preceding
  • amino acid sequence of the at least one IL13R ECD is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
  • Embodiment 9 The heterodimeric protein of any one of the preceding
  • amino acid sequence of the at least one IL13R ECD comprises a cysteine at a position corresponding to position 18 of SEQ ID NO: 22, corresponding to position 18 of SEQ ID NO: 24, or corresponding to position 18 of SEQ ID NO: 26.
  • Embodiment 10 The heterodimeric protein of any one of the preceding
  • amino acid sequence of the at least one IL13R ECD comprises a cysteine at position 18 of SEQ ID NO: 22, at position 18 of SEQ ID NO: 24, at position 18 of SEQ ID NO: 26, at position 15 of SEQ ID NO: 32, at position 15 of SEQ ID NO: 34, or at position 15 of SEQ ID NO: 36.
  • Embodiment 11 The heterodimeric protein of any one of the preceding
  • the at least one IL13R ECD comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36.
  • Embodiment 12 The heterodimeric protein of any one of the preceding
  • amino acid sequence of the at least one IL4R ECD is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • Embodiment 13 The heterodimeric protein of any one of the preceding
  • the at least one IL4R ECD comprises an amino acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37.
  • Embodiment 14 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide and/or the second Fc polypeptide is:
  • Embodiment 15 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises a knob mutation.
  • Embodiment 16 The heterodimeric protein of any one of the preceding embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises a hole mutation.
  • Embodiment 17 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 18 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 19 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 20 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 21 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 22 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 24 The heterodimeric protein of any one of the preceding
  • first Fc polypeptide or the second Fc polypeptide comprises:
  • Embodiment 25 The heterodimeric protein of any one of the preceding
  • the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83
  • Embodiment 26 The heterodimeric protein of any one of the preceding
  • the first contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113.
  • Embodiment 27 The heterodimeric protein of any one of the preceding
  • the second contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
  • Embodiment 28 An isolated nucleic acid encoding: a) the first contiguous polypeptide of any one of embodiments 1 to 27; b) the second contiguous polypeptide of any one of embodiments 1 to 27; or c) the first contiguous polypeptide and the second contiguous polypeptide of any one of embodiments 1 to 27.
  • Embodiment 29 A host cell comprising the nucleic acid of embodiment 28.
  • Embodiment 30 A host cell expressing: a) the first contiguous polypeptide of any one of embodiments 1 to 27; b) the second contiguous polypeptide of any one of embodiments 1 to 27; or c) the first contiguous polypeptide and the second contiguous polypeptide of any one of embodiments 1 to 27.
  • Embodiment 31 A method comprising culturing the host cell of embodiment 29 or embodiment 30 and isolating the first contiguous polypeptide, or the second contiguous polypeptide, or the first contiguous polypeptide and the second contiguous polypeptide.
  • Embodiment 32 A pharmaceutical composition comprising the heterodimeric protein of any one of embodiments 1 to 27 and a pharmaceutically acceptable carrier.
  • Embodiment 33 A method of treating a companion animal species having an IL13 and/or IL4-induced condition, the method comprising administering to the companion animal species a therapeutically effective amount of the heterodimeric protein of any one of
  • Embodiment 34 The method of embodiment 33, wherein the companion animal species is canine, feline, or equine.
  • Embodiment 35 The method of embodiment 33 or embodiment 34, wherein the
  • IL13 and /or IL4-induced condition is a pruritic or allergic condition, such as atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema.
  • Embodiment 36 The method of any one of embodiments 33 to 35, wherein the heterodimeric protein or the pharmaceutical composition is administered parenterally.
  • Embodiment 37 The method of any one of embodiments 33 to 36, wherein the heterodimeric protein or the pharmaceutical composition is administered by an intramuscular route, an intraperitoneal route, an intracerebrospinal route, a subcutaneous route, an intra-arterial route, an intrasynovial route, an intrathecal route, or an inhalation route.
  • Embodiment 38 The method of any one of embodiments 33 to 37, wherein the method further comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor.
  • Embodiment 39 The method of any one of embodiments 33 to 38, wherein the method further comprises administering one or more antibodies selected from an anti-ILl7 antibody, an anti-IL3 l antibody, an anti-TNFa antibody, an anti-CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-ILl3 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CDl la antibody, anti-IL6R antibody, anti-a4-Intergrin antibody, an anti-ILl2 antibody, an anti-IL 1 b antibody, and an anti-BlyS antibody.
  • an anti-ILl7 antibody an anti-IL3 l antibody, an anti-TNFa antibody, an anti-CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-ILl3 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CDl la antibody, anti-IL6R antibody, anti-a4-
  • Embodiment 40 A method of reducing IL13 and /or IL4 signaling activity in a cell, the method comprising exposing the cell to the heterodimeric protein of any one of
  • embodiments 1 to 27 or the pharmaceutical composition of embodiment 32 under conditions permissive for binding of the heterodimeric protein to IL13 and/or IL4, thereby (a) reducing binding of IL/4 and/or IL-13 to native IL13 receptor and/or native IL-4 receptor and reducing IL13- and/or IL-4-mediated signaling.
  • Embodiment 41 The method of embodiment 40, wherein the cell is exposed to the heterodimeric protein or the pharmaceutical composition ex vivo.
  • Embodiment 42 The method of embodiment 41, wherein the cell is exposed to the heterodimeric protein or the pharmaceutical composition in vivo.
  • Embodiment 43 The method of any one of embodiments 40 to 42, wherein the cell is a canine cell, a feline cell, or an equine cell.
  • Embodiment 44 A method for detecting IL13 or IL4 in a sample from a companion animal species comprising contacting the sample with the heterodimeric protein of any one of embodiments 1 to 27 or the pharmaceutical composition of embodiment 32 under conditions permissive for binding of the heterodimeric protein to IL13 and/or IL4, and detecting whether a complex is formed between the heterodimeric protein and IL13 and/or IL4 in the sample.
  • Embodiment 45 The method of embodiment 44, wherein the sample is a biological sample obtained from a canine, a feline, or an equine.
  • FIG. 1 is a graph of canine IL4RECD-ILl3RECD-Fc sequential binding to canine
  • IL4 and IL13 or canine IL13 and IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS.
  • FIG. 2 is a graph of canine ILl3RECD-IL4RECD-Fc sequential binding to canine IL4 and IL13 or canine IL13 and IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS.
  • FIG. 3 is a graph of canine IL4RECD-ILl3RECD-Fc neutralizing canine IL4 activity in a TF1 cell proliferation assay.
  • Canine IL4 50 ng/mL or 3.85 nM was used in the assay.
  • Table 1 provides a listing of certain sequences referenced herein.
  • IL13R/IL4R heterodimeric proteins that bind canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4 are provided.
  • the IL13R/IL4R heterodimeric protein comprises a first contiguous polypeptide comprising an extracellular domain of an IL13R polypeptide and an Fc polypeptide and a second contiguous polypeptide comprising an extracellular domain of an IL4R polypeptide and an Fc polypeptide.
  • Methods of producing or purifying IL13R/IL4R heterodimeric proteins and contiguous polypeptides are also provided.
  • Methods of treatment using IL13R/IL4R heterodimeric proteins to bind IL13 and/or IL4 and inhibit IL13- and/or IL-4-mediated signaling are provided. Such methods include, but are not limited to, methods of treating IL13- and/or IL4-induced conditions in companion animal species. Methods of detecting IL13 and/or IL4 in a sample from a companion animal species are also provided.
  • “a” or“an” means“at least one” or“one or more” unless otherwise specified.
  • the term“or” means“and/or” unless specified otherwise. In the context of a multiple dependent claim, the use of“or” when referring back to other claims refers to those claims in the alternative only.
  • Novel IL13R/IL4R heterodimeric proteins are provided, for example, heterodimeric proteins that bind canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4.
  • amino acid sequence means a sequence of amino acids residues in a peptide or protein.
  • polypeptide and“protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a“polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • contiguous polypeptide herein is used to mean an uninterrupted sequence of amino acids.
  • a contiguous polypeptide is typically translated from a single continuous DNA sequence. It can be made by genetic engineering, for example, by removing the stop codon from the DNA sequence of the first protein, then appending the DNA sequence of the second protein in frame, so that the DNA sequence is expressed as a single protein. Typically, this is accomplished by cloning a cDNA into an expression vector in frame with an existing gene [0017] “IL4R,” as used herein, is a polypeptide comprising the entirety or a fragment of
  • IL4 receptor subunit alpha that bind to IL-4.
  • IL4R refers to an IL4R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated.
  • IL4R is an extracellular domain fragment that binds IL4.
  • the IL4R may be referred to as an IL4R extracellular domain (ECD).
  • IL4R comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • IL13R is a polypeptide comprising the entirety or a portion of
  • IL13 receptor subunit alpha- 1 that binds to IL-13.
  • IL13R refers to an IL13R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated.
  • IL13R is an extracellular domain fragment that binds IL13.
  • the IL13R may be referred to as an IL13R extracellular domain (ECD).
  • the IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
  • a companion animal species refers to an animal suitable to be a companion to humans.
  • a companion animal species is a small mammal, such as a canine, feline, dog, cat, horse, rabbit, ferret, guinea pig, rodent, etc.
  • a companion animal species is a farm animal, such as a horse, cow, pig, etc.
  • An“extracellular domain” (“ECD”) is the portion of a polypeptide that extends beyond the transmembrane domain into the extracellular space.
  • the term“extracellular domain,” as used herein, may comprise a complete extracellular domain or may comprise a truncated extracellular domain missing one or more amino acids, that binds to its ligand.
  • the composition of the extracellular domain may depend on the algorithm used to determine which amino acids are in the membrane. Different algorithms may predict, and different systems may express, different extracellular domains for a given protein.
  • An extracellular domain of an IL4R polypeptide may comprise a complete extracellular domain or a truncated extracellular domain of IL4R that binds IL4.
  • the terms“extracellular domain of an IL4R polypeptide,”“IL4R ECD,” and similar terms refer to an IL4R polypeptide that does not comprise a transmembrane domain or cytoplasmic domain, even if the term follows an open transitional word, such as“comprising,”“comprises,” and the like.
  • an extracellular domain of an IL4R polypeptide is an extracellular domain of an IL4R polypeptide derived from a companion species animal.
  • an extracellular domain of an IL4R polypeptide is derived from canine IL4R, feline IL4R or equine IL4R.
  • an extracellular domain of an IL4R polypeptide comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or any fragment thereof.
  • an extracellular domain of an IL4R polypeptide comprises the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, or any fragment thereof.
  • An extracellular domain of an IL13R polypeptide may comprise a complete extracellular domain or a truncated extracellular domain of IL13R that binds IL13.
  • the terms“extracellular domain of an IL13R polypeptide,”“IL13R ECD,” and similar terms refer to an IL13R polypeptide that does not comprise a transmembrane domain or cytoplasmic domain, even if the term follows an open transitional word, such as“comprising,”“comprises,” and the like.
  • an extracellular domain of an IL13R polypeptide is an extracellular domain of an IL13R polypeptide derived from a companion species animal.
  • an extracellular domain of an IL13R polypeptide is derived from canine IL13R, feline IL13R or equine IL13R.
  • an extracellular domain of an IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26, or any fragment thereof.
  • an extracellular domain of an IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36, or any fragment thereof.
  • IL13R/IL4R heterodimeric protein and“IL4R/IL13R heterodimeric protein” are used interchangeably to refer to a heterodimeric protein comprising a first contiguous polypeptide comprising an IL13R polypeptide and a second contiguous polypeptide comprising an IL4R polypeptide.
  • the first contiguous polypeptide and/or second contiguous polypeptide comprises an Fc polypeptide.
  • the IL13R/IL4R heterodimeric protein of the invention may comprise an extracellular domain of a IL13R polypeptide and/or an extracellular domain of a IL4R polypeptide, wherein the polypeptides are derived from a companion animal species.
  • a heterodimeric protein may comprise an extracellular domain of an IL4R polypeptide from a dog, cat, or horse and/or may comprise an extracellular domain of an IL13R polypeptide from a dog, cat, or horse.
  • Wild-type refers to a non-mutated version of a polypeptide that occurs in nature, or a fragment thereof.
  • a wild-type polypeptide may be produced recombinant!y.
  • A“wildtype 1L13R ECD” or a“wildtype IL4R ECD” refers to a protein having an amino acid sequence that is identical to the same portion of an extracellular domain of an 11.13 R or IL4R that occurs in nature.
  • A“variant” is a nucleic acid molecule or polypeptide that differs from a referent nucleic acid molecule or polypeptide by single or multiple amino acid substitutions, deletions, and/or additions and substantially retains at least one biological activity of the referent nucleic acid molecule or polypeptide.
  • A“biologically active” entity, or an entity having“biological activity,” is an entity having any function related to or associated with a metabolic or physiological process, and/or having structural, regulatory, or biochemical functions of a naturally-occurring molecule.
  • Biologically active polynucleotide fragments are those exhibiting similar activity, but not necessarily identical, to an activity of a polynucleotide of the present invention.
  • a biologically active polypeptide or fragment thereof includes one that can participate in a biological reaction, including, but not limited to, a ligand-receptor interaction or antigen-antibody binding. The biological activity can include an improved desired activity, or a decreased undesirable activity.
  • An entity may demonstrate biological activity when it participates in a molecular interaction with another molecule, such as hybridization, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide that may be detected as unique for the polynucleotide molecule, and when it can be used as a primer in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • “percent (%) amino acid sequence identity” and“homology” with respect to a nucleic acid molecule or polypeptide sequence are defined as the percentage of nucleotide or amino acid residues in a referent sequence that are identical with the nucleotide or amino acid residues in the specific nucleic acid molecule or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINETM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared.
  • a variant has at least about 50% sequence identity with the referent nucleic acid molecule or polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide.
  • a variant has at least about 50% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 98% sequence identity with the sequence of the referent nucleic acid or polypeptide.
  • a contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide having at least 85%, at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
  • a contiguous polypeptide comprises an extracellular domain of an IL4R polypeptide having at least 85%, at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • position corresponding to position n refers to an amino acid position of a subject polypeptide that aligns with position n of a reference polypeptide after aligning the amino acid sequences of the subject and reference polypeptides and introducing gaps. Alignment for purposes of whether a position of a subject polypeptide corresponds with position n of a reference polypeptide can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any parameters needed to achieve maximal alignment over the full length of two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.
  • the contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine at a position corresponding to position 18 of SEQ ID NO: 22, at a position corresponding to position 18 of SEQ ID NO: 24, or at a position corresponding to position 18 of SEQ ID NO: 26.
  • the contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine at position 18 of SEQ ID NO: 22, at position 18 of SEQ ID NO: 24, at position 18 of SEQ ID NO: 26, at position 15 of SEQ ID NO: 32, at position 15 of SEQ ID NO: 34, or at position 15 of SEQ ID NO: 36.
  • A“point mutation” is a mutation that involves a single nucleotide or amino acid residue.
  • the mutation may be the loss of a nucleotide or amino acid, substitution of one nucleotide or amino acid residue for another, or the insertion of an additional nucleotide or amino acid residue.
  • An amino acid substitution may include but is not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into a molecule of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.
  • Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
  • Non-conservative substitutions will entail exchanging a member of one of these classes with another class.
  • A“fusion partner,” as used herein, refers to an additional component of an
  • IL13R/IL4R contiguous polypeptide such as an additional polypeptide, such as albumin, an albumin binding fragment, or a fragment of an immunoglobulin molecule.
  • a fusion partner may comprise an oligomerization domain such as an Fc domain of a heavy chain immunoglobulin
  • IgX Fc or“IgX Fc polypeptide” means the Fc region is derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where“X” denotes the antibody isotype.
  • “IgG” or“IgG Fc” denotes the Fc region of a g chain
  • “IgA” or“IgA Fc” denotes the Fc region of an a chain
  • “IgD” or“IgD Fc” denotes the Fc region of a d chain
  • “IgE” or“IgE Fc” denotes the Fc region of an e chain
  • “IgM” or“IgM Fc” denotes the Fc region of a m chain, etc.
  • the Fc polypeptide or the IgG Fc region comprises CH1, hinge, CH2, CH3, and CL1. In some embodiments, the IgG Fc polypeptide comprises the hinge, CH2, and CH3, but does not comprise CH1 or CL. In some embodiments, the IgG Fc polypeptide comprises CH2 and CH3, but does not comprise CH1, the hinge, or CL. In some embodiments, the IgG Fc polypeptide comprises CH1, hinge, CH2, and CH3, with or without CL1.
  • IgXN Fc or“IgXN Fc polypeptide” denotes that the Fc region is derived from a particular subclass of antibody isotype (such as canine IgG subclass A, B, C, or D; feline IgG subclass 1, 2a, or 2b; or equine IgG subclass IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where“N” denotes the subclass.
  • a particular subclass of antibody isotype such as canine IgG subclass A, B, C, or D; feline IgG subclass 1, 2a, or 2b; or equine IgG subclass IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.
  • IgX or IgXN regions are derived from a companion animal, such as a dog, a cat, or a horse.
  • IgG regions are isolated from canine g heavy chains, such as IgGA, IgGB, IgGC, or IgGD.
  • IgGFc regions are isolated from feline g heavy chains, such as IgGl a, IgGlb, or IgG2.
  • IgG regions are isolated from equine g heavy chains, such as IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7.
  • Polypeptides comprising an Fc region of IgGA, IgGB, IgGC, or IgGD may provide for higher expression levels in recombination production systems.
  • a contiguous polypeptide comprises a first variant IgG Fc polypeptide comprising a“knob” mutation and a second variant IgG Fc polypeptide comprising a “hole” mutation.
  • knob and hole mutations are described, for example, in Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat Biotechnol, 16(7):677-81 (1998).
  • A“knob” mutation,” as used herein, refers to an interfacing mutation of a molecule
  • an Fc polypeptide that comprises a bulky amino acid.
  • A“hole mutation,” as used herein, refers to an interfacing mutation of a molecule
  • an Fc polypeptide that comprises one or more smaller amino acids.
  • a variant IgG Fc polypeptide comprises a knob mutation.
  • a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO:
  • SEQ ID NO: 42 SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
  • a variant IgG Fc polypeptide comprises an amino acid substitution at position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO:
  • SEQ ID NO: 44 SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
  • a variant IgG Fc polypeptide comprises a tryptophan at a position corresponding to position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41, or position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
  • a variant IgG Fc polypeptide comprises a tryptophan at position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
  • a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, 66, 67, 68, 69, 70, 81, 82, 83, 84, 85, 86, or 87.
  • a variant IgG Fc polypeptide comprises a hole mutation.
  • a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40; position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or
  • a variant IgG Fc polypeptide comprises an amino acid substitution at position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40; position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
  • a variant IgG Fc polypeptide comprises a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 38; a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 39; a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position corresponding to position
  • a variant IgG Fc polypeptide comprises a serine at position corresponding to position 181 of SEQ ID NO: 41; a serine at a position corresponding to position 154 and/or an alanine at a position corresponding to position 156 and/or a threonine at a position corresponding to position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or a serine at a position corresponding to position 130 and/or an alanine at a position corresponding to position 132 and/or a threonine at a position corresponding to position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
  • a variant IgG Fc polypeptide comprises a serine at position corresponding to position 181 of SEQ ID
  • a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101.
  • A“signal sequence” refers to a sequence of amino acid residues or polynucleotides encoding such, which facilitates secretion of a polypeptide of interest and is typically cleaved upon export of the polypeptide to the outside of the cell surface membrane.
  • A“linker” refers to one or more amino acid residues that connects a first polypeptide with a second polypeptide.
  • the linker is a glycine-rich and/or serine-rich, flexible, non- structural linker.
  • a linker comprises the amino acids G (Gly) and/or S (Ser).
  • a linker may comprise G or a repeat of G (e.g., GG, GGG, etc.), GS or a repeat of GS (e.g., GSGS (SEQ ID NO: 114), GSGSGS (SEQ ID NO: 115), etc ); GGS or a repeat of GGS (e g., GGSGGS (SEQ ID NO: 116), GGS GGS GGS (SEQ ID NO: 117), etc ); GGGS (SEQ ID NO: 118) or a repeat of GGGS (SEQ ID NO: 119) (e.g., GGGS GGGS (SEQ ID NO: 120), GGGSGGGSGGGS (SEQ ID NO: 121), etc ); GSS or
  • the contiguous polypeptide comprises at least one linker. In some embodiments, the contiguous polypeptide comprises an optional signal sequence, and at least one optional linker. In some embodiments, the contiguous polypeptide does not comprise a signal sequence, or a linker. In some embodiments, the contiguous polypeptide is translated with a signal sequence, but the signal sequence is cleaved from the contiguous polypeptide.
  • a heterodimeric protein comprises a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide and b) a second contiguous polypeptide comprising at least one IL4R ECD and a second Fc polypeptide, wherein the IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
  • a first contiguous polypeptide or a second contiguous polypeptide has the formula:
  • ILl3R(n) is at least one IL13R extracellular domain (ECD) polypeptide derived from a companion animal species
  • IL4R(n) is at least one IL4R ECD polypeptide derived from a companion animal species
  • (n) is one, two, three, four, or more ECD polypeptides
  • L is an optional linker
  • Fc is a variant Fc polypeptide, such as a variant Fc polypeptide comprising knob or a hole mutation.
  • binding partner(s) may be included in the contiguous polypeptide before, after, and/or between any one or more IL13R or IL4R ECD polypeptide(s).
  • Other potential binding partners include: IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
  • the heterodimeric protein comprises a first contiguous polypeptide comprising the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113.
  • the heterodimeric protein comprises a second contiguous polypeptide comprising the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
  • Polynucleotide sequences that encode all or part (e.g., the extracellular domain) of a contiguous polypeptide with or without a signal sequence are provided. If a homologous signal sequence (i.e., a signal sequence of native IL-4R or IL13R) is not used in the construction of the nucleic acid molecule, then another signal sequence may be used, for example, any one of the signal sequences described in PCT/US06/02951.
  • nucleotide sequence encoding the polypeptide of interest is inserted into an expression vector, suitable for expression in a selected host cell.
  • A“vector” is a plasmid that can be used to transfer DNA sequences from one organism to another or to express a gene of interest.
  • a vector typically includes an origin of replication and regulatory sequences which regulate the expression of the gene of interest, and may or may not carry a selective marker gene, such as an antibiotic resistance gene.
  • a vector is suitable for the host cell in which it is to be expressed.
  • a vector may be termed a“recombinant vector” when the gene of interest is present in the vector.
  • A“host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide.
  • Host cells may be prokaryotic cells or eukaryotic cells.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Nonlimiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, and their derivatives, such as 293-6E, DG44, CHO-S, and CHO-K cells.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected in vivo with a polynucleotide(s) encoding an amino acid sequence(s) provided herein.
  • isolated refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced.
  • a polypeptide is referred to as“isolated” when it is separated from at least some of the components of the cell in which it was produced.
  • a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be“isolating” the polypeptide.
  • a polynucleotide is referred to as“isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide.
  • a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as“isolated.”
  • the heterodimeric protein or contiguous polypeptide is isolated using chromatography, such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.
  • chromatography such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.
  • label and“detectable label” mean a moiety attached to a IL13R/IL4R contiguous polypeptide to render it detectable.
  • the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (for example, 3 H, 14 C, 35 S, 90 Y, "Tc, U1 ln, 125 I, 131 I, 177 Lu, 166 HO, or 153 Sm); chromogens, fluorescent labels (for example, FITC, rhodamine, lanthanide phosphors), enzymatic labels (for example, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.
  • radioisotopes or radionuclides for example, 3 H, 14 C, 35 S, 90 Y, "Tc, U1
  • labels commonly employed for immunoassays include moieties that produce light, for example, acridinium compounds, and moieties that produce fluorescence, for example, fluorescein.
  • the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety.
  • the IL13R/IL4R heterodimeric proteins of the invention can function as decoy receptors for trapping IL13 and/or IL4 and inhibiting their interaction with IL13R and/or IL4R on cell surfaces.
  • Decoy receptors such as those of the invention, recognize their ligands with high affinity and specificity but are structurally incapable of signaling. They compete with wild-type receptors for ligand binding and participate in ligand/receptor interactions, thus modulating the activity of or the number of functioning receptors and/or the cellular activity downstream from the receptors. Decoy receptors can act as molecular traps for agonist ligands and thereby inhibit ligand-induced receptor activation.
  • IL13 refers to any native IL13 that results from expression and processing of IL13 in a cell.
  • the term includes IL13 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated.
  • the term also includes naturally occurring variants of IL13, e.g., splice variants or allelic variants.
  • a canine IL13 comprises the amino acid sequence of SEQ
  • a feline IL13 comprises the amino acid sequence of SEQ ID NO: 5.
  • an equine IL13 comprises the amino acid sequence of SEQ ID NO: 6.
  • IL4 refers to any native IL4 that results from expression and processing of IL4 in a cell.
  • the term includes IL4 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated.
  • the term also includes naturally occurring variants of IL4, e.g., splice variants or allelic variants.
  • a canine IL4 comprises the amino acid sequence of SEQ ID NO: 1
  • a feline IL4 comprises the amino acid sequence of SEQ ID NO: 2.
  • an equine IL4 comprises the amino acid sequence of SEQ ID NO: 3.
  • the invention provides IL13R/IL4R heterodimeric proteins as therapeutic agents.
  • IL13R/IL4R heterodimeric proteins of the invention bind to IL13 and/or IL4, described in more detail herein, which have been demonstrated to be associated with allergic diseases.
  • IL13R/IL4R heterodimeric proteins can bind IL13 and/or IL4 with very high affinity.
  • the IL13R/IL4R heterodimeric proteins can interfere with IL13 and/or IL4 signaling.
  • affinity means the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, a receptor) and its binding partner (for example, a ligand).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).
  • KD dissociation constant
  • Affinity can be measured by common methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.
  • KD The terms“KD,”“Kd,”“Kd” or“Kd value” as used interchangeably to refer to the equilibrium dissociation constant of a receptor fusion - ligand interaction.
  • the Kd of the fusion molecule to its ligand is measured by using biolayer interferometry assays using a biosensor, such as an Octet ® System (Pall ForteBio LLC, Fremont, CA) according to the supplier’s instructions. Briefly, biotinylated antigen is bound to the sensor tip and the association of fusion molecule is monitored for ninety seconds and the dissociation is monitored for 600 seconds.
  • the buffer for dilutions and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer only blank curve is subtracted to correct for any drift.
  • the data are fit to a 2: 1 binding model using ForteBio data analysis software to determine association rate constant (k 0n ), dissociation rate constant (k 0ff ), and the Kd.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio of k 0ff /k 0n.
  • the term“kon” refers to the rate constant for association of a molecule X to its partner Y and the term“koff’ refers to the rate constant for dissociation of a molecule X or partner Y from the molecule X / partner Y complex.
  • binding to a substance is a term that is well understood in the art, and methods to determine such binding are also well known in the art.
  • a molecule is said to exhibit “binding” if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, or etc.
  • Surface plasmon resonance denotes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcoreTM system (BIAcore International AB, a GE Healthcare company, ETppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al. (1993) Ann. Biol. Clin.51: 19-26.
  • Biolayer interferometry refers to an optical analytical technique that analyzes the interference pattern of light reflected from a layer of immobilized protein on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real-time.
  • a nonlimiting exemplary device for biolayer interferometry is an Octet ® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem.377: 209-277.
  • an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, or equine IL13 and/or IL4 with a dissociation constant (Kd) of less than 5 x 10 6 M, less than 1 x 10 6 M, less than 5 x 10 7 M, less than 1 x 10 7 M, less than 5 x 10 8 M, less than 1 x 10 8 M, less than 5 x 10 9 M, less than 1 x 10 9 M, less than 5 x 10 10 M, less than 1 x 10 10 M, less than 5 x 10 11 M, less than 1 x 10 11 M, less than 5 x 10 12 M, or less than 1 x 10 12 M, as measured by biolayer interferometry.
  • Kd dissociation constant
  • an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, or equine IL13 and/or IL4 with a Kd of between 5 x 10 6 M and 1 x 10 6 M, between 5 x 10 6 M and 5 x 10 7 M, between 5 x 10 6 M and 1 x 10 7 M, between 5 x 10 6 M and 5 x 10 8 M, 5 x 10 6 M and 1 x 10 8 M, between 5 x 10 6 M and 5 x 10 9 M, between 5 x 10 6 M and lx 10 9 M, between 5 x 10 6 M and 5 x 10 10 M, between 5 x 10 6 M and 1 x 10 10 M, between 5 x 10 6 M and 5 x 10 11 M, between
  • To“reduce” or“inhibit” means to decrease, reduce, or arrest an activity, function, or amount as compared to a reference.
  • by“reduce” or“inhibit” is meant the ability to cause an overall decrease of 20% or greater.
  • by“reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
  • by“reduce” or“inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time.
  • A“reference” as used herein refers to any sample, standard, or level that is used for comparison purposes.
  • a reference may be obtained from a healthy or non-diseased sample.
  • a reference is obtained from a non-diseased or non-treated sample of a companion animal.
  • a reference is obtained from one or more healthy animals of a particular species, which are not the animal being tested or treated.
  • the term“substantially reduced,” as used herein, denotes a sufficiently high degree of reduction between a numeric value and a reference numeric value such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values.
  • the substantially reduced numeric values is reduced by greater than about any one of 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.
  • an IL13R/IL4R heterodimeric protein may reduce IL13 and/or IL4 signaling in a companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL13 and/or IL4 signaling in the absence of the fusion molecule.
  • signaling is measured by a reduction in IL4-depedent TF-l cell proliferation.
  • the reduction in IL13 and/or IL4 signaling or the reduction in proliferation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 70%, between 25% and
  • pharmaceutical formulation and“pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.
  • A“pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a“pharmaceutical composition” for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinyl pyrrolidone, cellulose- based substances; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.
  • the pharmaceutical composition can be stored in lyophilized form.
  • the preparation process includes a lyophilization step.
  • the lyophilized composition may then be reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to the dog, cat, or horse.
  • the pharmaceutical composition can be stored as a liquid, i.e., as an aqueous composition, which may be administered directly, or with appropriate dilution, to the dog, cat, or horse.
  • a lyophilized composition can be reconstituted with sterile Water for Injection (WFI). Bacteriostatic reagents, such benzyl alcohol, may be included.
  • WFI sterile Water for Injection
  • Bacteriostatic reagents such benzyl alcohol, may be included.
  • the invention provides pharmaceutical compositions in solid or liquid form.
  • the pH of the pharmaceutical compositions may be in the range of from about pH
  • compositions of the invention are sterile if they are to be used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Sterility may be maintained with or without anti -bacterial agents.
  • an“IL13 or IL4-induced condition” means a disease associated with, caused by, or characterized by, elevated levels or altered distribution of IL13 or IL4.
  • Such IL13 and/or IL4-induced conditions include, but are not limited to, a pruritic or an allergic disease.
  • the IL13- and/or IL4-induced condition is atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema.
  • An IL13- or IL4-induced condition may be exhibited in a companion animal, including, but not limited to, canine, feline, or equine.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • Treatment covers any administration or application of a therapeutic for disease in a mammal, including a companion animal.
  • beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).
  • treatment is a reduction of pathological consequence of a proliferative disease.
  • the methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one- hundred percent removal of all aspects of the disorder.
  • an IL13R/IL4R heterodimeric proteins or pharmaceutical compositions comprising it can be utilized in accordance with the methods herein to treat IL13- or IL4-induced conditions.
  • an IL13R/IL4R heterodimeric proteins or pharmaceutical compositions is administered to a companion animal, such as a canine, a feline, or equine, to treat an IL13- and IL4-induced condition.
  • a “therapeutically effective amount” of a substance/molecule, agonist or antagonist may vary according to factors such as the type of disease to be treated, the disease state, the severity and course of the disease, the type of therapeutic purpose, any previous therapy, the clinical history, the response to prior treatment, the discretion of the attending veterinarian, age, sex, and weight of the animal, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the animal.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount may be delivered in one or more administrations.
  • a therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered parenterally, by subcutaneous administration, intravenous infusion, or intramuscular injection.
  • an IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered as a bolus injection or by continuous infusion over a period of time.
  • an IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered by an intramuscular, an intraperitoneal, an intracerebrospinal, a subcutaneous, an intra-arterial, an intrasynovial, an intrathecal, or an inhalation route.
  • An IL13R/IL4R heterodimeric protein described herein may be administered in an amount in the range of 0.1 mg/kg body weight to 100 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 0.1 mg/kg body weight to 50 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 1 mg/kg body weight to 10 mg/kg body weight per dose.
  • an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 0.5 mg/kg body weight to 100 mg/kg body, in the range of 1 mg/kg body weight to 100 mg/kg body weight, in the range of 5 mg/kg body weight to 100 mg/kg body weight, in the range of 10 mg/kg body weight to 100 mg/kg body weight, in the range of 20 mg/kg body weight to 100 mg/kg body weight, in the range of 50 mg/kg body weight to 100 mg/kg body weight, in the range of 1 mg/kg body weight to 10 mg/kg body weight, in the range of 5 mg/kg body weight to 10 mg/kg body weight, in the range of 0.5 mg/kg body weight to 10 mg/kg body weight, or in the range of 5 mg/kg body weight to 50 mg/kg body weight.
  • An IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein can be administered to a companion animal at one time or over a series of treatments.
  • IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein may be administered at least once, more than once, at least twice, at least three times, at least four times, or at least five times.
  • the dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more weeks of no treatment.
  • the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more days or weeks of no treatment.
  • Administration“in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.
  • the term“concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent.
  • the two or more therapeutic agents are administered with a time separation of no more than about a specified number of minutes.
  • the term“sequentially” is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s), or wherein administration of one or more agent(s) begins before the administration of one or more other agent(s).
  • administration of the two or more therapeutic agents are administered with a time separation of more than about a specified number of minutes.
  • “in conjunction with” refers to administration of one treatment modality in addition to another treatment modality.
  • “in conjunction with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the animal.
  • the method comprises administering in combination with an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein, a Jak inhibitor, a PI3K inhibitor, an ART inhibitor, or a MAPK inhibitor.
  • the method comprises administering in combination with an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R contiguous polypeptide, an anti-ILl7 antibody, an anti-TNFa antibody, an anti- CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL3 l antibody, an anti- IL23 antibody, an anti-IgE antibody, an anti-CDl la antibody, anti-IL6R antibody, anti-a4- Intergrin antibody, an anti-ILl2 antibody, an anti-IL 1 b antibody, or an anti-BlyS antibody.
  • an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R contiguous polypeptide, an anti-ILl7 antibody, an anti-TNFa antibody, an anti- CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL3 l antibody, an anti- IL23 antibody, an anti-I
  • the cell is exposed to the IL13R/IL4R heterodimeric protein or pharmaceutical composition ex vivo.
  • the cell is exposed to the IL13R/IL4R heterodimeric protein or pharmaceutical composition in vivo.
  • a cell is exposed to the IL13R/IL4R heterodimeric protein.
  • a cell is exposed to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition under conditions permissive for binding of the heterodimeric protein to extracellular IL13 and/or IL4.
  • a cell may be exposed in vivo to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition by any one or more of the administration methods described herein, including but not limited to, intraperitoneal, intramuscular, intravenous injection into the subject.
  • a cell may be exposed ex vivo to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition by exposing the cell to a culture medium comprising the heterodimeric protein or the pharmaceutical composition.
  • the permeability of the cell membrane may be affected using any number of methods understood by those of skill in the art (such as electroporating the cells or exposing the cells to a solution containing calcium chloride) before exposing the cell to a culture medium comprising the fusion molecule or the pharmaceutical composition.
  • an IL13R/IL4R heterodimeric protein may reduce IL13 and/or IL4 signaling in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL13 and/or IL4 signaling function in the absence of the IL13R/IL4R heterodimeric protein.
  • the reduction in IL13 and/or IL4 signaling and/or the reduction in TF-l proliferation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 40%, between 25%
  • the method comprises detecting whether the animal has cells that express IL13 or IL4 using an IL13R/IL4R heterodimeric protein.
  • the method of detection comprises contacting the sample with an antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from that of a reference or comparison sample (such as a control).
  • the method may be useful to determine whether the IL13R/IL4R heterodimeric protein described herein are an appropriate treatment for the subject animal.
  • the sample is a biological sample.
  • biological sample means a quantity of a substance from a living thing or formerly living thing.
  • the biological sample is a cell or cell/tissue lysate.
  • the biological sample includes, but is not limited to, blood, (for example, whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.
  • the cells or cell/tissue lysate are contacted with an
  • test cells show binding activity as compared to a reference cell of the same tissue type, it may indicate that the subject would benefit from treatment with an IL13R/IL4R heterodimeric protein.
  • the test cells are from tissue of a companion animal.
  • Various methods known in the art for detecting specific antibody-antigen binding can be used.
  • Exemplary immunoassays which can be conducted include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).
  • FPIA fluorescence polarization immunoassay
  • FIA fluorescence immunoassay
  • EIA enzyme immunoassay
  • NIA nephelometric inhibition immunoassay
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • An indicator moiety, or label group can be attached to the subject antibodies and is selected to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures.
  • Appropriate labels include, without limitation, radionuclides (for example 125 I, 131 I, 35 S, 3 H, or 32 P), enzymes (for example, alkaline phosphatase, horseradish peroxidase, luciferase, or p-galactosidase), fluorescent moieties or proteins (for example, fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (for example, QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.).
  • radionuclides for example 125 I, 131 I, 35 S, 3 H, or 32 P
  • enzymes for example, alkaline phosphatase, horseradish peroxidase, luciferase, or p-galactosidase
  • fluorescent moieties or proteins for example, fluorescein, rhodamine, phycoerythrin, G
  • the IL13R/IL4R heterodimeric protein can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to polypeptides are known in the art.
  • the IL13R/IL4R heterodimeric protein need not be labeled, and the presence thereof can be detected, for example, using an antibody that binds to the IL13R/IL4R heterodimeric protein.
  • the IL13R/IL4R heterodimeric protein can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
  • the anti-ILl3 and IL4 antibodies and polypeptides can also be used for in vivo diagnostic assays, such as in vivo imaging.
  • the antibody or the polypeptide is labeled with a radionuclide (such as U1 ln, "Tc, 14 C, 131 I, 125 I, 3 H, or any other radionuclide label, including those outlined herein) so that the cells or tissue of interest can be localized using immunoscintiography.
  • a radionuclide such as U1 ln, "Tc, 14 C, 131 I, 125 I, 3 H, or any other radionuclide label, including those outlined herein
  • the IL13R/IL4R heterodimeric protein may also be used as staining reagent in pathology using techniques well known in the art.
  • a IL13R/IL4R heterodimeric protein is used for a diagnostic and a IL13R/IL4R heterodimeric protein is used as a therapeutic.
  • the first and second IL13R/IL4R heterodimeric proteins are different.
  • a nucleotide sequence encoding canine IL13 protein (SEQ ID NO: 4) was synthesized with poly-His tag on the C-terminal end and cloned into a mammalian expression vector and transfected to 293 cells or CHOS. The same method was used to clone and express a nucleotide sequence encoding canine IL4 protein (SEQ ID NO: 1) with a poly-His tag on the C- terminal end.
  • Canine IL13 was affinity purified using Ni-NTA column (CaptivA ® Protein A Affinity Resin, Repligen). The same method was used to purify canine IL4.
  • Extracellular domains of canine, feline, and equine IL4R that are responsible for binding canine, feline and equine IL4 and/or IL13 were identified and boundaries were defined.
  • Full-length extracellular domains of canine IL4R, feline IL4R, and equine IL4 were identified as SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27, respectively.
  • Extracellular domain fragments of canine IL4R, feline IL4R, and equine IL4R postulated to retain biological activity were identified as SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, respectively.
  • Extracellular domains of canine, feline, and equine IL13R that are responsible for binding canine, feline, and equine IL4 and/or IL13 were identified and boundaries were defined.
  • Full length extracellular domains of canine IL13R, feline IL13R, and equine IL13R were identified as SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively.
  • Extracellular domain fragments of canine IL13R, feline IL13R, and equine IL13R postulated to retain biological activity were identified as SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively.
  • Cys An unpaired cysteine (Cys) in canine IL13R (at position 18 of SEQ ID NO: 22), feline IL13R (at position 18 of SEQ ID NO: 24), and equine IL13R (at position 18 of SEQ ID NO: 26) was identified informatically and determined as embedded (unexposed) based on 3-D modeling. It is unlikely that the unpaired cysteine will form disulfide bonds and the likelihood of aggregation is low. Thus, site-directed mutagenesis of this Cys residue was not introduced.
  • Nucleotide sequences encoding canine IL13R ECD/IL4R ECD contiguous polypeptides linked to an IgGB Fc polypeptide were designed with a signal sequence.
  • contiguous polypeptide“ILl3RECD-IL4RECD-IgGB Fc” (SEQ ID NO: 20)
  • an extracellular domain of IL13R (SEQ ID NO: 22) precedes an extracellular domain of IL4R (SEQ ID NO: 23).
  • SEQ ID NO: 21 For contiguous polypeptide“IL4RECD-ILl3RECD-IgGB Fc” (SEQ ID NO: 21), an extracellular domain of IL4R precedes an extracellular domain of IL13R.
  • nucleotide sequences were synthesized chemically and inserted into an expression vector suitable for transfection into a CHO host cell. After transfection into CHO cells, the fusion proteins were secreted from the cell. For example, fusion protein was purified by single step Protein A column chromatography.
  • Each of IL 13RECD-IL4RECD-IgGB Fc and IL4RECD-IL 13RECD-IgGB Fc may be expressed and purified in a single step with a protein A column or other chromatographic methods, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed mode column chromatography such as CHT, or multimodal mode column chromatography such as CaptoMMC. Low pH or other viral inactivation and viral removal steps can be applied.
  • the purified protein may be admixed with excipients, and sterilized by filtration to prepare a pharmaceutical composition of the invention.
  • the pharmaceutical composition may be administered to a dog with an atopic dermatitis or asthma in an amount sufficient to bind and/or inhibit either IL13 and/or IL4.
  • the vectors were then used to perform pilot-scale transfection in CHO-S cells using the FreestyleMaxTM transfection reagent (Life Technologies). The supernatant was harvested by clarifying the conditioned media. Protein was purified with a single pass Protein A chromatography step and used for further investigation.
  • the binding analysis was performed using a biosensor Octet as follows. Briefly, canine IL4 (produced using 293 cells) was biotinylated. The free unreacted biotin was removed from biotinylated IL4 by extensive dialysis. Biotinylated canine IL4 was captured on streptavidin sensor tips. The IL4 association with various concentrations (12, 16, and 44 nM) of IL13RECD- IL4RECD-IgGB Fc (SEQ ID NO:20) was monitored for ninety seconds. Dissociation was monitored for 600 seconds. A buffer only blank curve was subtracted to correct for any drift.
  • the data were fit to a 1 : 1 binding model using ForteBioTM data analysis software to determine the k 0 n, koff, and the Kd.
  • the buffer for dilutions and all binding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2.
  • the Kd for ILl3RECD-IL4RECD-IgGB Fc and ligand IL4 was 8 xlO 11 .
  • Canine IL4 and canine IL13 with C-terminal polyHis tag was expressed and purified from 293 cells.
  • EZ-Link NHS-LC-biotin was obtained from Thermo Scientific (Cat. #21336), and Streptavidin biosensors was obtained from ForteBio (Cat. #18-509).
  • IL4 and IL13 sequential binding experiments with ILl3RECD-IL4RECD-IgGB Fc (SEQ ID NO:20) were performed. Biotinylated canine ILl3RECD-IL4RECD-IgGB Fc was captured on streptavidin sensor tips. Canine ILl3RECD-IL4RECD-IgGB Fc was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS ( Figure 1). The experiments demonstrated that once ILl3RECD-IL4RECD-IgGB Fc bound to IL13, it may not bind to IL4, and that once bound to IL4, its ability to bind IL13 is reduced.
  • IL4 and IL13 sequential binding experiments with IL4RECD-ILl3RECD-IgGB Fc (SEQ ID NO:2l) were performed. Biotinylated canine IL4RECD-ILl3RECD-IgGB Fc was captured on streptavidin sensor tips. Canine IL4RECD-ILl3RECD-IgGB Fc was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS ( Figure 2). These experiments demonstrated that once IL4RECD-ILl3RECD-IgGB Fc bound to IL13, it may not bind to IL4, and that once bound to IL4, its ability to bind IL13 is reduced.
  • TF1 cells ATCC cat# CRL-2003
  • a human Erythroleukemic cell line which expresses endogenous interleukin 4 receptors on cell surface
  • RPMI1640 Gibco, Cat#l 1875
  • Fetal Bovine Serum heat inactivated
  • 2 nM/ml Human GM-CSF R&D System, Cat# 215-GM-010
  • 20,000 cells per well were plated in a 96-well plate (Corning, Cat# 3610).
  • Canine IL4RECD-ILl3RECD-IgGB Fc (SINK) was added at a series of dilutions followed by addition of canine IL4 (Sino Biological Inc, Cat# 70021-DNAE-5) at 50 ng/ml.
  • the cells were incubated in 37°C, 5% C02 for 48 hours in a total volume of 100 pl. At the end of the incubation, the cells were cooled in room temperature and assayed for proliferation/variability by measuring cellular ATP content using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat# G7570).
  • Pairs of variant canine IgG Fc polypeptides, variant feline IgG Fc polypeptides, and variant equine IgG Fc polypeptides were designed such that a knob-in-hole heterodimerization approach may be used to prepare heterodimeric proteins comprising at least one IL31R ECD and at least one IL4R ECD.
  • pairing of two Fc polypeptides was designed by introducing CH3 interfacing mutations so that a first Fc polypeptide comprises a bulky amino acid (knob) and a second Fc polypeptide comprises smaller amino acids in the same general location (hole).
  • An amino acid substitution of threonine to tryptophan at a position corresponding to position 138 of canine IgG-A (SEQ ID NO: 38), at a position corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 39), at a position corresponding to position 137 of canine IgG-C Fc (SEQ ID NO: 40), or at a position corresponding to position 138 of canine IgG-D Fc (SEQ ID NO: 41) (T138W or T137W) can be introduced as a knob.
  • amino acid sequences of a first variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptide comprising a knob mutation are SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively.
  • An amino acid substitution of threonine to serine at a position corresponding to position 138 and/or of leucine to alanine at a position corresponding to position 140 and/or of tyrosine to threonine at a position corresponding to position 180 of canine IgG-A (SEQ ID NO: 38) or of IgG-D (SEQ ID NO: 41) (T138S, L140A, and/or Y180T); or of threonine to serine at a position corresponding to position 137 and/or of leucine to alanine at a position corresponding to position 139 and/or of tyrosine to threonine at a position corresponding to position 179 of canine IgG-B Fc (SEQ ID NO: 39) or of IgG-C (SEQ ID NO: 40) (T137S, L139A, and/or Y179T) can be introduced as a hole.
  • amino acid sequences of a second variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptides comprising a hole mutation are SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65.
  • [00135] An amino acid substitution of threonine to tryptophan at a position corresponding to position 154 of feline IgGla Fc (SEQ ID NO: 42 or SEQ ID NO: 43), feline IgGlb Fc (SEQ ID NO: 44 or SEQ ID NO: 45), or of feline IgG2 (SEQ ID NO: 46) (T154W) can be introduced as a knob.
  • Examples of amino acid sequences of a first variant feline IgGla, and IgGlb, and IgG2 Fc polypeptide comprising a knob mutation are SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.
  • amino acid sequences of a second variant feline IgGla, IgGlb, IgG2 Fc polypeptide comprising a hole mutation are SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.
  • amino acid sequences of a first variant equine IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides comprising a knob mutation are SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively.
  • amino acid sequences of a second variant equine IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides comprising a hole mutation are SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101.
  • heterodimeric protein pairs may have the following formats:
  • Polypeptide 1 ILl3R(n)-L-Fcl and
  • Polypeptide 2 IL4R(n)-L-Fc2; or
  • Polypeptide 1 IL4R(n)-L-Fcl and
  • Polypeptide 2 ILl3R(n)-L-Fc2,
  • ILl3R(n) is at least one IL13R extracellular domain (ECD) polypeptide derived from a companion animal species
  • IL4R(n) is at least one IL4R ECD polypeptide derived from a companion animal species
  • (n) is one, two, three, four, or more ECD polypeptides
  • L is an optional linker
  • Fcl is a variant Fc polypeptide, such as a variant Fc polypeptide comprising knob mutation
  • Fc2 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a hole mutation.
  • An optional linker could also be used between multiple ECD polypeptides.
  • binding partner(s) may be included before, after, and/or between any one or more ECD polypeptide(s).
  • Other potential binding partners include: IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
  • pairs of contiguous polypeptide 1 and 2 that may form a heterodimeric protein include SEQ ID NOs 102 and 103, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 108 and 109, SEQ ID NOs: 110 and 111, and SEQ ID NOs: 112 and 113.
  • a host cell may be cotransfected with vectors expressing these contiguous polypeptide pairs to produce the heterodimeric proteins described.

Abstract

Provided are various embodiments relating to IL13R/IL4R heterodimeric proteins derived from companion animal species and that bind to IL13 and/or IL4. Such heterodimeric proteins can be used in methods to treat IL13 and/or IL4-induced conditions in companion animals, such as canines, felines, and equines.

Description

IL4/IL13 RECEPTOR MOLECULE FOR VETERINARY USE
[0001] This application claims the benefit of U.S. Provisional Application No.
62/750,782, filed October 25, 2018, which is incorporated by reference herein in its entirety for any purpose.
FIELD
[0002] This present disclosure relates to heterodimeric proteins comprising interleukin 4 receptor and interleukin 13 receptor fragments derived from companion animal species that bind to IL4 and/or IL13 of a companion animal species, for example, canine IL4 and canine IL13. The present disclosure also relates to methods of using the heterodimeric proteins, for example, for treating IL4 and/or ILl3-induced conditions or reducing IL4 and/or IL13 signaling activity in cells, for instance in companion animals, such as canines, felines, and equines.
SEQUENCE LISTING
[0003] The present application is filed with a Sequence Listing in electronic format. The
Sequence Listing is provided as a file entitled “20l9-l0-24_0l l57-0027-00PCT_ST25.txt” created on October 25, 2019, which is 334 kilobytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND
[0004] Interleukin 4 (IL4) is a cytokine promoting differentiation of naive helper T cells to Th2 cells. Interleukin 13 (IL13) has similar effects on immune cells. Both IL4 and IL13 play important roles in T cell-mediated immune responses that are directly associated with allergy, for example, atopic dermatitis and asthma. It is generally understood that IL4 can form a signaling complex either with heterodimeric receptors IL4 receptor subunit alpha (IL4R) and yc or IL4R and IL13 receptor subunit alpha-l (IL13R). IL13 can form a signaling complex with heterodimeric receptors IL4Ra and ILl3Ral . Extracellular domains of IL4Ra or ILl3Ral may bind to IL4 and/or IL13 and reduce the free concentrations of the cytokines, thus diminishing the clinical signs and symptoms associated with dermatitis, asthma and other disorders.
[0005] Companion species animals, such as cats, dogs, and horses, suffer from many allergic diseases similar to human allergic diseases, including atopic dermatitis and asthma. There remains a need, therefore, for methods and compounds that can be used specifically to bind companion animal IL4 and/or IL13 for treating IL4/IL 13 -induced conditions and for reducing IL4/IL13 signaling activity.
SUMMARY
Embodiment 1. A heterodimeric protein comprising:
a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide, and
b) a second contiguous polypeptide comprising at least one IL4R ECD and a second Fc polypeptide,
wherein the IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
Embodiment 2. The heterodimeric protein of embodiment 1, wherein the first contiguous polypeptide and/or the second contiguous polypeptide comprises one, two, three, or four IL4R ECDs and/or one, two, three, or four IL13R ECDs.
Embodiment 3. The heterodimeric protein of any one of the preceding
embodiments, wherein the first contiguous polypeptide and/or the second contiguous polypeptide further comprises at least one binding partner other than IL4R ECD or IL13R ECD.
Embodiment 4. The heterodimeric protein of embodiment 3, wherein the at least one binding partner comprises IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
Embodiment 5. The heterodimeric protein of any one of the preceding
embodiments, wherein the heterodimeric protein binds to IL13 and/or IL4 with a dissociation constant (Kd) of less than 5 x 10 6 M, less than 1 x 10 6 M, less than 5 x 10 7 M, less than 1 x 10 7 M, less than 5 x 10 8 M, less than 1 x 10 8 M, less than 5 x 10 9 M, less than 1 x 10 9 M, less than 5 x 10 10 M, less than 1 x 10 10 M, less than 5 x 10 11 M, less than 1 x 10 11 M, less than 5 x 10 12 M, or less than 1 x 10 12 M, as measured by biolayer interferometry.
Embodiment 6. The heterodimeric protein of any one of the preceding
embodiments, wherein the heterodimeric protein reduces IL13 and/or IL4 signaling in a companion animal species.
Embodiment 7. The heterodimeric protein of any one of the preceding
embodiments, wherein the companion animal species is canine, feline, or equine.
Embodiment 8. The heterodimeric protein of any one of the preceding
embodiments, wherein the amino acid sequence of the at least one IL13R ECD is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
Embodiment 9. The heterodimeric protein of any one of the preceding
embodiments, wherein the amino acid sequence of the at least one IL13R ECD comprises a cysteine at a position corresponding to position 18 of SEQ ID NO: 22, corresponding to position 18 of SEQ ID NO: 24, or corresponding to position 18 of SEQ ID NO: 26.
Embodiment 10. The heterodimeric protein of any one of the preceding
embodiments, wherein the amino acid sequence of the at least one IL13R ECD comprises a cysteine at position 18 of SEQ ID NO: 22, at position 18 of SEQ ID NO: 24, at position 18 of SEQ ID NO: 26, at position 15 of SEQ ID NO: 32, at position 15 of SEQ ID NO: 34, or at position 15 of SEQ ID NO: 36.
Embodiment 11. The heterodimeric protein of any one of the preceding
embodiments, wherein the at least one IL13R ECD comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36.
Embodiment 12. The heterodimeric protein of any one of the preceding
embodiments, wherein the amino acid sequence of the at least one IL4R ECD is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
Embodiment 13. The heterodimeric protein of any one of the preceding
embodiments, wherein the at least one IL4R ECD comprises an amino acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37.
Embodiment 14. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide and/or the second Fc polypeptide is:
a) a canine IgG-A, IgG-B, IgG-C, or IgG-D Fc polypeptide;
b) a feline IgGla, IgGlb, or IgG2 Fc polypeptide; and/or
c) an equine IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7 Fc polypeptide.
Embodiment 15. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises a knob mutation. Embodiment 16. The heterodimeric protein of any one of the preceding embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises a hole mutation.
Embodiment 17. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at a position corresponding to position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or
b) an amino acid substitution at a position corresponding to position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) an amino acid substitution at a position corresponding to position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 18. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a tryptophan at a position corresponding to position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or b) a tryptophan at a position corresponding to position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) a tryptophan at a position corresponding to position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 19. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or
b) an amino acid substitution at position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) an amino acid substitution at position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 20. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a tryptophan at position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or b) a tryptophan at position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) a tryptophan at position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 21. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at a position corresponding to position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40, or position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; and/or
b) an amino acid substitution at a position corresponding to position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) an amino acid substitution at a position corresponding to position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 22. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 38, a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 39, a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 40, or a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position
corresponding to position 181 of SEQ ID NO: 41; and/or
b) a serine at a position corresponding to position 154 and/or an alanine at a position corresponding to position 156 and/or a threonine at a position corresponding to position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or c) a serine at a position corresponding to position 130 and/or an alanine at a position corresponding to position 132 and/or a threonine at a position corresponding to position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. Embodiment 23. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40, or position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; and/or
b) an amino acid substitution at position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or c) an amino acid substitution at position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 24. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 38, a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 39, a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 40, or a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 41; and/or b) a serine at position 154 and/or an alanine at position 156 and/or a threonine at position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) a serine at position 130 and/or an alanine at position 132 and/or a threonine at position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
Embodiment 25. The heterodimeric protein of any one of the preceding
embodiments, wherein the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101
Embodiment 26. The heterodimeric protein of any one of the preceding
embodiments, wherein the first contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113.
Embodiment 27. The heterodimeric protein of any one of the preceding
embodiments, wherein the second contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
Embodiment 28. An isolated nucleic acid encoding: a) the first contiguous polypeptide of any one of embodiments 1 to 27; b) the second contiguous polypeptide of any one of embodiments 1 to 27; or c) the first contiguous polypeptide and the second contiguous polypeptide of any one of embodiments 1 to 27.
Embodiment 29. A host cell comprising the nucleic acid of embodiment 28.
Embodiment 30. A host cell expressing: a) the first contiguous polypeptide of any one of embodiments 1 to 27; b) the second contiguous polypeptide of any one of embodiments 1 to 27; or c) the first contiguous polypeptide and the second contiguous polypeptide of any one of embodiments 1 to 27.
Embodiment 31. A method comprising culturing the host cell of embodiment 29 or embodiment 30 and isolating the first contiguous polypeptide, or the second contiguous polypeptide, or the first contiguous polypeptide and the second contiguous polypeptide.
Embodiment 32. A pharmaceutical composition comprising the heterodimeric protein of any one of embodiments 1 to 27 and a pharmaceutically acceptable carrier.
Embodiment 33. A method of treating a companion animal species having an IL13 and/or IL4-induced condition, the method comprising administering to the companion animal species a therapeutically effective amount of the heterodimeric protein of any one of
embodiments 1 to 27 or the pharmaceutical composition of embodiment 32.
Embodiment 34. The method of embodiment 33, wherein the companion animal species is canine, feline, or equine.
Embodiment 35. The method of embodiment 33 or embodiment 34, wherein the
IL13 and /or IL4-induced condition is a pruritic or allergic condition, such as atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema. Embodiment 36. The method of any one of embodiments 33 to 35, wherein the heterodimeric protein or the pharmaceutical composition is administered parenterally.
Embodiment 37. The method of any one of embodiments 33 to 36, wherein the heterodimeric protein or the pharmaceutical composition is administered by an intramuscular route, an intraperitoneal route, an intracerebrospinal route, a subcutaneous route, an intra-arterial route, an intrasynovial route, an intrathecal route, or an inhalation route.
Embodiment 38. The method of any one of embodiments 33 to 37, wherein the method further comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor.
Embodiment 39. The method of any one of embodiments 33 to 38, wherein the method further comprises administering one or more antibodies selected from an anti-ILl7 antibody, an anti-IL3 l antibody, an anti-TNFa antibody, an anti-CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-ILl3 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CDl la antibody, anti-IL6R antibody, anti-a4-Intergrin antibody, an anti-ILl2 antibody, an anti-IL 1 b antibody, and an anti-BlyS antibody.
Embodiment 40. A method of reducing IL13 and /or IL4 signaling activity in a cell, the method comprising exposing the cell to the heterodimeric protein of any one of
embodiments 1 to 27 or the pharmaceutical composition of embodiment 32 under conditions permissive for binding of the heterodimeric protein to IL13 and/or IL4, thereby (a) reducing binding of IL/4 and/or IL-13 to native IL13 receptor and/or native IL-4 receptor and reducing IL13- and/or IL-4-mediated signaling.
Embodiment 41. The method of embodiment 40, wherein the cell is exposed to the heterodimeric protein or the pharmaceutical composition ex vivo.
Embodiment 42. The method of embodiment 41, wherein the cell is exposed to the heterodimeric protein or the pharmaceutical composition in vivo.
Embodiment 43. The method of any one of embodiments 40 to 42, wherein the cell is a canine cell, a feline cell, or an equine cell.
Embodiment 44. A method for detecting IL13 or IL4 in a sample from a companion animal species comprising contacting the sample with the heterodimeric protein of any one of embodiments 1 to 27 or the pharmaceutical composition of embodiment 32 under conditions permissive for binding of the heterodimeric protein to IL13 and/or IL4, and detecting whether a complex is formed between the heterodimeric protein and IL13 and/or IL4 in the sample.
Embodiment 45. The method of embodiment 44, wherein the sample is a biological sample obtained from a canine, a feline, or an equine. BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a graph of canine IL4RECD-ILl3RECD-Fc sequential binding to canine
IL4 and IL13 or canine IL13 and IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS.
[0007] FIG. 2 is a graph of canine ILl3RECD-IL4RECD-Fc sequential binding to canine IL4 and IL13 or canine IL13 and IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS.
[0008] FIG. 3 is a graph of canine IL4RECD-ILl3RECD-Fc neutralizing canine IL4 activity in a TF1 cell proliferation assay. Canine IL4 (50 ng/mL or 3.85 nM) was used in the assay.
DESCRIPTION OF CERTAIN SEQUENCES
[0009] Table 1 provides a listing of certain sequences referenced herein.
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
DESCRIPTION OF THE EMBODIMENTS
[0010] IL13R/IL4R heterodimeric proteins that bind canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4 are provided. In some embodiments, the IL13R/IL4R heterodimeric protein comprises a first contiguous polypeptide comprising an extracellular domain of an IL13R polypeptide and an Fc polypeptide and a second contiguous polypeptide comprising an extracellular domain of an IL4R polypeptide and an Fc polypeptide. Methods of producing or purifying IL13R/IL4R heterodimeric proteins and contiguous polypeptides are also provided. Methods of treatment using IL13R/IL4R heterodimeric proteins to bind IL13 and/or IL4 and inhibit IL13- and/or IL-4-mediated signaling are provided. Such methods include, but are not limited to, methods of treating IL13- and/or IL4-induced conditions in companion animal species. Methods of detecting IL13 and/or IL4 in a sample from a companion animal species are also provided.
[0011] For the convenience of the reader, the following definitions of terms used herein are provided. [0012] As used herein, numerical terms such as Kd are calculated based upon scientific measurements and, thus, are subject to appropriate measurement error. In some instances, a numerical term may include numerical values that are rounded to the nearest significant figure.
[0013] As used herein,“a” or“an” means“at least one” or“one or more” unless otherwise specified. As used herein, the term“or” means“and/or” unless specified otherwise. In the context of a multiple dependent claim, the use of“or” when referring back to other claims refers to those claims in the alternative only.
Exemplary IL13R/IL4R Heterodimeric Proteins
[0014] Novel IL13R/IL4R heterodimeric proteins are provided, for example, heterodimeric proteins that bind canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4.
[0015] “Amino acid sequence,” means a sequence of amino acids residues in a peptide or protein. The terms“polypeptide” and“protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a“polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
[0016] The term “contiguous polypeptide” herein is used to mean an uninterrupted sequence of amino acids. A contiguous polypeptide is typically translated from a single continuous DNA sequence. It can be made by genetic engineering, for example, by removing the stop codon from the DNA sequence of the first protein, then appending the DNA sequence of the second protein in frame, so that the DNA sequence is expressed as a single protein. Typically, this is accomplished by cloning a cDNA into an expression vector in frame with an existing gene [0017] “IL4R,” as used herein, is a polypeptide comprising the entirety or a fragment of
IL4 receptor subunit alpha that bind to IL-4.
[0018] For example,“IL4R” refers to an IL4R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. In some embodiments, IL4R is an extracellular domain fragment that binds IL4. In some such embodiments, the IL4R may be referred to as an IL4R extracellular domain (ECD). In some embodiments, IL4R comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
[0019] “IL13R,” as used herein, is a polypeptide comprising the entirety or a portion of
IL13 receptor subunit alpha- 1 that binds to IL-13.
[0020] For example,“IL13R” refers to an IL13R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. In some embodiments, IL13R is an extracellular domain fragment that binds IL13. In some such embodiments, the IL13R may be referred to as an IL13R extracellular domain (ECD). In some embodiments, the IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
[0021] The term “companion animal species” refers to an animal suitable to be a companion to humans. In some embodiments, a companion animal species is a small mammal, such as a canine, feline, dog, cat, horse, rabbit, ferret, guinea pig, rodent, etc. In some embodiments, a companion animal species is a farm animal, such as a horse, cow, pig, etc.
[0022] An“extracellular domain” (“ECD”) is the portion of a polypeptide that extends beyond the transmembrane domain into the extracellular space. The term“extracellular domain,” as used herein, may comprise a complete extracellular domain or may comprise a truncated extracellular domain missing one or more amino acids, that binds to its ligand. The composition of the extracellular domain may depend on the algorithm used to determine which amino acids are in the membrane. Different algorithms may predict, and different systems may express, different extracellular domains for a given protein.
[0023] An extracellular domain of an IL4R polypeptide may comprise a complete extracellular domain or a truncated extracellular domain of IL4R that binds IL4. As used herein, the terms“extracellular domain of an IL4R polypeptide,”“IL4R ECD,” and similar terms refer to an IL4R polypeptide that does not comprise a transmembrane domain or cytoplasmic domain, even if the term follows an open transitional word, such as“comprising,”“comprises,” and the like. In some embodiments, an extracellular domain of an IL4R polypeptide is an extracellular domain of an IL4R polypeptide derived from a companion species animal. For example, in some embodiments, an extracellular domain of an IL4R polypeptide is derived from canine IL4R, feline IL4R or equine IL4R. In some embodiments, an extracellular domain of an IL4R polypeptide comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or any fragment thereof. In some embodiments, an extracellular domain of an IL4R polypeptide comprises the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, or any fragment thereof.
[0024] An extracellular domain of an IL13R polypeptide may comprise a complete extracellular domain or a truncated extracellular domain of IL13R that binds IL13. As used herein, the terms“extracellular domain of an IL13R polypeptide,”“IL13R ECD,” and similar terms refer to an IL13R polypeptide that does not comprise a transmembrane domain or cytoplasmic domain, even if the term follows an open transitional word, such as“comprising,”“comprises,” and the like. In some embodiments, an extracellular domain of an IL13R polypeptide is an extracellular domain of an IL13R polypeptide derived from a companion species animal. For example, in some embodiments, an extracellular domain of an IL13R polypeptide is derived from canine IL13R, feline IL13R or equine IL13R. In some embodiments, an extracellular domain of an IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26, or any fragment thereof. In some embodiments, an extracellular domain of an IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36, or any fragment thereof.
[0025] The terms“IL13R/IL4R heterodimeric protein” and“IL4R/IL13R heterodimeric protein” are used interchangeably to refer to a heterodimeric protein comprising a first contiguous polypeptide comprising an IL13R polypeptide and a second contiguous polypeptide comprising an IL4R polypeptide.
[0026] In some embodiments, the first contiguous polypeptide and/or second contiguous polypeptide comprises an Fc polypeptide.
[0027] The IL13R/IL4R heterodimeric protein of the invention may comprise an extracellular domain of a IL13R polypeptide and/or an extracellular domain of a IL4R polypeptide, wherein the polypeptides are derived from a companion animal species. For example, a heterodimeric protein may comprise an extracellular domain of an IL4R polypeptide from a dog, cat, or horse and/or may comprise an extracellular domain of an IL13R polypeptide from a dog, cat, or horse.
[0028] “Wild-type” refers to a non-mutated version of a polypeptide that occurs in nature, or a fragment thereof. A wild-type polypeptide may be produced recombinant!y. A“wildtype 1L13R ECD” or a“wildtype IL4R ECD” refers to a protein having an amino acid sequence that is identical to the same portion of an extracellular domain of an 11.13 R or IL4R that occurs in nature.
[0029] A“variant” is a nucleic acid molecule or polypeptide that differs from a referent nucleic acid molecule or polypeptide by single or multiple amino acid substitutions, deletions, and/or additions and substantially retains at least one biological activity of the referent nucleic acid molecule or polypeptide.
[0030] A“biologically active” entity, or an entity having“biological activity,” is an entity having any function related to or associated with a metabolic or physiological process, and/or having structural, regulatory, or biochemical functions of a naturally-occurring molecule. Biologically active polynucleotide fragments are those exhibiting similar activity, but not necessarily identical, to an activity of a polynucleotide of the present invention. A biologically active polypeptide or fragment thereof includes one that can participate in a biological reaction, including, but not limited to, a ligand-receptor interaction or antigen-antibody binding. The biological activity can include an improved desired activity, or a decreased undesirable activity. An entity may demonstrate biological activity when it participates in a molecular interaction with another molecule, such as hybridization, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide that may be detected as unique for the polynucleotide molecule, and when it can be used as a primer in a polymerase chain reaction (PCR).
[0031] As used herein,“percent (%) amino acid sequence identity” and“homology” with respect to a nucleic acid molecule or polypeptide sequence are defined as the percentage of nucleotide or amino acid residues in a referent sequence that are identical with the nucleotide or amino acid residues in the specific nucleic acid molecule or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared.
[0032] In some embodiments, a variant has at least about 50% sequence identity with the referent nucleic acid molecule or polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 50% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 98% sequence identity with the sequence of the referent nucleic acid or polypeptide.
[0033] In some embodiments, a contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide having at least 85%, at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36. In some embodiments, a contiguous polypeptide comprises an extracellular domain of an IL4R polypeptide having at least 85%, at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
[0034] As used herein,“position corresponding to position n,” wherein n is any number, refers to an amino acid position of a subject polypeptide that aligns with position n of a reference polypeptide after aligning the amino acid sequences of the subject and reference polypeptides and introducing gaps. Alignment for purposes of whether a position of a subject polypeptide corresponds with position n of a reference polypeptide can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any parameters needed to achieve maximal alignment over the full length of two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.
[0035] In some embodiments, the contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine at a position corresponding to position 18 of SEQ ID NO: 22, at a position corresponding to position 18 of SEQ ID NO: 24, or at a position corresponding to position 18 of SEQ ID NO: 26. In some embodiments, the contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine at position 18 of SEQ ID NO: 22, at position 18 of SEQ ID NO: 24, at position 18 of SEQ ID NO: 26, at position 15 of SEQ ID NO: 32, at position 15 of SEQ ID NO: 34, or at position 15 of SEQ ID NO: 36. [0036] A“point mutation” is a mutation that involves a single nucleotide or amino acid residue. The mutation may be the loss of a nucleotide or amino acid, substitution of one nucleotide or amino acid residue for another, or the insertion of an additional nucleotide or amino acid residue.
[0037] An amino acid substitution may include but is not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into a molecule of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.
[0038] Table 2
Figure imgf000040_0001
[0039] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[0040] Non-conservative substitutions will entail exchanging a member of one of these classes with another class.
[0041] A“fusion partner,” as used herein, refers to an additional component of an
IL13R/IL4R contiguous polypeptide, such as an additional polypeptide, such as albumin, an albumin binding fragment, or a fragment of an immunoglobulin molecule. A fusion partner may comprise an oligomerization domain such as an Fc domain of a heavy chain immunoglobulin
[0042] The term“IgX Fc” or“IgX Fc polypeptide” means the Fc region is derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where“X” denotes the antibody isotype. Thus,“IgG” or“IgG Fc” denotes the Fc region of a g chain,“IgA” or“IgA Fc” denotes the Fc region of an a chain,“IgD” or“IgD Fc” denotes the Fc region of a d chain,“IgE” or“IgE Fc” denotes the Fc region of an e chain,“IgM” or“IgM Fc” denotes the Fc region of a m chain, etc.
[0043] In some embodiments, the Fc polypeptide or the IgG Fc region comprises CH1, hinge, CH2, CH3, and CL1. In some embodiments, the IgG Fc polypeptide comprises the hinge, CH2, and CH3, but does not comprise CH1 or CL. In some embodiments, the IgG Fc polypeptide comprises CH2 and CH3, but does not comprise CH1, the hinge, or CL. In some embodiments, the IgG Fc polypeptide comprises CH1, hinge, CH2, and CH3, with or without CL1.
[0044] “IgXN Fc” or“IgXN Fc polypeptide” denotes that the Fc region is derived from a particular subclass of antibody isotype (such as canine IgG subclass A, B, C, or D; feline IgG subclass 1, 2a, or 2b; or equine IgG subclass IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where“N” denotes the subclass.
[0045] In some embodiments, IgX or IgXN regions are derived from a companion animal, such as a dog, a cat, or a horse. In some embodiments, IgG regions are isolated from canine g heavy chains, such as IgGA, IgGB, IgGC, or IgGD. In some instances, IgGFc regions are isolated from feline g heavy chains, such as IgGl a, IgGlb, or IgG2. In other instances, IgG regions are isolated from equine g heavy chains, such as IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7. Polypeptides comprising an Fc region of IgGA, IgGB, IgGC, or IgGD may provide for higher expression levels in recombination production systems. [0046] In some embodiments, a contiguous polypeptide comprises a first variant IgG Fc polypeptide comprising a“knob” mutation and a second variant IgG Fc polypeptide comprising a “hole” mutation. Nonlimiting exemplary knob and hole mutations are described, for example, in Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat Biotechnol, 16(7):677-81 (1998).
[0047] A“knob” mutation,” as used herein, refers to an interfacing mutation of a molecule
(e.g., an Fc polypeptide) that comprises a bulky amino acid.
[0048] A“hole mutation,” as used herein, refers to an interfacing mutation of a molecule
(e.g., an Fc polypeptide) that comprises one or more smaller amino acids.
[0049] In some embodiments, a variant IgG Fc polypeptide comprises a knob mutation.
In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
[0050] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO:
43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
[0051] In some embodiments, a variant IgG Fc polypeptide comprises a tryptophan at a position corresponding to position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41, or position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
[0052] In some embodiments, a variant IgG Fc polypeptide comprises a tryptophan at position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. [0053] In some embodiments, a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, 66, 67, 68, 69, 70, 81, 82, 83, 84, 85, 86, or 87.
[0054] In some embodiments, a variant IgG Fc polypeptide comprises a hole mutation. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40; position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
[0055] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40; position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
[0056] In some embodiments, a variant IgG Fc polypeptide comprises a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 38; a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 39; a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position
139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 40; a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position
140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 41; a serine at a position corresponding to position 154 and/or an alanine at a position corresponding to position 156 and/or a threonine at a position corresponding to position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or a serine at a position corresponding to position 130 and/or an alanine at a position corresponding to position 132 and/or a threonine at a position corresponding to position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. [0057] In some embodiments, a variant IgG Fc polypeptide comprises a serine at position
138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 38; a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 39; a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 40; a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 41; a serine at position 154 and/or an alanine at position 156 and/or a threonine at position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or a serine at position 130 and/or an alanine at position 132 and/or a threonine at position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
[0058] In some embodiments, a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101.
[0059] A“signal sequence” refers to a sequence of amino acid residues or polynucleotides encoding such, which facilitates secretion of a polypeptide of interest and is typically cleaved upon export of the polypeptide to the outside of the cell surface membrane.
[0060] A“linker” refers to one or more amino acid residues that connects a first polypeptide with a second polypeptide.
[0061] In some embodiments, the linker is a glycine-rich and/or serine-rich, flexible, non- structural linker. In some embodiments, a linker comprises the amino acids G (Gly) and/or S (Ser). For example, a linker may comprise G or a repeat of G (e.g., GG, GGG, etc.), GS or a repeat of GS (e.g., GSGS (SEQ ID NO: 114), GSGSGS (SEQ ID NO: 115), etc ); GGS or a repeat of GGS (e g., GGSGGS (SEQ ID NO: 116), GGS GGS GGS (SEQ ID NO: 117), etc ); GGGS (SEQ ID NO: 118) or a repeat of GGGS (SEQ ID NO: 119) (e.g., GGGS GGGS (SEQ ID NO: 120), GGGSGGGSGGGS (SEQ ID NO: 121), etc ); GSS or a repeat of GSS (e.g., GSSGSS (SEQ ID NO: 122), GSSGSSGSS (SEQ ID NO: 123), etc ); or GGSS (SEQ ID NO: 124) or a repeat of GGSS (SEQ ID NO: 125) (e.g., GGSSGGSS (SEQ ID NO: 126), GGSSGGSSGGSS (SEQ ID NO: 127), etc,).
[0062] In some embodiments, the contiguous polypeptide comprises at least one linker. In some embodiments, the contiguous polypeptide comprises an optional signal sequence, and at least one optional linker. In some embodiments, the contiguous polypeptide does not comprise a signal sequence, or a linker. In some embodiments, the contiguous polypeptide is translated with a signal sequence, but the signal sequence is cleaved from the contiguous polypeptide. [0063] In some embodiments, a heterodimeric protein comprises a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide and b) a second contiguous polypeptide comprising at least one IL4R ECD and a second Fc polypeptide, wherein the IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
[0064] In some embodiments, a first contiguous polypeptide or a second contiguous polypeptide has the formula:
ILl3R(n)-L-Fc or
IL4R(n)-L-Fc,
wherein ILl3R(n) is at least one IL13R extracellular domain (ECD) polypeptide derived from a companion animal species, IL4R(n) is at least one IL4R ECD polypeptide derived from a companion animal species, (n) is one, two, three, four, or more ECD polypeptides, L is an optional linker, Fc is a variant Fc polypeptide, such as a variant Fc polypeptide comprising knob or a hole mutation.
[0065] In addition, other binding partner(s) may be included in the contiguous polypeptide before, after, and/or between any one or more IL13R or IL4R ECD polypeptide(s). Other potential binding partners include: IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
[0066] In some embodiments, the heterodimeric protein comprises a first contiguous polypeptide comprising the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113.
[0067] In some embodiments, the heterodimeric protein comprises a second contiguous polypeptide comprising the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
Exemplary Expression and Production
[0068] Polynucleotide sequences that encode all or part (e.g., the extracellular domain) of a contiguous polypeptide with or without a signal sequence are provided. If a homologous signal sequence (i.e., a signal sequence of native IL-4R or IL13R) is not used in the construction of the nucleic acid molecule, then another signal sequence may be used, for example, any one of the signal sequences described in PCT/US06/02951.
[0069] Typically, nucleotide sequence encoding the polypeptide of interest, such as a contiguous polypeptide, is inserted into an expression vector, suitable for expression in a selected host cell.
[0070] A“vector” is a plasmid that can be used to transfer DNA sequences from one organism to another or to express a gene of interest. A vector typically includes an origin of replication and regulatory sequences which regulate the expression of the gene of interest, and may or may not carry a selective marker gene, such as an antibiotic resistance gene. A vector is suitable for the host cell in which it is to be expressed. A vector may be termed a“recombinant vector” when the gene of interest is present in the vector.
[0071] A“host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Nonlimiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, and their derivatives, such as 293-6E, DG44, CHO-S, and CHO-K cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) encoding an amino acid sequence(s) provided herein.
[0072] The term“isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as“isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be“isolating” the polypeptide. Similarly, a polynucleotide is referred to as“isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as“isolated.”
[0073] In some embodiments, the heterodimeric protein or contiguous polypeptide is isolated using chromatography, such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.
[0074] The terms“label” and“detectable label” mean a moiety attached to a IL13R/IL4R contiguous polypeptide to render it detectable. In some embodiments, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (for example, 3H, 14C, 35S, 90Y, "Tc, U1ln, 125I, 131I, 177Lu, 166HO, or 153Sm); chromogens, fluorescent labels (for example, FITC, rhodamine, lanthanide phosphors), enzymatic labels (for example, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, for example, acridinium compounds, and moieties that produce fluorescence, for example, fluorescein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety.
Exemplary IL13R/IL4R Heterodimeric Proteins as Decoy Receptor Traps
[0075] The IL13R/IL4R heterodimeric proteins of the invention can function as decoy receptors for trapping IL13 and/or IL4 and inhibiting their interaction with IL13R and/or IL4R on cell surfaces. Decoy receptors, such as those of the invention, recognize their ligands with high affinity and specificity but are structurally incapable of signaling. They compete with wild-type receptors for ligand binding and participate in ligand/receptor interactions, thus modulating the activity of or the number of functioning receptors and/or the cellular activity downstream from the receptors. Decoy receptors can act as molecular traps for agonist ligands and thereby inhibit ligand-induced receptor activation.
[0076] “IL13” as used herein refers to any native IL13 that results from expression and processing of IL13 in a cell. The term includes IL13 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. The term also includes naturally occurring variants of IL13, e.g., splice variants or allelic variants.
[0077] In some embodiments, a canine IL13 comprises the amino acid sequence of SEQ
ID NO: 4. In some embodiments, a feline IL13 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, an equine IL13 comprises the amino acid sequence of SEQ ID NO: 6.
[0078] “IL4” as used herein refers to any native IL4 that results from expression and processing of IL4 in a cell. The term includes IL4 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. The term also includes naturally occurring variants of IL4, e.g., splice variants or allelic variants.
[0079] In some embodiments, a canine IL4 comprises the amino acid sequence of SEQ ID
NO: 1. In some embodiments, a feline IL4 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, an equine IL4 comprises the amino acid sequence of SEQ ID NO: 3.
[0080] The invention provides IL13R/IL4R heterodimeric proteins as therapeutic agents.
The IL13R/IL4R heterodimeric proteins of the invention bind to IL13 and/or IL4, described in more detail herein, which have been demonstrated to be associated with allergic diseases. In various embodiments, IL13R/IL4R heterodimeric proteins can bind IL13 and/or IL4 with very high affinity. In various embodiments, the IL13R/IL4R heterodimeric proteins can interfere with IL13 and/or IL4 signaling.
[0081] The term“affinity” means the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, a receptor) and its binding partner (for example, a ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.
[0082] The terms“KD,”“Kd,”“Kd” or“Kd value” as used interchangeably to refer to the equilibrium dissociation constant of a receptor fusion - ligand interaction. In some embodiments, the Kd of the fusion molecule to its ligand is measured by using biolayer interferometry assays using a biosensor, such as an Octet® System (Pall ForteBio LLC, Fremont, CA) according to the supplier’s instructions. Briefly, biotinylated antigen is bound to the sensor tip and the association of fusion molecule is monitored for ninety seconds and the dissociation is monitored for 600 seconds. The buffer for dilutions and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer only blank curve is subtracted to correct for any drift. The data are fit to a 2: 1 binding model using ForteBio data analysis software to determine association rate constant (k0n), dissociation rate constant (k0ff), and the Kd. The equilibrium dissociation constant (Kd) is calculated as the ratio of k0ff/k0n. The term“kon” refers to the rate constant for association of a molecule X to its partner Y and the term“koff’ refers to the rate constant for dissociation of a molecule X or partner Y from the molecule X / partner Y complex.
[0083] The term“binds” to a substance is a term that is well understood in the art, and methods to determine such binding are also well known in the art. A molecule is said to exhibit “binding” if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, or etc.
[0084] Surface plasmon resonance” denotes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (BIAcore International AB, a GE Healthcare company, ETppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al. (1993) Ann. Biol. Clin.51: 19-26.
[0085] “Biolayer interferometry” refers to an optical analytical technique that analyzes the interference pattern of light reflected from a layer of immobilized protein on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real-time. A nonlimiting exemplary device for biolayer interferometry is an Octet® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem.377: 209-277.
[0086] In some embodiments, an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, or equine IL13 and/or IL4 with a dissociation constant (Kd) of less than 5 x 106 M, less than 1 x 106 M, less than 5 x 107 M, less than 1 x 107 M, less than 5 x 108 M, less than 1 x 108 M, less than 5 x 109 M, less than 1 x 109 M, less than 5 x 1010 M, less than 1 x 1010 M, less than 5 x 1011 M, less than 1 x 1011 M, less than 5 x 1012 M, or less than 1 x 1012 M, as measured by biolayer interferometry. In some embodiments, an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, or equine IL13 and/or IL4 with a Kd of between 5 x 106 M and 1 x 106 M, between 5 x 106 M and 5 x 107 M, between 5 x 106 M and 1 x 107 M, between 5 x 106 M and 5 x 108 M, 5 x 106 M and 1 x 108 M, between 5 x 106 M and 5 x 109 M, between 5 x 106 M and lx 109 M, between 5 x 106 M and 5 x 1010 M, between 5 x 106 M and 1 x 1010 M, between 5 x 106 M and 5 x 1011 M, between
5 x 106 M and 1 x 1011 M, between 5 x 106 M and 5 x 1012 M, between 5 x 106 M and 1x10 12 M, between 1 x 106 M and 5 x 107 M, between 1 x 106 M and 1 x 107 M, between 1 x 106 M and 5 x 108 M, 1 x 106 M and 1 x 108 M, between 1 x 106 M and 5 x 109 M, between 1 x 10
6 M and lx 109 M, between 1 x 106 M and 5 x 1010 M, between 1 x 106 M and 1 x 1010 M, between 1 x 106 M and 5 x 1011 M, between 1 x 106 M and 1 x 1011 M, between 1 x 106 M and 5 x 1012 M, between 1 x 106 M and 1 x 1012 M, between 5 x 107 M and 1 x 107 M, between 5 x 107 M and 5 x 108 M, 5 x 107 M and 1 x 108 M, between 5 x 107 M and 5 x 109 M, between 5 x 107 M and 1 x 109 M, between 5 x 107 M and 5 x 1010 M, between 5 x 107 M and 1 x 1010 M, between 5 x 107 M and 5 x 1011 M, between 5 x 107 M and 1 x 1011 M, between 5 x 107 M and 5 x 1012 M, between 5 x 107 M and 1 x 1012 M, between 1 x 107 M and 5 x 108 M, 1x10 7 M and lx 108 M, between 1 x 107 M and 5 x 109 M, between 1 x 107 M and lx 109 M, between 1 x 107 M and 5 x 1010 M, between 1 x 107 M and 1 x 1010 M, between 1 x 107 M and 5 x 1011 M, between 1 x 107 M and 1 x 1011 M, between 1 x 107 M and 5 x 1012 M, between 1 x 107 M and 1 x 1012 M, between 5 x 108 M and 1 x 108 M, between 5 x 108 M and 5 x 109 M, between 5 x 108 M and 1 x 109 M, between 5 x 108 M and 5 x 1010 M, between 5 x 108 M and 1 x 1010 M, between 5 x 108 M and 5 x 1011 M, between 5 x 108 M and 1 x 1011 M, between 5 x 108 M and 5 x 1012 M, between 5 x 108 M and 1 x 1012 M, 1 x 108 M and 5 x 109 M, between
I x 108 M and 1 x 109 M, between 1 x 108 M and 5 x 1010 M, between 1 x 108 M and 1 x 1010 M, between 1 x 108 M and 5 x 1011 M, between 1 x 108 M and 1 x 1011 M, between 1 x 108 M and 5 x 1012 M, between 1 x 108 M and 1 x 1012 M, between 5 x 109 M and 1 x 109 M, between 5 x 109 M and 5 x 1010 M, between 5 x 109 M and 1 x 1010 M, between 5 x 109 M and 5x10
II M, between 5 x 109 M and 1 x 1011 M, between 5 x 109 M and 5 x 1012 M, between 5 x 109 M and 1 x 1012 M, between 1 x 109 M and 5 x 1010 M, between 1 x 109 M and 1 x 1010 M, between 1 x 109 M and 5 x 1011 M, between 1 x 109 M and 1 x 1011 M, between 1 x 109 M and 5 x 1012 M, between 1 x 109 M and 1 x 1012 M, between 5 x 1010 M and 1 x 1010 M, between 5 x 1010 M and 5 x 1011 M, between, 1 x 1010 M and 5 x 1011 M, 1 x 1010 M and 1 x 1011 M, between 1 x 1010 M and 5 x 1012 M, between 1 x 1010 M and 1 x 1012 M, between 5 x 1011 M and 1 x 1012 M, between 5 x 1011 M and 5 x 1012 M, between 5 x 1011 M and 1 x 1012 M, between 1 x 1011 M and 5 x 1012 M, or between 1 x 1011 M and 1 x 1012 M, as measured by biolayer interferometry. In some embodiments, an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4.
[0087] To“reduce” or“inhibit” means to decrease, reduce, or arrest an activity, function, or amount as compared to a reference. In some embodiments, by“reduce” or“inhibit” is meant the ability to cause an overall decrease of 20% or greater. In some embodiments, by“reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In some embodiments, by“reduce” or“inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time. A“reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy or non-diseased sample. In some examples, a reference is obtained from a non-diseased or non-treated sample of a companion animal. In some examples, a reference is obtained from one or more healthy animals of a particular species, which are not the animal being tested or treated. [0088] The term“substantially reduced,” as used herein, denotes a sufficiently high degree of reduction between a numeric value and a reference numeric value such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. In some embodiments, the substantially reduced numeric values is reduced by greater than about any one of 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.
[0089] In some embodiments, an IL13R/IL4R heterodimeric protein may reduce IL13 and/or IL4 signaling in a companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL13 and/or IL4 signaling in the absence of the fusion molecule. In some embodiments, signaling is measured by a reduction in IL4-depedent TF-l cell proliferation. In some embodiments, the reduction in IL13 and/or IL4 signaling or the reduction in proliferation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.
Exemplary Pharmaceutical Compositions
[0090] The terms“pharmaceutical formulation” and“pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.
[0091] A“pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a“pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinyl pyrrolidone, cellulose- based substances; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.
[0092] The pharmaceutical composition can be stored in lyophilized form. Thus, in some embodiments, the preparation process includes a lyophilization step. The lyophilized composition may then be reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to the dog, cat, or horse. In other embodiments, particularly where the fusion molecule is highly stable to thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., as an aqueous composition, which may be administered directly, or with appropriate dilution, to the dog, cat, or horse. A lyophilized composition can be reconstituted with sterile Water for Injection (WFI). Bacteriostatic reagents, such benzyl alcohol, may be included. Thus, the invention provides pharmaceutical compositions in solid or liquid form.
[0093] The pH of the pharmaceutical compositions may be in the range of from about pH
5 to about pH 8, when administered. The compositions of the invention are sterile if they are to be used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Sterility may be maintained with or without anti -bacterial agents.
Exemplary Uses of IL13R/IL4R Heterodimeric Proteins and Pharmaceutical Compositions
[0094] The IL13R/IL4R heterodimeric proteins or pharmaceutical compositions comprising the IL13R/IL4R heterodimeric proteins of the invention may be useful for treating an IL13- and/or IL4-induced condition. As used herein, an“IL13 or IL4-induced condition” means a disease associated with, caused by, or characterized by, elevated levels or altered distribution of IL13 or IL4. Such IL13 and/or IL4-induced conditions include, but are not limited to, a pruritic or an allergic disease. In some embodiments, the IL13- and/or IL4-induced condition is atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema. An IL13- or IL4-induced condition may be exhibited in a companion animal, including, but not limited to, canine, feline, or equine.
[0095] As used herein,“treatment” is an approach for obtaining beneficial or desired clinical results. “Treatment” as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a companion animal. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by“treatment” is a reduction of pathological consequence of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one- hundred percent removal of all aspects of the disorder.
[0096] In some embodiments, an IL13R/IL4R heterodimeric proteins or pharmaceutical compositions comprising it can be utilized in accordance with the methods herein to treat IL13- or IL4-induced conditions. In some embodiments, an IL13R/IL4R heterodimeric proteins or pharmaceutical compositions is administered to a companion animal, such as a canine, a feline, or equine, to treat an IL13- and IL4-induced condition.
[0097] A “therapeutically effective amount” of a substance/molecule, agonist or antagonist may vary according to factors such as the type of disease to be treated, the disease state, the severity and course of the disease, the type of therapeutic purpose, any previous therapy, the clinical history, the response to prior treatment, the discretion of the attending veterinarian, age, sex, and weight of the animal, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the animal. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
[0098] In some embodiments, IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered parenterally, by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, an IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, an IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered by an intramuscular, an intraperitoneal, an intracerebrospinal, a subcutaneous, an intra-arterial, an intrasynovial, an intrathecal, or an inhalation route.
[0099] An IL13R/IL4R heterodimeric protein described herein may be administered in an amount in the range of 0.1 mg/kg body weight to 100 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 0.1 mg/kg body weight to 50 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 1 mg/kg body weight to 10 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 0.5 mg/kg body weight to 100 mg/kg body, in the range of 1 mg/kg body weight to 100 mg/kg body weight, in the range of 5 mg/kg body weight to 100 mg/kg body weight, in the range of 10 mg/kg body weight to 100 mg/kg body weight, in the range of 20 mg/kg body weight to 100 mg/kg body weight, in the range of 50 mg/kg body weight to 100 mg/kg body weight, in the range of 1 mg/kg body weight to 10 mg/kg body weight, in the range of 5 mg/kg body weight to 10 mg/kg body weight, in the range of 0.5 mg/kg body weight to 10 mg/kg body weight, or in the range of 5 mg/kg body weight to 50 mg/kg body weight.
[00100] An IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein can be administered to a companion animal at one time or over a series of treatments. For example, IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein may be administered at least once, more than once, at least twice, at least three times, at least four times, or at least five times.
[00101] In some embodiments, the dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more weeks of no treatment. In other embodiments, the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more days or weeks of no treatment.
[00102] Administration“in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order. The term“concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered with a time separation of no more than about a specified number of minutes. The term“sequentially” is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s), or wherein administration of one or more agent(s) begins before the administration of one or more other agent(s). For example, administration of the two or more therapeutic agents are administered with a time separation of more than about a specified number of minutes. As used herein,“in conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the animal.
[00103] In some embodiments, the method comprises administering in combination with an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein, a Jak inhibitor, a PI3K inhibitor, an ART inhibitor, or a MAPK inhibitor. In some embodiments, the method comprises administering in combination with an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R contiguous polypeptide, an anti-ILl7 antibody, an anti-TNFa antibody, an anti- CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL3 l antibody, an anti- IL23 antibody, an anti-IgE antibody, an anti-CDl la antibody, anti-IL6R antibody, anti-a4- Intergrin antibody, an anti-ILl2 antibody, an anti-IL 1 b antibody, or an anti-BlyS antibody. [00104] Provided herein are methods of exposing to a cell an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein under conditions permissive for binding to IL13 and/or IL4. In some embodiments, the cell is exposed to the IL13R/IL4R heterodimeric protein or pharmaceutical composition ex vivo. In some embodiments, the cell is exposed to the IL13R/IL4R heterodimeric protein or pharmaceutical composition in vivo. In some embodiments, a cell is exposed to the IL13R/IL4R heterodimeric protein. In some embodiments, a cell is exposed to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition under conditions permissive for binding of the heterodimeric protein to extracellular IL13 and/or IL4. In some embodiments, a cell may be exposed in vivo to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition by any one or more of the administration methods described herein, including but not limited to, intraperitoneal, intramuscular, intravenous injection into the subject. In some embodiments, a cell may be exposed ex vivo to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition by exposing the cell to a culture medium comprising the heterodimeric protein or the pharmaceutical composition. In some embodiments, the permeability of the cell membrane may be affected using any number of methods understood by those of skill in the art (such as electroporating the cells or exposing the cells to a solution containing calcium chloride) before exposing the cell to a culture medium comprising the fusion molecule or the pharmaceutical composition.
[00105] In some embodiments, the exposure results in a reduction of IL13 and/or IL4 signaling function by the cell. In some embodiments, an IL13R/IL4R heterodimeric protein may reduce IL13 and/or IL4 signaling in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL13 and/or IL4 signaling function in the absence of the IL13R/IL4R heterodimeric protein. In some embodiments, the reduction in IL13 and/or IL4 signaling and/or the reduction in TF-l proliferation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.
[00106] Provided herein are methods of using the IL13R/IL4R heterodimeric protein, polypeptides and polynucleotides for detection, diagnosis and monitoring of an IL13- or IL4- induced condition. Provided herein are methods of determining whether a companion animal will respond to IL13R/IL4R heterodimeric protein therapy. In some embodiments, the method comprises detecting whether the animal has cells that express IL13 or IL4 using an IL13R/IL4R heterodimeric protein. In some embodiments, the method of detection comprises contacting the sample with an antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from that of a reference or comparison sample (such as a control). In some embodiments, the method may be useful to determine whether the IL13R/IL4R heterodimeric protein described herein are an appropriate treatment for the subject animal.
[00107] In some embodiments, the sample is a biological sample. The term“biological sample” means a quantity of a substance from a living thing or formerly living thing. In some embodiments, the biological sample is a cell or cell/tissue lysate. In some embodiments, the biological sample includes, but is not limited to, blood, (for example, whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.
[00108] In some embodiments, the cells or cell/tissue lysate are contacted with an
IL13R/IL4R heterodimeric protein and the binding between the IL13R/IL4R heterodimeric protein and the cell is determined. When the test cells show binding activity as compared to a reference cell of the same tissue type, it may indicate that the subject would benefit from treatment with an IL13R/IL4R heterodimeric protein. In some embodiments, the test cells are from tissue of a companion animal.
[00109] Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays which can be conducted include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An indicator moiety, or label group, can be attached to the subject antibodies and is selected to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Appropriate labels include, without limitation, radionuclides (for example 125I, 131I, 35S, 3H, or 32P), enzymes (for example, alkaline phosphatase, horseradish peroxidase, luciferase, or p-galactosidase), fluorescent moieties or proteins (for example, fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (for example, Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). General techniques to be used in performing the various immunoassays noted above are known to those of ordinary skill in the art.
[00110] For purposes of diagnosis, the IL13R/IL4R heterodimeric protein can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to polypeptides are known in the art. In some embodiments, the IL13R/IL4R heterodimeric protein need not be labeled, and the presence thereof can be detected, for example, using an antibody that binds to the IL13R/IL4R heterodimeric protein. In some embodiments, the IL13R/IL4R heterodimeric protein can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). The anti-ILl3 and IL4 antibodies and polypeptides can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody or the polypeptide is labeled with a radionuclide (such as U1ln, "Tc, 14C, 131I, 125I, 3H, or any other radionuclide label, including those outlined herein) so that the cells or tissue of interest can be localized using immunoscintiography. The IL13R/IL4R heterodimeric protein may also be used as staining reagent in pathology using techniques well known in the art.
[00111] In some embodiments, a IL13R/IL4R heterodimeric protein is used for a diagnostic and a IL13R/IL4R heterodimeric protein is used as a therapeutic. In some embodiments, the first and second IL13R/IL4R heterodimeric proteins are different.
[00112] The following examples illustrate particular aspects of the disclosure and are not intended in any way to limit the disclosure. EXAMPLES
Example 1
Expression and purification of canine IL4 and IL13
[00113] A nucleotide sequence encoding canine IL13 protein (SEQ ID NO: 4) was synthesized with poly-His tag on the C-terminal end and cloned into a mammalian expression vector and transfected to 293 cells or CHOS. The same method was used to clone and express a nucleotide sequence encoding canine IL4 protein (SEQ ID NO: 1) with a poly-His tag on the C- terminal end.
[00114] The supernatant containing canine IL13 protein was collected and filtered. Canine IL13 was affinity purified using Ni-NTA column (CaptivA® Protein A Affinity Resin, Repligen). The same method was used to purify canine IL4.
Example 2
Extracellular Domains of IL13R and IL4R
[00115] Extracellular domains of canine, feline, and equine IL4R that are responsible for binding canine, feline and equine IL4 and/or IL13 were identified and boundaries were defined. Full-length extracellular domains of canine IL4R, feline IL4R, and equine IL4 were identified as SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27, respectively. Extracellular domain fragments of canine IL4R, feline IL4R, and equine IL4R postulated to retain biological activity were identified as SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, respectively.
[00116] Extracellular domains of canine, feline, and equine IL13R that are responsible for binding canine, feline, and equine IL4 and/or IL13 were identified and boundaries were defined. Full length extracellular domains of canine IL13R, feline IL13R, and equine IL13R were identified as SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively. Extracellular domain fragments of canine IL13R, feline IL13R, and equine IL13R postulated to retain biological activity were identified as SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively.
[00117] An unpaired cysteine (Cys) in canine IL13R (at position 18 of SEQ ID NO: 22), feline IL13R (at position 18 of SEQ ID NO: 24), and equine IL13R (at position 18 of SEQ ID NO: 26) was identified informatically and determined as embedded (unexposed) based on 3-D modeling. It is unlikely that the unpaired cysteine will form disulfide bonds and the likelihood of aggregation is low. Thus, site-directed mutagenesis of this Cys residue was not introduced.
Example 3 Expression and Purification of canine IL13R/IL4R contiguous polypeptides from CHO Cells
[00118] Nucleotide sequences encoding canine IL13R ECD/IL4R ECD contiguous polypeptides linked to an IgGB Fc polypeptide were designed with a signal sequence. For contiguous polypeptide“ILl3RECD-IL4RECD-IgGB Fc” (SEQ ID NO: 20), an extracellular domain of IL13R (SEQ ID NO: 22) precedes an extracellular domain of IL4R (SEQ ID NO: 23). For contiguous polypeptide“IL4RECD-ILl3RECD-IgGB Fc” (SEQ ID NO: 21), an extracellular domain of IL4R precedes an extracellular domain of IL13R.
[00119] The nucleotide sequences were synthesized chemically and inserted into an expression vector suitable for transfection into a CHO host cell. After transfection into CHO cells, the fusion proteins were secreted from the cell. For example, fusion protein was purified by single step Protein A column chromatography.
[00120] Each of IL 13RECD-IL4RECD-IgGB Fc and IL4RECD-IL 13RECD-IgGB Fc may be expressed and purified in a single step with a protein A column or other chromatographic methods, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed mode column chromatography such as CHT, or multimodal mode column chromatography such as CaptoMMC. Low pH or other viral inactivation and viral removal steps can be applied. The purified protein may be admixed with excipients, and sterilized by filtration to prepare a pharmaceutical composition of the invention. The pharmaceutical composition may be administered to a dog with an atopic dermatitis or asthma in an amount sufficient to bind and/or inhibit either IL13 and/or IL4.
[00121] The vectors were then used to perform pilot-scale transfection in CHO-S cells using the FreestyleMax™ transfection reagent (Life Technologies). The supernatant was harvested by clarifying the conditioned media. Protein was purified with a single pass Protein A chromatography step and used for further investigation.
Example 4
Demonstration of IL13 and IL4 Binding Activity
[00122] This example demonstrates that both ILl3RECD-IL4RECD-IgGB Fc (SEQ ID NO:20) and IL4RECD-ILl3RECD-IgGB Fc (SEQ ID NO:2l) bind canine IL4 and IL13 with kinetics requisite for therapeutic activity.
[00123] The binding analysis was performed using a biosensor Octet as follows. Briefly, canine IL4 (produced using 293 cells) was biotinylated. The free unreacted biotin was removed from biotinylated IL4 by extensive dialysis. Biotinylated canine IL4 was captured on streptavidin sensor tips. The IL4 association with various concentrations (12, 16, and 44 nM) of IL13RECD- IL4RECD-IgGB Fc (SEQ ID NO:20) was monitored for ninety seconds. Dissociation was monitored for 600 seconds. A buffer only blank curve was subtracted to correct for any drift. The data were fit to a 1 : 1 binding model using ForteBio™ data analysis software to determine the k0n, koff, and the Kd. The buffer for dilutions and all binding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd for ILl3RECD-IL4RECD-IgGB Fc and ligand IL4 was 8 xlO 11.
[00124] The canine IL4 association with various concentrations (40.7, and 140 nM) of IL4RECD-ILl3RECD-IgGB Fc (SEQ ID NO:2l) was monitored for ninety seconds. Dissociation was monitored for 600 seconds. A buffer only blank curve was subtracted to correct for any drift. The data were fit to a 1 : 1 binding model using ForteBio™ data analysis software to determine the kon, koff, and the Kd. The buffer for dilutions and all binding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd for IL4RECD -IL 13 RECD-IgGB Fc and ligand IL4 was 1.1 xlO 11.
[00125] Canine IL4 and canine IL13 with C-terminal polyHis tag was expressed and purified from 293 cells. EZ-Link NHS-LC-biotin was obtained from Thermo Scientific (Cat. #21336), and Streptavidin biosensors was obtained from ForteBio (Cat. #18-509).
[00126] IL4 and IL13 sequential binding experiments with ILl3RECD-IL4RECD-IgGB Fc (SEQ ID NO:20) were performed. Biotinylated canine ILl3RECD-IL4RECD-IgGB Fc was captured on streptavidin sensor tips. Canine ILl3RECD-IL4RECD-IgGB Fc was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS (Figure 1). The experiments demonstrated that once ILl3RECD-IL4RECD-IgGB Fc bound to IL13, it may not bind to IL4, and that once bound to IL4, its ability to bind IL13 is reduced.
[00127] IL4 and IL13 sequential binding experiments with IL4RECD-ILl3RECD-IgGB Fc (SEQ ID NO:2l) were performed. Biotinylated canine IL4RECD-ILl3RECD-IgGB Fc was captured on streptavidin sensor tips. Canine IL4RECD-ILl3RECD-IgGB Fc was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4 using concentrations of 30pg/mL of IL4 and IL13 in PBS (Figure 2). These experiments demonstrated that once IL4RECD-ILl3RECD-IgGB Fc bound to IL13, it may not bind to IL4, and that once bound to IL4, its ability to bind IL13 is reduced.
[00128] The tight binding of IL 13 RECD-IL4RECD -IgGB Fc and IL4RECD-IL13RECD- IgGB Fc to IL4 or IL13 is thought to be due to simultaneous binding contributions made by both IL4RECD and IL13RECD. Example 5
Cellular functional activity of canine IL4RECD-ILl3RECD-Fc (SINK)
[00129] TF1 cells (ATCC cat# CRL-2003), a human Erythroleukemic cell line which expresses endogenous interleukin 4 receptors on cell surface, was used in a proliferation assay. Cells grown in RPMI1640 (Gibco, Cat#l 1875) supplemented with 10% Fetal Bovine Serum, heat inactivated (Sigma, Cat#2868) and 2 nM/ml Human GM-CSF (R&D System, Cat# 215-GM-010) at exponential growth phase were used for the assay. Cells were washed with PBS twice and resuspended in above medium without GM-CSF. 20,000 cells per well were plated in a 96-well plate (Corning, Cat# 3610). Canine IL4RECD-ILl3RECD-IgGB Fc (SINK) was added at a series of dilutions followed by addition of canine IL4 (Sino Biological Inc, Cat# 70021-DNAE-5) at 50 ng/ml. The cells were incubated in 37°C, 5% C02 for 48 hours in a total volume of 100 pl. At the end of the incubation, the cells were cooled in room temperature and assayed for proliferation/variability by measuring cellular ATP content using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat# G7570).
[00130] In this assay, 100 pl premixed reagent A and B were added to each well. After shaking on an orbital shaker for 2 mins, the cells were lysed. Mono-oxygenation of luciferin was catalyzed by luciferase in the presence of Mg2+ and ATP that presented in cells, resulting in the generation of a luminescent signal proportional to the amount of ATP in the cells. The amount of ATP is directly proportional to the number of cells present in culture. The plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal and luminescence was detected using a Synergy HT microplate reader (Biotek, Winooski, VT).
[00131] The data were analyzed using 4 parameter logistic fit and IC50 is 2.0 nM. See Figure 3.
Example 6
Canine, feline, and equine IgG Fc polypeptides for IL31R and IL4R heterodimeric proteins
[00132] Pairs of variant canine IgG Fc polypeptides, variant feline IgG Fc polypeptides, and variant equine IgG Fc polypeptides were designed such that a knob-in-hole heterodimerization approach may be used to prepare heterodimeric proteins comprising at least one IL31R ECD and at least one IL4R ECD. First, pairing of two Fc polypeptides was designed by introducing CH3 interfacing mutations so that a first Fc polypeptide comprises a bulky amino acid (knob) and a second Fc polypeptide comprises smaller amino acids in the same general location (hole). [00133] An amino acid substitution of threonine to tryptophan at a position corresponding to position 138 of canine IgG-A (SEQ ID NO: 38), at a position corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 39), at a position corresponding to position 137 of canine IgG-C Fc (SEQ ID NO: 40), or at a position corresponding to position 138 of canine IgG-D Fc (SEQ ID NO: 41) (T138W or T137W) can be introduced as a knob. Examples of amino acid sequences of a first variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptide comprising a knob mutation are SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively.
[00134] An amino acid substitution of threonine to serine at a position corresponding to position 138 and/or of leucine to alanine at a position corresponding to position 140 and/or of tyrosine to threonine at a position corresponding to position 180 of canine IgG-A (SEQ ID NO: 38) or of IgG-D (SEQ ID NO: 41) (T138S, L140A, and/or Y180T); or of threonine to serine at a position corresponding to position 137 and/or of leucine to alanine at a position corresponding to position 139 and/or of tyrosine to threonine at a position corresponding to position 179 of canine IgG-B Fc (SEQ ID NO: 39) or of IgG-C (SEQ ID NO: 40) (T137S, L139A, and/or Y179T) can be introduced as a hole. Examples of amino acid sequences of a second variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptides comprising a hole mutation are SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65.
[00135] An amino acid substitution of threonine to tryptophan at a position corresponding to position 154 of feline IgGla Fc (SEQ ID NO: 42 or SEQ ID NO: 43), feline IgGlb Fc (SEQ ID NO: 44 or SEQ ID NO: 45), or of feline IgG2 (SEQ ID NO: 46) (T154W) can be introduced as a knob. Examples of amino acid sequences of a first variant feline IgGla, and IgGlb, and IgG2 Fc polypeptide comprising a knob mutation are SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.
[00136] An amino acid substitution of threonine to serine at a position corresponding to position 154 and/or of leucine to alanine at a position corresponding to position 156 and/or of tyrosine to threonine at a position corresponding to position 197 of feline IgGla (SEQ ID NO: 42 or SEQ ID NO: 43), feline IgG-b Fc (SEQ ID NO: 44 or SEQ ID NO: 45), or feline IgG2 Fc (SEQ ID NO: 46) (T154S, L156A, and/or Y(l97)T) can be introduced as a hole. Examples of amino acid sequences of a second variant feline IgGla, IgGlb, IgG2 Fc polypeptide comprising a hole mutation are SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.
[00137] An amino acid substitution of threonine to tryptophan at a position corresponding to position 130 of equine IgGl Fc (SEQ ID NO: 47), of equine IgG2 Fc (SEQ ID NO: 48), of equine IgG3 Fc (SEQ ID NO: 49), of equine IgG4 Fc (SEQ ID NO: 50), of equine IgG5 Fc (SEQ ID NO: 51), ofequine IgG6 Fc (SEQ ID NO: 52), or ofequine IgG7 Fc (SEQ ID NO: 53) (Tl30W) can be introduced as a knob. Examples of amino acid sequences of a first variant equine IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides comprising a knob mutation are SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively.
[00138] An amino acid substitution of threonine to serine at a position corresponding to position 130 and/or of leucine to alanine at a position corresponding to position 132 and/or of tyrosine to threonine at a position corresponding to position 173 of equine IgGl Fc (SEQ ID NO: 47), of equine IgG2 Fc (SEQ ID NO: 48), of equine IgG3 Fc (SEQ ID NO: 49), of equine IgG4 Fc (SEQ ID NO: 50), of equine IgG5 Fc (SEQ ID NO: 51), of equine IgG6 Fc (SEQ ID NO: 52), or of equine IgG7 Fc (SEQ ID NO: 53) (T130W, L(l32)A, and/or Y(l73)T) can be introduced as a hole. Examples of amino acid sequences of a second variant equine IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides comprising a hole mutation are SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101.
Example 7
IL13R/IL4R ECD Heterodimeric Proteins
[00139] In addition to contiguous IL13R/IL4R ECD polypeptide formats, heterodimeric protein pairs may have the following formats:
Heterodimeric protein A:
Polypeptide 1 : ILl3R(n)-L-Fcl and
Polypeptide 2: IL4R(n)-L-Fc2; or
Heterodimeric protein B:
Polypeptide 1 : IL4R(n)-L-Fcl and
Polypeptide 2: ILl3R(n)-L-Fc2,
wherein ILl3R(n) is at least one IL13R extracellular domain (ECD) polypeptide derived from a companion animal species, IL4R(n) is at least one IL4R ECD polypeptide derived from a companion animal species, (n) is one, two, three, four, or more ECD polypeptides, L is an optional linker, Fcl is a variant Fc polypeptide, such as a variant Fc polypeptide comprising knob mutation, Fc2 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a hole mutation. An optional linker could also be used between multiple ECD polypeptides. In addition, other binding partner(s) may be included before, after, and/or between any one or more ECD polypeptide(s). Other potential binding partners include: IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
[00140] Examples of pairs of contiguous polypeptide 1 and 2 that may form a heterodimeric protein include SEQ ID NOs 102 and 103, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 108 and 109, SEQ ID NOs: 110 and 111, and SEQ ID NOs: 112 and 113. A host cell may be cotransfected with vectors expressing these contiguous polypeptide pairs to produce the heterodimeric proteins described.

Claims

1. A heterodimeric protein comprising:
a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide, and
b) a second contiguous polypeptide comprising at least one IL4R ECD and a second Fc polypeptide,
wherein the IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
2. The heterodimeric protein of claim 1, wherein the first contiguous polypeptide and/or the second contiguous polypeptide comprises one, two, three, or four IL4R ECDs and/or one, two, three, or four IL13R ECDs.
3. The heterodimeric protein of any one of the preceding claims, wherein the first contiguous polypeptide and/or the second contiguous polypeptide further comprises at least one binding partner other than IL4R ECD or IL13R ECD.
4. The heterodimeric protein of claim 3, wherein the at least one binding partner comprises IL5, IL6, IL17, IL22, IL31, LFA-l, TNF-a, TSLP, and/or IgE.
5. The heterodimeric protein of any one of the preceding claims, wherein the heterodimeric protein binds to IL13 and/or IL4 with a dissociation constant (Kd) of less than 5 x 10 6 M, less than 1 x 10 6 M, less than 5 x 10 7 M, less than 1 x 10 7 M, less than 5 x 10 8 M, less than 1 x 10 8 M, less than 5 x 10 9 M, less than 1 x 10 9 M, less than 5 x 10 10 M, less than 1 x 10 10 M, less than 5 x 10 11 M, less than 1 x 10 11 M, less than 5 x 10 12 M, or less than 1 x 10 12 M, as measured by biolayer interferometry.
6. The heterodimeric protein of any one of the preceding claims, wherein the heterodimeric protein reduces IL13 and/or IL4 signaling in a companion animal species.
7. The heterodimeric protein of any one of the preceding claims, wherein the companion animal species is canine, feline, or equine.
8. The heterodimeric protein of any one of the preceding claims, wherein the amino acid sequence of the at least one IL13R ECD is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
9. The heterodimeric protein of any one of the preceding claims, wherein the amino acid sequence of the at least one IL13R ECD comprises a cysteine at a position corresponding to position 18 of SEQ ID NO: 22, corresponding to position 18 of SEQ ID NO: 24, or
corresponding to position 18 of SEQ ID NO: 26.
10. The heterodimeric protein of any one of the preceding claims, wherein the amino acid sequence of the at least one IL13R ECD comprises a cysteine at position 18 of SEQ ID NO: 22, at position 18 of SEQ ID NO: 24, at position 18 of SEQ ID NO: 26, at position 15 of SEQ ID NO: 32, at position 15 of SEQ ID NO: 34, or at position 15 of SEQ ID NO: 36.
11. The heterodimeric protein of any one of the preceding claims, wherein the at least one IL13R ECD comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36.
12. The heterodimeric protein of any one of the preceding claims, wherein the amino acid sequence of the at least one IL4R ECD is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
13. The heterodimeric protein of any one of the preceding claims, wherein the at least one IL4R ECD comprises an amino acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37.
14. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide and/or the second Fc polypeptide is: a) a canine IgG-A, IgG-B, IgG-C, or IgG-D Fc polypeptide; b) a feline IgGla, IgGlb, or IgG2 Fc polypeptide; and/or c) an equine IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7 Fc polypeptide.
15. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises a knob mutation.
16. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises a hole mutation.
17. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at a position corresponding to position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or
b) an amino acid substitution at a position corresponding to position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) an amino acid substitution at a position corresponding to position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
18. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a tryptophan at a position corresponding to position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or b) a tryptophan at a position corresponding to position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) a tryptophan at a position corresponding to position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
19. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or
b) an amino acid substitution at position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) an amino acid substitution at position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
20. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a tryptophan at position 138 of SEQ ID NO: 38, position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40, or position 138 of SEQ ID NO: 41; and/or
b) a tryptophan at position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or c) a tryptophan at position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
21. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at a position corresponding to position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40, or position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; and/or
b) an amino acid substitution at a position corresponding to position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) an amino acid substitution at a position corresponding to position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
22. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 38, a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 39, a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 40, or a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position
corresponding to position 181 of SEQ ID NO: 41; and/or
b) a serine at a position corresponding to position 154 and/or an alanine at a position corresponding to position 156 and/or a threonine at a position corresponding to position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or c) a serine at a position corresponding to position 130 and/or an alanine at a position corresponding to position 132 and/or a threonine at a position corresponding to position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
23. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) an amino acid substitution at position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39, position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40, or position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; and/or
b) an amino acid substitution at position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or c) an amino acid substitution at position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
24. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises:
a) a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 38, a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 39, a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 40, or a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 41; and/or b) a serine at position 154 and/or an alanine at position 156 and/or a threonine at position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or
c) a serine at position 130 and/or an alanine at position 132 and/or a threonine at position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
25. The heterodimeric protein of any one of the preceding claims, wherein the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:
54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101.
26. The heterodimeric protein of any one of the preceding claims, wherein the first contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113.
27. The heterodimeric protein of any one of the preceding claims, wherein the second contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
28. An isolated nucleic acid encoding: a) the first contiguous polypeptide of any one of claims 1 to 27; b) the second contiguous polypeptide of any one of claims 1 to 27; or c) the first contiguous polypeptide and the second contiguous polypeptide of any one of claims 1 to 27.
29. A host cell comprising the nucleic acid of claim 28.
30. A host cell expressing: a) the first contiguous polypeptide of any one of claims 1 to 27; b) the second contiguous polypeptide of any one of claims 1 to 27; or c) the first contiguous polypeptide and the second contiguous polypeptide of any one of claims 1 to 27.
31. A method comprising culturing the host cell of claim 29 or 30 and isolating the first contiguous polypeptide, or the second contiguous polypeptide, or the first contiguous polypeptide and the second contiguous polypeptide.
32. A pharmaceutical composition comprising the heterodimeric protein of any one of claims 1 to 27 and a pharmaceutically acceptable carrier.
33. A method of treating a companion animal species having an IL13 and/or IL4-induced condition, the method comprising administering to the companion animal species a
therapeutically effective amount of the heterodimeric protein of any one of claims 1 to 27 or the pharmaceutical composition of claim 32.
34. The method of claim 33, wherein the companion animal species is canine, feline, or equine.
35. The method of claim 33 or 34, wherein the IL13 and /or IL4-induced condition is a pruritic or allergic condition, such as atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema.
36. The method of any one of claims 33 to 35, wherein the heterodimeric protein or the pharmaceutical composition is administered parenterally.
37. The method of any one of claims 33 to 36, wherein the heterodimeric protein or the pharmaceutical composition is administered by an intramuscular route, an intraperitoneal route, an intracerebrospinal route, a subcutaneous route, an intra-arterial route, an intrasynovial route, an intrathecal route, or an inhalation route.
38. The method of any one of claims 33 to 37, wherein the method further comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor.
39. The method of any one of claims 33 to 38, wherein the method further comprises administering one or more antibodies selected from an anti-ILl7 antibody, an anti-IL3 l antibody, an anti-TNFa antibody, an anti-CD20 antibody, an anti-CD 19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-ILl3 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CDl la antibody, anti-IL6R antibody, anti-a4-Intergrin antibody, an anti-ILl2 antibody, an anti-IL 1 b antibody, and an anti-BlyS antibody.
40. A method of reducing IL13 and /or IL4 signaling activity in a cell, the method comprising exposing the cell to the heterodimeric protein of any one of claims 1 to 27 or the pharmaceutical composition of claim 32 under conditions permissive for binding of the heterodimeric protein to IL13 and/or IL4, thereby (a) reducing binding of IL/4 and/or IL-13 to native IL13 receptor and/or native IL-4 receptor and reducing IL13- and/or IL-4-mediated signaling.
41. The method of claim 40, wherein the cell is exposed to the heterodimeric protein or the pharmaceutical composition ex vivo.
42. The method of claim 41, wherein the cell is exposed to the heterodimeric protein or the pharmaceutical composition in vivo.
43. The method of any one of claims 40 to 42, wherein the cell is a canine cell, a feline cell, or an equine cell.
44. A method for detecting IL13 or IL4 in a sample from a companion animal species comprising contacting the sample with the heterodimeric protein of any one of claims 1 to 27 or the pharmaceutical composition of claim 32 under conditions permissive for binding of the heterodimeric protein to IL13 and/or IL4, and detecting whether a complex is formed between the heterodimeric protein and IL13 and/or IL4 in the sample.
45. The method of claim 44, wherein the sample is a biological sample obtained from a canine, a feline, or an equine.
PCT/US2019/057922 2018-10-25 2019-10-24 Il4/il13 receptor molecule for veterinary use WO2020086886A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3902564A4 (en) * 2018-12-27 2022-09-28 Kindred Biosciences, Inc. Igg fc variants for veterinary use
WO2023021170A2 (en) 2021-08-20 2023-02-23 Intervet International B.V. Fusion proteins for treating atopic dermatitis
US11970526B2 (en) 2017-04-21 2024-04-30 Elanco Us Inc. IL4/IL13 receptor molecule for veterinary use

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10093731B2 (en) 2017-02-24 2018-10-09 Kindred Biosciences, Inc. Anti-IL31 antibodies for veterinary use

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050191730A1 (en) * 2004-02-27 2005-09-01 Regeneron Pharmaceuticals, Inc. IL-4/IL-13 specific polypeptides and therapeutic uses thereof
US20090156421A1 (en) * 2007-11-06 2009-06-18 Wyeth Assays and methods for evaluating multimeric complexes
US20100278827A1 (en) * 2001-07-26 2010-11-04 Medexgen Co., Ltd. Concatameric immunoadhesion molecule
US20110151515A1 (en) * 2007-11-13 2011-06-23 Sapphire Energy, Inc. Production of fc-fusion polypeptides in eukaryotic algae
US20130089893A1 (en) * 2005-12-13 2013-04-11 Suppremol Gmbh MULTIMERIC Fc RECEPTOR POLYPEPTIDES
EP2674440A2 (en) * 2005-12-16 2013-12-18 IBC Pharmaceuticals, Inc. Multivalent immunoglobulin-based bioactive assemblies
WO2018064190A1 (en) * 2016-09-27 2018-04-05 Epicentrx, Inc. Immunomodulatory fusion proteins

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005032399A2 (en) * 2003-05-30 2005-04-14 The Regents Of The University Of California Il4 receptor antagonists for horse, dog and cat
US9487587B2 (en) * 2013-03-05 2016-11-08 Macrogenics, Inc. Bispecific molecules that are immunoreactive with immune effector cells of a companion animal that express an activating receptor and cells that express B7-H3 and uses thereof
WO2018195388A1 (en) * 2017-04-21 2018-10-25 Kindred Biosciences, Inc. Il4/il13 receptor molecule for veterinary use

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100278827A1 (en) * 2001-07-26 2010-11-04 Medexgen Co., Ltd. Concatameric immunoadhesion molecule
US20050191730A1 (en) * 2004-02-27 2005-09-01 Regeneron Pharmaceuticals, Inc. IL-4/IL-13 specific polypeptides and therapeutic uses thereof
US20130089893A1 (en) * 2005-12-13 2013-04-11 Suppremol Gmbh MULTIMERIC Fc RECEPTOR POLYPEPTIDES
EP2674440A2 (en) * 2005-12-16 2013-12-18 IBC Pharmaceuticals, Inc. Multivalent immunoglobulin-based bioactive assemblies
US20090156421A1 (en) * 2007-11-06 2009-06-18 Wyeth Assays and methods for evaluating multimeric complexes
US20110151515A1 (en) * 2007-11-13 2011-06-23 Sapphire Energy, Inc. Production of fc-fusion polypeptides in eukaryotic algae
WO2018064190A1 (en) * 2016-09-27 2018-04-05 Epicentrx, Inc. Immunomodulatory fusion proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3870601A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11970526B2 (en) 2017-04-21 2024-04-30 Elanco Us Inc. IL4/IL13 receptor molecule for veterinary use
EP3902564A4 (en) * 2018-12-27 2022-09-28 Kindred Biosciences, Inc. Igg fc variants for veterinary use
WO2023021170A2 (en) 2021-08-20 2023-02-23 Intervet International B.V. Fusion proteins for treating atopic dermatitis
WO2023021171A1 (en) 2021-08-20 2023-02-23 Intervet International B.V. Homodimer fusion proteins for treating atopic dermatitis
WO2023021170A3 (en) * 2021-08-20 2023-03-30 Intervet International B.V. Fusion proteins for treating atopic dermatitis

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