IL4 / IL13 receptor molecules for veterinary use

Abstract

To provide compositions and methods for treating conditions induced by IL13 and / or IL4 in companion animals such as dogs, cats, and horses.SOLUTION: Provided are a contiguous polypeptide comprising an extracellular domain of an IL13R polypeptide and an extracellular domain of an IL4R polypeptide, the IL13R and IL4R polypeptides being derived from a companion animal species; and a pharmaceutical composition comprising the contiguous polypeptide and a pharmaceutically acceptable carrier. Also provided is a therapeutic method comprising administering a therapeutically effective amount of the contiguous polypeptide or the pharmaceutical composition.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 488,509, filed April 21, 2018, which is incorporated herein by reference in its entirety for all purposes.

[0002] Field The present disclosure relates to contiguous polypeptides comprising interleukin-4 receptor and interleukin-13 receptor fragments derived from companion animal species that bind IL4 and / or IL13 of companion animal species, such as canine IL4 and canine IL13. The invention also relates to methods of using the contiguous polypeptides to treat IL4- and / or IL13-induced conditions or reduce cellular IL4- and / or IL13-signaling activity in companion animals, e.g., dogs, cats, and horses. [Background technology]

[0003] Interleukin 4 (IL4) is a cytokine that promotes the differentiation of naive helper T cells into Th2 cells. Interleukin 13 (IL13) has similar effects on immune cells. Both IL4 and IL13 play important roles in T cell-mediated immune responses, which are directly related to allergies, such as atopic dermatitis and asthma. IL4 is generally understood to be able to form signaling complexes with either the heterodimeric receptor IL4 receptor subunit alpha (IL4R) and γc or the IL4R and IL13 receptor subunit alpha-1 (IL13R). IL13 can form signaling complexes with the heterodimeric receptors IL4Ra and IL13Ra1. The extracellular domains of IL4Ra or IL13Ra1 can bind to IL4 and / or IL13, reducing the free cytokine concentrations and thus reducing the clinical signs and symptoms associated with dermatitis, asthma, and other disorders.

[0004] Companion species animals, such as cats, dogs, and horses, suffer from many allergic diseases similar to those in humans, including atopic dermatitis and asthma. Thus, there remains a need for methods and compounds that can be used to specifically bind companion animal IL4 and / or IL13 for treating IL4 / IL13-induced conditions and for reducing IL4 / IL13 signaling activity. Summary of the Invention

[0005] In some embodiments, an IL13R / IL4R contiguous polypeptide is provided that comprises the extracellular domain of an IL13R polypeptide and the extracellular domain of an IL4R polypeptide derived from a companion animal species.

[0006] In some embodiments, the IL13R / IL4R sequential polypeptide comprises formula (I) IL13R-L1-IL4R-L2-FP or formula (II) IL4R-L1-IL13R-L2-FP, wherein: a. IL13R is the extracellular domain of an IL13R polypeptide from a companion animal species; b. IL4R is the extracellular domain of an IL4R polypeptide from a companion animal species; c. L1 is a first optional linker; d. L2 is a second optional linker, and e. FP is a fusion partner.

[0007] In some embodiments, the IL13R / IL4R sequential polypeptide exhibits a 5×10 affinity to IL13 of a companion animal species as measured by biolayer interferometry. -6 Less than M, 1 x 10 -6 Less than M, 5 x 10 -7 Less than M, 1 x 10 -7 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 -11 Less than M, 1 x 10 -11 Less than M, 5 x 10 -12 Less than M or 1 x 10 -12 It binds with a dissociation constant (Kd) less than M.

[0008] In some embodiments, the IL13R / IL4R sequential polypeptide exhibits a 5×10 affinity to IL4 of a companion animal species, as measured by biolayer interferometry. -6 Less than M, 1 x 10 -6 Less than M, 5 x 10 -7 Less than M, 1 x 10 -7 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 -11 Less than M, 1 x 10 -11 Less than M, 5 x 10 -12 Less than M or 1 x 10 -12 It binds with a dissociation constant (Kd) less than M.

[0009] In some embodiments, the IL13R / IL4R continuous polypeptide reduces IL13 and / or IL4 signaling in a companion animal species.

[0010] In some embodiments, the companion animal species is a dog, cat, or horse.

[0011] In some embodiments, the extracellular domain of an IL13R polypeptide is at least 85% 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. In some embodiments, the extracellular domain of an IL13R polypeptide is at least 90% 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. In some embodiments, the extracellular domain of an IL13R polypeptide is at least 95% 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. In some embodiments, the extracellular domain of an IL13R polypeptide is 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.

[0012] In some embodiments, the extracellular domain of an IL13R polypeptide 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. In some embodiments, the extracellular domain of an IL13R polypeptide comprises a cysteine ​​at position 18 of SEQ ID NO:22, position 18 of SEQ ID NO:24, position 18 of SEQ ID NO:26, position 15 of SEQ ID NO:32, position 15 of SEQ ID NO:34, or position 15 of SEQ ID NO:36. In some embodiments, the extracellular domain of an IL13R polypeptide comprises an amino acid sequence selected from SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36. In some embodiments, the extracellular domain of an IL13R polypeptide comprises an amino acid sequence selected from SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.

[0013] In some embodiments, the extracellular domain of an IL4R polypeptide is at least 85% 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. In some embodiments, the extracellular domain of an IL4R polypeptide is at least 90% 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. In some embodiments, the extracellular domain of an IL4R polypeptide is at least 95% 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. In some embodiments, the extracellular domain of an IL4R polypeptide is 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. In some embodiments, the extracellular domain of an IL4R polypeptide comprises an amino acid sequence selected from SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37. In some embodiments, the extracellular domain of the IL4R polypeptide comprises an amino acid sequence selected from SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27.

[0014] In some embodiments, L1 and L2, if present, each independently comprise an amino acid sequence selected from G, GG, GGG, S, SS, SSS, GS, GSGS (SEQ ID NO: 38), GSGSGS (SEQ ID NO: 39), GGS, GGSGGS (SEQ ID NO: 40), GGSGGSGGS (SEQ ID NO: 41), GGGS (SEQ ID NO: 42), GGGSGGGS (SEQ ID NO: 43), GGGSGGGSGGGS (SEQ ID NO: 44), GSS, GSSGSS (SEQ ID NO: 45), GSSGSSGSS (SEQ ID NO: 46), GGSS (SEQ ID NO: 47), GGSSGGSS (SEQ ID NO: 48), and GGSSGGSSGGSS (SEQ ID NO: 49).

[0015] In some embodiments, the fusion partner is selected from Fc, albumin, and an albumin-binding fragment. In some embodiments, Fc is (a) a dog heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region; (b) a feline heavy chain constant region selected from an IgG1, IgG2a, and IgG2b constant region; or (c) an horse heavy chain constant region selected from an IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant region.

[0016] In some embodiments, the IL13R / IL4R continuous polypeptide comprises a sequence selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0017] In some embodiments, an isolated nucleic acid is provided, which encodes an IL13R / IL4R sequential polypeptide described herein above. In some embodiments, a host cell is provided, which comprises a nucleic acid encoding an IL13R / IL4R sequential polypeptide described herein above. In some embodiments, a method of producing an IL13R / IL4R sequential polypeptide is provided, which comprises culturing such a host cell containing a nucleic acid encoding an IL13R / IL4R sequential polypeptide described herein above and isolating the sequential polypeptide. In some embodiments, a pharmaceutical composition is provided, which comprises an IL13R / IL4R sequential polypeptide described herein and a pharmaceutically acceptable carrier.

[0018] In some embodiments, methods of treating a companion animal species having an IL13- and / or IL4-induced condition are provided, comprising administering to the companion animal species a therapeutically effective amount of an IL13R / IL4R sequential polypeptide described herein or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide described herein. In some embodiments, the companion animal species is a dog, cat, or horse. In some embodiments, the IL13- and / or IL4-induced condition is a pruritic or allergic condition, such as atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema.

[0019] In some embodiments, an IL13R / IL4R sequential polypeptide or pharmaceutical composition is administered parenterally, hi some embodiments, an IL13R / IL4R sequential polypeptide or pharmaceutical composition is administered intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intra-arterially, intrasynovially, intrathecally, or by inhalation.

[0020] In some embodiments, the method further comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor. In some embodiments, the method further comprises administering one or more antibodies selected from an anti-IL17 antibody, an anti-IL31 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-Intergrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, and an anti-BlyS antibody.

[0021] In some embodiments, methods are provided for reducing IL13 and / or IL4 signaling activity in cells, comprising exposing the cells to an IL13R / IL4R sequential polypeptide or pharmaceutical composition described herein under conditions permissive for binding of the sequential polypeptide to IL13 and / or IL4, thereby (a) reducing binding of IL13 and / or IL-4 to the native IL13 receptor and / or native IL-4 receptor and reducing IL13- and / or IL-4-mediated signaling. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide or pharmaceutical composition ex vivo. In some embodiments, the cells are exposed to the sequential polypeptide or pharmaceutical composition in vivo. In some embodiments, the cells are canine, feline, or equine cells.

[0022] In some embodiments, methods are provided for detecting IL13 or IL4 in a sample from a companion animal species, comprising contacting the sample with an IL13R / IL4R sequential polypeptide or pharmaceutical composition described herein under conditions permissive for binding of the sequential polypeptide to IL13 and / or IL4, and detecting whether a complex is formed between the sequential polypeptide and IL13 and / or IL4 in the sample. In some embodiments, the sample is a biological sample obtained from a dog, cat, or horse. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a graph of the sequential binding of canine IL4R-IL13R-Fc to canine IL4 and IL13 or canine IL13 and IL4 using IL4 and IL13 at a concentration of 30 μg / mL in PBS. [Figure 2] FIG. 2 is a graph of the sequential binding of canine IL4 and IL13 or canine IL13R-IL4R-Fc to canine IL4 and IL13, using IL4 and IL13 at a concentration of 30 μg / mL in PBS. [Figure 3]3 is a graph of canine IL4R-IL13R-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. DETAILED DESCRIPTION OF THE INVENTION

[0024] Array Description Table 1 provides a sequence listing of certain sequences referenced herein. TIFF0007776461000001.tif251170TIFF0007776461000002.tif251170TIFF0007776461000003.tif252170TIFF000 7776461000004.tif250170TIFF0007776461000005.tif251170TIFF0007776461000006.tif247170TIFF0007776461 000007.tif252170TIFF0007776461000008.tif249170TIFF0007776461000009.tif251170TIFF0007776461000010. tif252170TIFF0007776461000011.tif251170TIFF0007776461000012.tif253170TIFF0007776461000013.tif75170

[0025] Description of the Preferred Embodiments Consecutive polypeptides 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 consecutive polypeptide comprises the extracellular domain of an IL13R polypeptide and the extracellular domain of an IL4R polypeptide. Methods for producing or purifying the consecutive polypeptides are also provided. Methods of treatment using consecutive polypeptides that 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 for treating IL13- and / or IL4-induced conditions in companion animal species. Methods for detecting IL13 and / or IL4 in a sample from a companion animal species are also provided.

[0026] For the convenience of the reader, the following definitions of terms used herein are provided.

[0027] Numerical terms used herein, such as Kd, are calculated based on scientific measurements and are therefore subject to appropriate measurement error. In some cases, numerical terms may include numerical values ​​that are rounded to the nearest significant figure.

[0028] 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 otherwise specified. In the context of multiple dependent claims, the use of "or" when referring back to other claims refers to any one of those claims only.

[0029] IL13R / IL4R continuous polypeptide For example, novel IL13R / IL4R contiguous polypeptides are provided that are contiguous polypeptides that bind to canine IL13 and / or IL4, feline IL13 and / or IL4, and / or equine IL13 and / or IL4.

[0030] An "amino acid sequence" refers to the sequence of amino acid 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 unnatural 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 term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein containing modifications to the native sequence, such as deletions, additions, and substitutions (generally conserved in nature), so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or accidental, such as due to mutations of hosts producing the protein or errors resulting from PCR amplification.

[0031] The term "contiguous polypeptide" as used herein refers to an uninterrupted sequence of amino acids. A contiguous polypeptide is typically translated from a single continuous DNA sequence. A contiguous polypeptide can be produced by genetic engineering, for example, by removing the stop codon from the DNA sequence of a first protein, and then adding the DNA sequence of a second protein in frame, so that the DNA sequence is expressed as a single protein. Typically, this is achieved by cloning a cDNA into an expression vector in frame with an existing gene.

[0032] As used herein, "IL4R" refers to a polypeptide comprising all or a fragment of the IL4 receptor subunit alpha, which binds to IL-4.

[0033] For example, "IL4R," unless otherwise indicated, refers to an IL4R polypeptide from any vertebrate source, including mammals, e.g., primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses). In some embodiments, an IL4R is an extracellular domain fragment that binds IL4. In some such embodiments, an IL4R may be referred to as an IL4R extracellular domain (ECD). In some embodiments, an 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.

[0034] As used herein, "IL13R" is a polypeptide comprising all or part of the IL13 receptor subunit alpha-1, which binds to IL-13.

[0035] For example, "IL13R," unless otherwise indicated, refers to an IL13R polypeptide from any vertebrate source, including mammals, e.g., primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses). In some embodiments, an IL13R is an extracellular domain fragment that binds to IL13. In some such embodiments, an IL13R may be referred to as an IL13R extracellular domain (ECD). In some embodiments, an 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.

[0036] The term "companion animal species" refers to animals that are suitable companions to humans. In some embodiments, companion animal species are small mammals, such as canines, felines, dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, etc. In some embodiments, companion animal species are livestock, such as horses, cows, pigs, etc.

[0037] An "extracellular domain" ("ECD") is a portion of a polypeptide that extends beyond the transmembrane domain into the extracellular space. As used herein, the term "extracellular domain" may include an entire extracellular domain or a truncated extracellular domain lacking one or more amino acids that bind to its ligand. The composition of an extracellular domain may depend on the algorithm used to determine which amino acids are in the membrane. Different algorithms may predict different extracellular domains for a given protein, and different systems may express different extracellular domains for a given protein.

[0038] The extracellular domain of an IL4R polypeptide may comprise the complete extracellular domain or a truncated extracellular domain of an 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 include the transmembrane or cytoplasmic domain, even when the terms are followed by open transitional phrases such as "comprising," "comprises," and similar terms. In some embodiments, the 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, the extracellular domain of an IL4R polypeptide is derived from a canine IL4R, a feline IL4R, or an equine IL4R. In some embodiments, the 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, the 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.

[0039] The extracellular domain of an IL13R polypeptide may comprise the entire extracellular domain or a truncated extracellular domain of IL13R that binds to 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 include the transmembrane domain or the cytoplasmic domain, even when the terms are followed by an open transitional phrase such as "comprising," "comprises," and the like. In some embodiments, the extracellular domain of an IL13R polypeptide is the extracellular domain of an IL13R polypeptide derived from a companion species animal. For example, in some embodiments, the extracellular domain of an IL13R polypeptide is derived from canine IL13R, feline IL13R, or equine IL13R. In some embodiments, the 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, the 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.

[0040] The terms "IL13R / IL4R sequential polypeptide" and "IL4R / IL13R sequential polypeptide" are used interchangeably and refer to a sequential polypeptide comprising an IL13R polypeptide and an IL4R polypeptide, and the terms are not indicative of the order in which the IL13R and IL4R polypeptides appear in the sequential polypeptide, unless otherwise specified. For example, an IL13R / IL4R sequential polypeptide or an IL4R / IL13R sequential polypeptide may refer to an IL4R polypeptide that precedes or follows a sequence of an IL13R polypeptide. In addition, an IL13R / IL4R sequential polypeptide or an IL4R / IL13R sequential polypeptide may refer to an IL13R polypeptide that precedes or follows a sequence of an IL4R polypeptide.

[0041] In some embodiments, an IL13R / IL4R continuous polypeptide comprises an IL13R polypeptide linked to an IL4R polypeptide at the C-terminus of the IL13R polypeptide or at the N-terminus of the IL13R polypeptide, In some embodiments, an IL13R / IL4R continuous polypeptide comprises an IL4R polypeptide linked to an IL13R polypeptide at the C-terminus of the IL4R polypeptide or at the N-terminus of the IL4R polypeptide.

[0042] The IL13R / IL4R sequential polypeptides of the present invention may comprise the extracellular domain of an IL13R polypeptide and / or the extracellular domain of an IL4R polypeptide derived from a companion animal species. For example, the sequential polypeptide may comprise the extracellular domain of an IL4R polypeptide from a dog, cat, or horse and / or the extracellular domain of an IL13R polypeptide from a dog, cat, or horse.

[0043] "Wild-type" refers to a non-mutated version of a naturally occurring polypeptide, or a fragment thereof. A wild-type polypeptide may be recombinantly produced. "Wild-type IL13R ECD" or "wild-type IL4R ECD" refers to a protein having an amino acid sequence identical to the same portion of the extracellular domain of a naturally occurring IL13R or IL4R.

[0044] A "variant" is a nucleic acid molecule or polypeptide that differs from the reference nucleic acid molecule or polypeptide by single or multiple amino acid substitutions, deletions, and / or additions, and that substantially retains at least one biological activity of the reference nucleic acid molecule or polypeptide.

[0045] A "biologically active" or "biologically active" entity is one that has any function related to or associated with a metabolic or physiological process and / or has a structural, regulatory, or biochemical function of a naturally occurring molecule. Biologically active polynucleotide fragments are those that exhibit similar, but not necessarily identical, activity to the polynucleotides of the present invention. Biologically active polypeptides or fragments thereof include, but are not limited to, those that can participate in a biological reaction, including a ligand-receptor interaction or antigen-antibody binding. Biological activity can include improved desired activity or decreased undesirable activity. An entity can demonstrate biological activity if it participates in a molecular interaction with another molecule, such as hybridization, has therapeutic value in alleviating a disease state, has preventative value in inducing an immune response, has diagnostic and / or prognostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide, that can be detected as unique to the polynucleotide molecule, and can be used as a primer in a polymerase chain reaction (PCR).

[0046] 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 nucleotides or amino acid residues in a reference sequence that are identical to the nucleotides or amino acid residues in a particular nucleic acid molecule or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved by various methods within the skill of the art, for example, using commercially 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 maximum alignment over the entire length of the sequences being compared.

[0047] In some embodiments, a variant has at least about 50% sequence identity with the reference nucleic acid molecule or polypeptide, after aligning the sequences and introducing gaps, if necessary, without considering any conservative substitutions as part of the sequence identity to achieve the maximum percent sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted to 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, or at least about 98% sequence identity with the reference nucleic acid or polypeptide sequence.

[0048] In some embodiments, the IL13R / IL4R continuous polypeptide comprises an extracellular domain of an IL13R polypeptide having at least 85%, at least 90%, at least 95%, or 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, the IL13R / IL4R continuous polypeptide comprises an extracellular domain of an IL4R polypeptide having at least 85%, at least 90%, at least 95%, or 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.

[0049] In some embodiments, an IL13R / IL4R continuous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine ​​at a position corresponding to position 18 of SEQ ID NO: 22, a position corresponding to position 18 of SEQ ID NO: 24, or a position corresponding to position 18 of SEQ ID NO: 26. In some embodiments, an IL13R / IL4R continuous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine ​​at position 18 of SEQ ID NO: 22, position 18 of SEQ ID NO: 24, position 18 of SEQ ID NO: 26, position 15 of SEQ ID NO: 32, position 15 of SEQ ID NO: 34, or position 15 of SEQ ID NO: 36.

[0050] A "point mutation" is a mutation involving a single nucleotide or amino acid residue. The mutation may be the deletion of one nucleotide or amino acid, the substitution of one nucleotide or amino acid residue for another, or the insertion of an additional nucleotide or amino acid residue.

[0051] Amino acid substitutions can include, but are not limited to, the replacement of one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into molecules of interest and products screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics. TIFF0007776461000014.tif222170

[0052] Amino acids are classified according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral and 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: Can be classified into Trp, Tyr, and Phe.

[0053] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0054] As used herein, "fusion partner" refers to an additional component of an IL13R / IL4R continuous polypeptide, such as an additional polypeptide, e.g., albumin, an albumin-binding fragment, or a fragment of an immunoglobulin molecule. The fusion partner may also include an oligomerization domain, e.g., the Fc domain of a heavy chain immunoglobulin.

[0055] A "fragment crystallizable (Fc) polypeptide" is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the C-terminal portion of an immunoglobulin heavy chain. As used herein, an Fc polypeptide includes fragments of the Fc domain that have one or more biological activities of the entire Fc polypeptide. An "effector function" of an Fc polypeptide is an action or activity carried out in whole or in part by any antibody in response to a stimulus and may include complement fixation or ADCC (antibody-dependent cellular cytotoxicity) induction.

[0056] The term "IgX" or "IgX Fc" means that the Fc region is derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" indicates the antibody isotype. Thus, "IgG" or "IgG Fc" indicates the Fc region of the gamma chain, "IgA" or "IgA Fc" indicates the Fc region of the alpha chain, "IgD" or "IgD Fc" indicates the Fc region of the delta chain, "IgE" or "IgE Fc" indicates the Fc region of the epsilon chain, "IgM" or "IgM Fc" indicates the Fc region of the mu chain, etc. In some embodiments, an IgG Fc region comprises a CH1, hinge, CH2, CH3, and CL1. "IgXN" or "IgXN Fc" indicates that the Fc region is derived from a particular subclass of antibody isotype (such as canine IgG subclasses A, B, C, or D; feline IgG subclasses 1, 2a, or 2b; or equine IgG subclasses IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7), where "N" indicates the subclass.

[0057] In some embodiments, the IgX or IgXN region is derived from a companion animal, such as a dog, cat, or horse. In some embodiments, the IgG region is isolated from a canine gamma heavy chain, such as IgGA, IgGB, IgGC, or IgGD. In some cases, the IgG Fc region is isolated from a feline gamma heavy chain, such as IgG1, IgG2a, or IgG2b. In other cases, the IgG region is isolated from an equine gamma heavy chain, such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7. Polypeptides comprising the Fc region of IgGA, IgGB, IgGC, or IgGD may provide higher expression levels in recombinant production systems.

[0058] "Signal sequence" refers to a sequence of amino acid residues or polynucleotides that encodes a polypeptide of interest that facilitates secretion and is typically cleaved upon transport of the polypeptide across the cell surface membrane.

[0059] "Linker" refers to one or more amino acid residues that connect a first polypeptide to a second polypeptide.

[0060] In some embodiments, the linker is a flexible, unstructured linker that is glycine-rich, serine-rich, or GS-rich. In some embodiments, the linker comprises the amino acids G (Gly) and / or S (Ser). For example, the linker may contain G or G repeats (e.g., GG, GGG, etc.); GS or GS repeats (e.g., GSGS (SEQ ID NO: 38), GSGSGS (SEQ ID NO: 39), etc.); GGS or GGS repeats (e.g., GGSGGS (SEQ ID NO: 40), GGSGGSGGS (SEQ ID NO: 41), etc.); GGGS (SEQ ID NO: 42) or GGGS repeats (SEQ ID NO: 42) (e.g., GGGSGGGS (SEQ ID NO: 43), GGGSGGGSGGGS (SEQ ID NO: 44), etc.); GSS or GSS repeats (e.g., GSSGSS (SEQ ID NO: 45), GSSGSSGSS (SEQ ID NO: 46), etc.); or GGSS (SEQ ID NO: 47) or GGSS repeats (SEQ ID NO: 47) (e.g., GGSSGGSS (SEQ ID NO: 48), GGSSGGSSGGSS (SEQ ID NO: 49), etc.).

[0061] In some embodiments, an IL13R / IL4R continuous polypeptide comprises at least one fusion partner and / or at least one linker. In some embodiments, an IL13R / IL4R continuous polypeptide comprises an optional signal sequence, an optional fusion partner, and at least one optional linker. In some embodiments, an IL13R / IL4R continuous polypeptide does not comprise a signal sequence, a fusion partner, or a linker. In some embodiments, an IL13 / IL4 continuous polypeptide is translated with a signal sequence, but the signal sequence is cleaved from the IL13R / IL4R continuous polypeptide.

[0062] In some embodiments, the IL13R / IL4R sequential polypeptide is: Formula (I): IL13R-L1-IL4R-L2-FP or Formula (II): IL4R-L1-IL13R-L2-FP, wherein IL13R is an IL13R extracellular domain (ECD) polypeptide derived from a companion animal species, IL4R is an IL4R ECD polypeptide derived from a companion animal species, L1 is a first optional linker, L2 is a second optional linker, and FP is an optional fusion partner.

[0063] In some embodiments, the IL13R / IL4R continuous polypeptide comprises an amino acid sequence selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31.

[0064] IL13R / IL4R sequential polypeptide expression and production Polynucleotide sequences encoding all or a portion (e.g., the extracellular domain) of the IL13R / IL4R contiguous polypeptide, with or without a signal sequence, are provided. If a homologous signal sequence (i.e., the signal sequence of the native IL-4R or IL13R) is not used in the construction of the nucleic acid molecule, an alternative signal sequence may be used, for example, any one of the signal sequences described in PCT US06 / 02951.

[0065] Typically, a nucleotide sequence encoding a nucleotide sequence of interest, eg, an IL13R / IL4R continuous polypeptide, is inserted into an appropriate expression vector for expression in a selected host cell.

[0066] 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. Typically, a vector contains an origin of replication and regulatory sequences that control the expression of the gene of interest and may or may not carry a selectable marker gene, e.g., an antibiotic resistance gene. The vector is appropriate for the host cell in which it is expressed. A vector may be called a "recombinant vector" when a gene of interest is present in the vector.

[0067] A "host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate cells; fungal cells, e.g., yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, as well as their derivatives, e.g., 293-6E, DG44, CHO-S, and CHO-K cells. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or in overall 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 encoding the amino acid sequences provided herein.

[0068] As used herein, the term "isolated" refers to a molecule that is separated from at least some components in which it is typically found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some components of the cell in which it is produced. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it is produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) that is separated from at least some components in which it is typically found in nature or the cell in which it was produced (e.g., in the case of an RNA polynucleotide). Thus, a DNA polynucleotide contained in a vector inside a host cell may be referred to as "isolated."

[0069] In some embodiments, the IL13R / IL4R continuous polypeptide is isolated using chromatography, for example, size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0070] The terms "label" and "detectable label" refer to a moiety attached to an IL13R / IL4R continuous polypeptide that renders it detectable. In some embodiments, the label is a detectable marker that can produce a detectable signal by visual or instrumental means, e.g., by incorporation of a radiolabeled amino acid or attachment of a biotinylated moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin, which contains a fluorescent marker or enzymatic activity that can be detected optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinylation groups; defined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, e.g., gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-generating moieties, e.g., acridinium compounds, and fluorescence-generating moieties, e.g., fluorescein. In this regard, the moiety itself need not be acceptably labeled but may become detectable upon reaction with yet another moiety.

[0071] IL13R / IL4R sequential polypeptides as decoy receptor traps The IL13R / IL4R continuous polypeptides of the present invention can function as decoy receptors to trap IL13 or IL4 and inhibit their interaction with IL13R and IL4R on the cell surface. Decoy receptors (e.g., those of the present invention) recognize their ligands with high affinity and specificity but are constitutively incapable of signal transduction. They compete with wild-type receptors for ligand binding and participate in ligand / receptor interactions, thus modulating the activity or number of functional receptors and / or downstream cellular activities of the receptors. Decoy receptors can function as molecular traps for agonist ligands, thereby inhibiting ligand-induced receptor activation.

[0072] As used herein, "IL13" refers to any native IL13 resulting from expression and processing of IL13 in cells. The term, unless otherwise indicated, includes IL13 from any vertebrate source, including mammals, e.g., primates (e.g., humans and cynomolgus monkeys), and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses). The term also includes naturally occurring variants of IL13, e.g., splice variants or allelic variants.

[0073] In some embodiments, canine IL13 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, feline IL13 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, equine IL13 comprises the amino acid sequence of SEQ ID NO: 6.

[0074] As used herein, "IL4" refers to any native IL4 resulting from expression and processing of IL4 in a cell. The term, unless otherwise indicated, includes IL4 from any vertebrate source, including mammals, e.g., primates (e.g., humans and cynomolgus monkeys), and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses). The term also includes naturally occurring variants of IL4, e.g., splice variants or allelic variants.

[0075] In some embodiments, canine IL4 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, feline IL4 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, equine IL4 comprises the amino acid sequence of SEQ ID NO: 3.

[0076] The present invention provides IL13R / IL4R sequential polypeptides as therapeutic agents. The IL13R / IL4R sequential polypeptides of the present invention bind to IL13 and / or IL4, which are described in more detail herein and have been demonstrated to be associated with allergic diseases. In various embodiments, the IL13R / IL4R sequential polypeptides can bind to IL13 and IL4 with very high affinity. In various embodiments, the IL13R / IL4R sequential polypeptides can interfere with IL13 and IL4 signaling.

[0077] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD). Affinity can be measured by common methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance.

[0078] The terms "KD", "K" and "K" are used interchangeably. d "," "Kd" or "Kd value" refers to the equilibrium dissociation constant of a receptor fusion-ligand interaction. In some embodiments, the K d is measured by using a biolayer interferometry assay using a biosensor such as the Octet® System (Pall ForteBio LLC, Fremont, CA) according to the supplier's instructions. Briefly, biotinylated antigen is bound to the sensor chip, and association of the fusion molecule is monitored for 90 seconds, and dissociation is monitored for 600 seconds. The buffer for the dilution and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer-only blank curve is drawn to correct for any variations. Data are fitted to a 2:1 binding model using ForteBio data analysis software to determine the association rate constant (k on ), dissociation rate constant (k off ), and Kd is determined. The equilibrium dissociation constant (K d ) is k off / k on The term "k" refers to the rate constant for the association of molecule X to its partner Y, and the term "k" refers to the rate constant for the dissociation of molecule X or partner Y from the molecule X / partner Y complex.

[0079] The term "binding" to a substance is a term well understood in the art, and methods for determining such binding are also well known in the art. A molecule is said to exhibit "binding" if it reacts with, associates with, or has affinity for a particular cell or substance, where the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance, etc.

[0080] "Surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting alterations in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

[0081] "Bio-layer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from a layer of proteins immobilized on a biosensor chip and an internal reference layer. Changes in the number of molecules bound to the biosensor chip cause shifts in the interference pattern that can be measured in real time. A non-limiting exemplary device for bio-layer interferometry is the Octet® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0082] In some embodiments, the IL13R / IL4R sequential polypeptide has a binding affinity to canine IL13 and / or IL4, feline IL13 and / or IL4, or equine IL13 and / or IL4, as measured by biolayer interferometry, of less than 5×10 -6 Less than M, 1 x 10 -6 Less than M, 5 x 10 -7 Less than M, 1 x 10 -7 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M, 5 x 10 -10 Less than M, 1 x 10 -10 Less than M, 5 x 10 -11 Less than M, 1 x 10 -11 Less than M, 5 x 10 -12 Less than M or 1 x 10 -12 In some embodiments, the IL13R / IL4R sequential polypeptide 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 5×10 M as measured by biolayer interferometry. -6 M to 1 x 10 -6 Between M, 5 x 10 -6 M to 5 x 10 -7 Between M, 5 x 10 -6 M to 1 x 10 -7 Between M, 5 x 10 -6 M to 5 x 10 -8 Between M, 5 x 10 -6M and 1 x 10 -8 M, 5 x 10 -6 M to 5 x 10 -9 Between M, 5 x 10 -6 M to 1 x 10 -9 Between M, 5 x 10 -6 M to 5 x 10 -10 Between M, 5 x 10 -6 M to 1 x 10 -10 Between M, 5 x 10 -6 M to 5 x 10 -11 Between M, 5 x 10 -6 M to 1 x 10 -11 Between M, 5 x 10 -6 M to 5 x 10 -12 Between M, 5 x 10 -6 M to 1 x 10 -12 Between M, 1 x 10 -6 M to 5 x 10 -7 Between M, 1 x 10 -6 M to 1 x 10 -7 Between M, 1 x 10 -6 M to 5 x 10 -8 Between M, 1 x 10 -6 M and 1 x 10 -8 M, 1 x 10 -6 M to 5 x 10 -9 Between M, 1 x 10 -6 M to 1 x 10 -9 Between M, 1 x 10 -6 M to 5 x 10 -10 Between M, 1 x 10 -6 M to 1 x 10 -10 Between M, 1 x 10 -6 M to 5 x 10 -11 Between M, 1 x 10 -6 M to 1 x 10 -11 Between M, 1 x 10 -6 M to 5 x 10 -12 Between M, 1 x 10 -6 M to 1 x 10 -12 Between M, 5 x 10 -7 M to 1 x 10 -7 Between M, 5 x 10 -7 M to 5 x 10 -8 Between M, 5 x 10 -7 M and 1 x 10 -8 M, 5 x 10 -7 M to 5 x 10 -9Between M, 5 x 10 -7 M to 1 x 10 -9 Between M, 5 x 10 -7 M to 5 x 10 -10 Between M, 5 x 10 -7 M to 1 x 10 -10 Between M, 5 x 10 -7 M to 5 x 10 -11 Between M, 5 x 10 -7 M to 1 x 10 -11 Between M, 5 x 10 -7 M to 5 x 10 -12 Between M, 5 x 10 -7 M to 1 x 10 -12 Between M, 1 x 10 -7 M to 5 x 10 -8 Between M, 1 x 10 -7 M and 1 x 10 -8 M, 1 x 10 -7 M to 5 x 10 -9 Between M, 1 x 10 -7 M to 1 x 10 -9 Between M, 1 x 10 -7 M to 5 x 10 -10 Between M, 1 x 10 -7 M to 1 x 10 -10 Between M, 1 x 10 -7 M to 5 x 10 -11 Between M, 1 x 10 -7 M to 1 x 10 -11 Between M, 1 x 10 -7 M to 5 x 10 -12 Between M, 1 x 10 -7 M to 1 x 10 -12 Between M, 5 x 10 -8 M to 1 x 10 -8 Between M, 5 x 10 -8 M to 5 x 10 -9 Between M, 5 x 10 -8 M to 1 x 10 -9 Between M, 5 x 10 -8 M to 5 x 10 -10 Between M, 5 x 10 -8 M to 1 x 10 -10 Between M, 5 x 10 -8 M to 5 x 10 -11 Between M, 5 x 10 -8 M to 1 x 10 -11 Between M, 5 x 10 -8M to 5 x 10 -12 Between M, 5 x 10 -8 M to 1 x 10 -12 Between M, 1 x 10 -8 M and 5 x 10 -9 M, 1 x 10 -8 M to 1 x 10 -9 Between M, 1 x 10 -8 M to 5 x 10 -10 Between M, 1 x 10 -8 M to 1 x 10 -10 Between M, 1 x 10 -8 M to 5 x 10 -11 Between M, 1 x 10 -8 M to 1 x 10 -11 Between M, 1 x 10 -8 M to 5 x 10 -12 Between M, 1 x 10 -8 M to 1 x 10 -12 Between M, 5 x 10 -9 M to 1 x 10 -9 Between M, 5 x 10 -9 M to 5 x 10 -10 Between M, 5 x 10 -9 M to 1 x 10 -10 Between M, 5 x 10 -9 M to 5 x 10 -11 Between M, 5 x 10 -9 M to 1 x 10 -11 Between M, 5 x 10 -9 M to 5 x 10 -12 Between M, 5 x 10 -9 M to 1 x 10 -12 Between M, 1 x 10 -9 M to 5 x 10 -10 Between M, 1 x 10 -9 M to 1 x 10 -10 Between M, 1 x 10 -9 M to 5 x 10 -11 Between M, 1 x 10 -9 M to 1 x 10 -11 Between M, 1 x 10 -9 M to 5 x 10 -12 Between M, 1 x 10 -9 M to 1 x 10 -12 Between M, 5 x 10 -10 M to 1 x 10 -10 Between M, 5 x 10 -10 M to 5 x 10 -11Between M, 1 x 10 -10 M to 5 x 10 -11 Between M, 1 x 10 -10 M and 1 x 10 -11 M, 1 x 10 -10 M to 5 x 10 -12 Between M, 1 x 10 -10 M to 1 x 10 -12 Between M, 5 x 10 -11 M to 1 x 10 -12 Between M, 5 x 10 -11 M to 5 x 10 -12 Between M, 5 x 10 -11 M to 1 x 10 -12 Between M, 1 x 10 -11 M to 5 x 10 -12 Between M or 1 x 10 -11 M to 1 x 10 -12 M. In some embodiments, the IL13R / IL4R transconjugate polypeptide binds to canine IL13 and / or IL4, feline IL13 and / or IL4, and / or equine IL13 and / or IL4.

[0083] "Reducing" or "inhibiting" means decreasing, lowering, or ceasing an activity, function, or amount compared to a reference. In some embodiments, by "reducing" or "inhibiting" is meant the ability to cause an overall decrease of 20% or more. In some embodiments, by "reducing" or "inhibiting" is meant the ability to cause an overall decrease of 50% or more. In some embodiments, by "reducing" or "inhibiting" is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount noted above is inhibited or reduced over a period of time compared to a control dose (such as a placebo) over the same period of time. As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference may be obtained from a healthy or disease-free sample. In some examples, a reference is obtained from a disease-free or untreated sample of a companion animal. In some examples, a reference is obtained from one or more healthy animals of a particular species that are not the animal being tested or treated.

[0084] As used herein, the term "substantially reduced" indicates that the reduction between a numerical value and a reference numerical value is sufficiently high that one of skill in the art would consider the difference between the two values ​​to be statistically significant within the context of the biological property measured by the values. In some embodiments, a substantially reduced numerical value is reduced by more 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.

[0085] In some embodiments, the IL13R / IL4R transconjugate polypeptide 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-dependent TF-1 cell proliferation. In some embodiments, the reduction in IL13 and / or IL4 signaling or 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 15%. Between 0% 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%.

[0086] Pharmaceutical Compositions The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that effectively potentiates the biological activity of the active ingredient and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered.

[0087] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with therapeutic agents 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. A pharmaceutically acceptable carrier is appropriate for the formulation with which it is used. Examples of pharmaceutically acceptable carriers include: alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin, dog or other animal albumin; buffers, such as phosphate, citrate, tromethamine or HEPES buffer; 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; polyvinylpyrrolidone, cellulose-based materials; polyethylene glycol; sucrose; mannitol; or amino acids, including, but not limited to, arginine.

[0088] 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 re-formulated as an aqueous composition suitable for parenteral administration, typically before administration to dogs, cats, or horses. In other embodiments, particularly when the fusion molecule is highly stable to heat and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., an aqueous composition that can be administered directly or after appropriate dilution to dogs, cats, or horses. The lyophilized composition can be reconstituted with sterile water for injection (WFI). A bacteriostatic agent, such as benzyl alcohol, may also be included. Thus, the present invention provides pharmaceutical compositions in solid or liquid form.

[0089] The pH of the pharmaceutical composition may range from about pH 5 to about pH 8 when administered. The compositions of the present invention are sterilized when used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Sterility can be maintained with or without the use of antibacterial agents.

[0090] Uses of IL13R / IL4R sequential polypeptides and pharmaceutical compositions The IL13R / IL4R sequential polypeptides or pharmaceutical compositions comprising the IL13R / IL4R sequential polypeptides of the present invention may be useful for treating IL13- and / or IL4-induced conditions. As used herein, "IL13- or IL4-induced conditions" refers to diseases 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, pruritic or allergic diseases. In some embodiments, the IL13- and / or IL4-induced condition is atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema. The IL13- or IL4-induced condition may be observed in companion animals, including, but not limited to, dogs, cats, or horses.

[0091] As used herein, "treatment" refers to an approach to obtain beneficial or desired clinical results. As used herein, "treatment" encompasses any administration or application of a disease treatment 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 the following: alleviation of one or more symptoms; reduction in the extent of the disease; prevention or delay of disease spread; prevention or delay of disease recurrence; delay or slowing of disease progression; amelioration of the condition; inhibition of the disease or disease progression; inhibition or slowing of the disease or its progression; arrest of its occurrence; and remission (whether partial or complete). Reduction of the pathological consequences of a proliferative disease is also encompassed by "treatment." The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require 100% elimination of all aspects of the disorder.

[0092] In some embodiments, an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising the same can be utilized in accordance with the methods herein to treat an IL13- or IL4-induced condition, hi some embodiments, an IL13R / IL4R sequential polypeptide or pharmaceutical composition is administered to a companion animal, e.g., a dog, cat, or horse, to treat an IL13- and IL4-induced condition.

[0093] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist can vary according to factors such as the type of disease being treated, the condition, the severity and course of the disease, the type of therapeutic objective, any previous treatments, medical history, response to previous treatments, the discretion of the attending veterinarian, the 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 may also be 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.

[0094] In some embodiments, an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide is administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide is administered intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation routes.

[0095] The IL13R / IL4R sequential polypeptides described herein may be administered in an amount ranging from 0.1 mg / kg to 100 mg / kg of body weight per dose. In some embodiments, the anti-IL13 / IL4 fusion may be administered in an amount ranging from 0.5 mg / kg to 50 mg / kg of body weight per dose. In some embodiments, the anti-IL13 / IL4 fusion may be administered in an amount ranging from 1 mg / kg to 10 mg / kg of body weight per dose. In some embodiments, the fusion molecule may be administered in an amount ranging from 0.5 mg / kg to 100 mg / kg body weight, from 1 mg / kg to 100 mg / kg body weight, from 5 mg / kg to 100 mg / kg body weight, from 10 mg / kg to 100 mg / kg body weight, from 20 mg / kg to 100 mg / kg body weight, from 50 mg / kg to 100 mg / kg body weight, from 1 mg / kg to 10 mg / kg body weight, from 5 mg / kg to 10 mg / kg body weight, from 0.5 mg / kg to 10 mg / kg body weight, or from 5 mg / kg to 50 mg / kg body weight.

[0096] The IL13R / IL4R sequential polypeptide or pharmaceutical composition comprising the IL13R / IL4R sequential polypeptide can be administered to the companion animal once or over a series of treatments. For example, the IL13R / IL4R sequential polypeptide or pharmaceutical composition comprising IL13R and IL4R 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.

[0097] 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 optionally repeated two or more times, separated by one or more weeks of treatment-free intervals. 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 optionally repeated two or more times, separated by one or more days or weeks of treatment-free intervals.

[0098] "Administration in combination" with one or more additional therapeutic agents includes simultaneous (concurrent) and sequential or consecutive administration in any order. The term "concurrent" is used herein to refer to the administration of two or more therapeutic agents, where at least some of the administration overlaps in time or where the administration of one therapeutic agent falls within a short time range compared to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered approximately a certain number of minutes or less apart. The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, where the administration of one or more agents is consecutive after the discontinuous administration of one or more other agents, or where the administration of one or more agents begins before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is administered approximately more than a certain number of minutes apart. As used herein, "in conjunction with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an animal.

[0099] In some embodiments, the method comprises administering a combination of an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide, a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor. In some embodiments, the method comprises administering a combination of an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide, an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL31 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-intergrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, or an anti-BlyS antibody.

[0100] Provided herein are methods of contacting cells with an IL13R / IL4R sequential polypeptide or a pharmaceutical composition comprising an IL13R / IL4R sequential polypeptide under conditions permissive for binding to IL13 or IL4. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide or pharmaceutical composition ex vivo. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide or pharmaceutical composition in vivo. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide or pharmaceutical composition under conditions permissive for binding of the fusion molecule to extracellular IL13 or IL4. In some embodiments, the cells may be exposed to the IL13R / IL4R sequential polypeptide or pharmaceutical composition in vivo by any one or more administration methods described herein, including, but not limited to, intraperitoneal, intramuscular, or intravenous injection into a subject. In some embodiments, cells may be exposed to the IL13R / IL4R sequential polypeptide or pharmaceutical composition ex vivo by exposing the cells to medium containing the fusion molecule or 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, prior to exposing the cells to medium containing the fusion molecule or pharmaceutical composition.

[0101] In some embodiments, exposure results in a decrease in IL13 or IL4 signaling function by the cell. In some embodiments, the IL13R / IL4R sequential polypeptide may reduce IL13 or IL4 signaling in the 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 or IL4 signaling in the absence of the IL13R / IL4R sequential polypeptide. In some embodiments, the reduction in IL13 or IL4 signaling or the reduction in TF-1 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%.

[0102] Provided herein are methods of using IL13R / IL4R sequential polypeptides, polypeptides, and polynucleotides for the detection, diagnosis, and monitoring of IL13- or IL4-induced conditions. Provided herein are methods of detecting whether a companion animal will respond to IL13R / IL4R sequential polypeptide therapy. In some embodiments, the method involves determining whether the animal has cells that express IL13 or IL4 using an IL13R / IL4R sequential polypeptide. In some embodiments, the method of detection involves contacting a sample with an antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from that of a reference or comparative sample (e.g., a control). In some embodiments, the method may be useful for determining whether the IL13R / IL4R sequential polypeptides described herein are an appropriate treatment for a subject animal.

[0103] In some embodiments, the sample is a biological sample. The term "biological sample" refers to a quantity of material from an organism or formerly living thing. In some embodiments, the biological sample is a cell or cell / tissue lysate. In some embodiments, biological samples include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.

[0104] In some embodiments, cells or cell / tissue lysates are contacted with an IL13R / IL4R sequential polypeptide and binding between the IL13R / IL4R sequential polypeptide and the cells is determined. If the test cells exhibit binding activity compared to reference cells of the same tissue type, this may indicate that the subject may benefit from treatment with an IL13R / IL4R sequential polypeptide. In some embodiments, the test cells are derived from tissue of a companion animal.

[0105] Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that can be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator moieties or label groups can be attached to the antibody of interest and are selected to meet the needs of various uses of the method, which is often dictated by the availability of assay equipment and compatible immunoassay procedures. Suitable labels include radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or p-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by Quantum Dot Corporation, Palo Alto, Calif.). General techniques used to perform the various immunoassays noted above are known to those of skill in the art.

[0106] For diagnostic purposes, IL13R / IL4R continuous polypeptides can be labeled with a detectable moiety, including, but not limited to, radioisotopes, fluorescent labels, and various enzyme substrate labels known in the art. Methods for conjugating labels to polypeptides are known in the art. In some embodiments, IL13R / IL4R continuous polypeptides need not be labeled, and their presence can be detected, for example, using an antibody that binds to the IL13R / IL4R continuous polypeptide. In some embodiments, IL13R / IL4R continuous polypeptides can be assayed using any known assay method, for example, 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). Anti-IL13 and IL4 antibodies and polypeptides can also be used in in vivo diagnostic assays, for example, in vivo imaging. Generally, antibodies or polypeptides can be labeled with a radionuclide (e.g., 111 In, 99 Tc, 14 C. 131 I, 125 I, 3When labeled with 3H, or any other radionuclide label, including those outlined herein, cells or tissues of interest can be localized using immunoscintigraphy. IL13R / IL4R continuous polypeptides can also be used as staining reagents in pathology using techniques well known in the art.

[0107] In some embodiments, the IL13R / IL4R sequential polypeptides are used for diagnosis and the IL13R / IL4R sequential polypeptides are used as therapeutic agents. In some embodiments, the first and second IL13R / IL4R sequential polypeptides are different.

[0108] The following examples illustrate certain aspects of the disclosure, but are not intended to limit the disclosure in any way. [Example]

[0109] Example 1 Expression and purification of canine IL4 and IL13 A nucleotide sequence encoding the canine IL13 protein (SEQ ID NO: 4) was synthesized with a C-terminal poly-His tag, cloned into a mammalian expression vector, and transfected into 293 or CHOS cells. The same method was used to clone and express a nucleotide sequence encoding the canine IL4 protein (SEQ ID NO: 1) with a C-terminal poly-His tag.

[0110] The supernatant containing canine IL13 protein was collected and filtered. Canine IL13 was affinity purified using a Ni-NTA column (CaptivA® Protein A affinity resin, Repligen). Canine IL4 was purified using the same method.

[0111] Example 2 Extracellular domains of IL13R and IL4R The extracellular domains of canine, feline, and equine IL4R responsible for binding canine, feline, and equine IL4 and / or IL13 have been identified and boundaries defined. The full-length extracellular domains of canine IL4R, feline IL4R, and equine IL4 are 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 that are predicted to retain biological activity are identified as SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, respectively.

[0112] The extracellular domains of canine, feline, and equine IL13R responsible for binding canine, feline, and equine IL4 and / or IL13 have been identified and boundaries defined. The full-length extracellular domains of canine IL13R, feline IL13R, and equine IL13R are 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 that are predicted to retain biological activity are identified as SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively.

[0113] The 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 informatics-based and determined to be buried (unexposed) based on 3-D modeling. Unpaired cysteines are unlikely to form disulfide bonds and are unlikely to aggregate. Therefore, site-directed mutagenesis of this Cys residue was not introduced.

[0114] Example 3 Expression and purification of canine IL13R / IL4R continuous polypeptides from CHO cells A nucleotide sequence encoding a canine IL13R / IL4R consecutive polypeptide linked to Fc IgGB was designed along with a signal sequence. For the consecutive polypeptide "IL13R-IL4R-IgGB" (SEQ ID NO: 20), the extracellular domain of IL13R (SEQ ID NO: 22) precedes the extracellular domain of IL4R (SEQ ID NO: 23). For the consecutive polypeptide IL4R-IL13R-IgGB (SEQ ID NO: 21), the extracellular domain of IL4R precedes the extracellular domain of IL13R.

[0115] The nucleotide sequence was chemically synthesized and inserted into an expression vector suitable for transfection into CHO host cells. After transfection into CHO cells, the fusion protein was secreted from the cells. For example, the fusion protein was purified by single-step protein A column chromatography.

[0116] Each of IL13R-IL4R-IgGB and IL4R-IL13R-IgGB can be expressed and purified in a single step on a protein A column or other chromatographic method, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed mode column chromatography (e.g., CHT), or multimodal mode column chromatography (e.g., CaptoMMC). Low pH or other viral inactivation and removal steps may be applied. The purified proteins may be mixed with excipients and sterilized by filtration to prepare pharmaceutical compositions of the invention. The pharmaceutical compositions may be administered to dogs with atopic dermatitis or asthma in an amount sufficient to bind to and / or inhibit either IL13 and / or IL4.

[0117] The vector was then used to perform pilot-scale transfections in CHO-S cells using FreestyleMax™ transfection reagent (Life Technologies). Supernatants were harvested by clarifying the conditioned medium. Proteins were purified with a single-pass Protein A chromatography step and used for further investigations.

[0118] Example 4 Demonstration of IL13 and IL4 binding activity This example demonstrates that both IL13R-IL4R-IgGB (SEQ ID NO: 20) and IL4R-IL13R-IgGB (SEQ ID NO: 21) bind to canine IL4 and IL13 with kinetics essential for therapeutic activity.

[0119] Binding analysis was performed using the biosensor Octet as follows. Briefly, canine IL4 (produced using 293 cells) was biotinylated. Free, unreacted biotin was removed from the biotinylated IL4 by extensive dialysis. Biotinylated canine IL4 was captured on a streptavidin sensor chip. IL4 association at various concentrations (12, 16, and 44 nM) of IL13R-IL4R-IgGB (SEQ ID NO: 20) was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was drawn to correct for any variations. Data were fitted to a 1:1 binding model using ForteBio™ data analysis software to obtain k on , k off The Kd for IL13R-IL4R-IgGB and the ligand IL4 was determined. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd for IL13R-IL4R-IgGB and the ligand IL4 was 8x10 -11 It was.

[0120] Canine IL4 association at various concentrations (40.7 and 140 nM) of IL4R-IL13R-IgGB (SEQ ID NO: 21) was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was drawn to correct for any variations. Data were fit to a 1:1 binding model using ForteBio™ data analysis software to obtain k on , k off The Kd for IL4R-IL13R-IgGB and the ligand IL4 was determined. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd for IL4R-IL13R-IgGB and the ligand IL4 was 1.1 x 10 -11 It was.

[0121] Canine IL4 and canine IL13 with C-terminal poly-His tags were expressed and purified from 293 cells. EZ-Link NHS-LC-Biotin was obtained from Thermo Scientific (catalog no. 21336) and streptavidin biosensor was obtained from ForteBio (catalog no. 18-509).

[0122] A sequential binding experiment of IL4 and IL13 with IL13R-IL4R-IgGB (SEQ ID NO: 20) was performed. Biotinylated canine IL13R-IL4R-IgGB was captured on a streptavidin sensor chip. Canine IL13R-IL4R-IgGB was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4, using IL4 and IL13 at a concentration of 30 μg / mL in PBS (FIG. 1). The experiment demonstrated that once IL13R-IL4R-IgGB bound to IL13, it may not bind to IL4, and once bound to IL4, its ability to bind IL13 was reduced.

[0123] Sequential binding experiments of IL4 and IL13 with IL4R-IL13R-IgGB (SEQ ID NO: 21) were performed. Biotinylated canine IL4R-IL13R-IgGB was captured on a streptavidin sensor chip. Canine IL4R-IL13R-IgGB was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4, using IL4 and IL13 at a concentration of 30 μg / mL in PBS (FIG. 2). These experiments demonstrated that once IL4R-IL13R-IgGB bound to IL13, it may not bind to IL4, and once bound to IL4, its ability to bind IL13 was reduced.

[0124] The tight binding of IL13R-IL4R-IgGB and IL4R-IL13R-IgGB to IL4 or IL13 is thought to be due to simultaneous binding contributions made by both IL4R and IL13R.

[0125] Example 5 Cellular functional activity of canine IL4R-IL13R-Fc (SINK) TF1 cells (ATCC catalog no. CRL-2003), a human erythroleukemia cell line expressing endogenous interleukin-4 receptors on the cell surface, were used for the proliferation assay. Cells were grown in exponential growth phase in RPMI 1640 (Gibco, catalog no. 11875) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, catalog no. 2868) and 2 nM / ml human GM-CSF (R&D System, catalog no. 215-GM-010). Cells were washed twice with PBS and resuspended in the above medium without GM-CSF. 20,000 cells were plated per well in a 96-well plate (Corning, catalog no. 3610). Canine IL4R-IL13R-Fc (SINK) was added in a dilution series, followed by canine IL4 (Sino Biological Inc, catalog no. 70021-DNAE-5) at 50 ng / ml. Cells were incubated at 37°C, 5% CO2, in a total volume of 100 μl for 48 hours. At the end of the incubation, cells were cooled to room temperature and assayed for proliferation / viability by measuring cellular ATP content using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, catalog no. G7570).

[0126] In this assay, 100 μl of a premix of Reagents A and B was added to each well. After 2 minutes of shaking on an orbital shaker, cells were lysed. Monooxygenation of luciferin is catalyzed by luciferase in the presence of Mg2+ and ATP present in the 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 the culture. Plates were 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).

[0127] The data was analyzed using a four parameter logistic fit and the IC50 is 2.0 nM. See Figure 3.

Claims

1. A polypeptide comprising an extracellular domain of an IL13R polypeptide and an extracellular domain of an IL4R polypeptide, wherein the IL13R and IL4R polypeptides are derived from a companion animal species; the companion animal species is a dog, a cat, or a horse; the polypeptide consists of an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; A polypeptide that reduces IL13 and / or IL4 signaling in a companion animal species compared to IL13 and / or IL4 signaling in the absence of said polypeptide.

2. 2. The polypeptide of claim 1, wherein the extracellular domain of the IL13R polypeptide 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.

3. 3. The polypeptide of claim 1 or 2, wherein the extracellular domain of the IL13R polypeptide comprises a cysteine ​​at position 18 of SEQ ID NO:22, position 18 of SEQ ID NO:24, position 18 of SEQ ID NO:26, position 15 of SEQ ID NO:32, position 15 of SEQ ID NO:34, or position 15 of SEQ ID NO:

36.

4. IL13 in companion animal species and 5 × 10 as measured by biolayer interferometry －６ Less than M, 1 x 10 －６ Less than M, 5 x 10 －７ Less than M, 1 x 10 －７ Less than M, 5 x 10 －８ Less than M, 1 x 10 －８ Less than M, 5 x 10 －９ Less than M, 1 x 10 －９ Less than M, 5 x 10 －１０ Less than M, 1 x 10 －１０ Less than M, 5 x 10 －１１ Less than M, 1 x 10 －１１ Less than M, 5 x 10 －１２ Less than M or 1 x 10 －１２ 4. The polypeptide of claim 1, which binds with a dissociation constant (Kd) of less than M.

5. IL4 in companion animal species and 5 × 10 as measured by biolayer interferometry. －６ Less than M, 1 x 10 －６ Less than M, 5 x 10 －７ Less than M, 1 x 10 －７ Less than M, 5 x 10 －８ Less than M, 1 x 10 －８ Less than M, 5 x 10 －９ Less than M, 1 x 10 －９ Less than M, 5 x 10 －１０ Less than M, 1 x 10 －１０ Less than M, 5 x 10 －１１ Less than M, 1 x 10 －１１ Less than M, 5 x 10 －１２ Less than M or 1 x 10 －１２ 5. The polypeptide of claim 1, which binds with a dissociation constant (Kd) of less than M.

6. The polypeptide of claim 1 , wherein the extracellular domain of the IL13R polypeptide consists of an amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO:

36.

7. The polypeptide of claim 1 , wherein the extracellular domain of the IL13R polypeptide consists of an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:

26.

8. The polypeptide of any one of claims 1 to 7, wherein the extracellular domain of the IL4R polypeptide consists of an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO:

37.

9. 9. The polypeptide of claim 1, wherein the extracellular domain of the IL4R polypeptide consists of an amino acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO:

27.

10. A polypeptide according to any one of claims 4 to 9, wherein the first linker (L1) and the second linker (L2), if present, each independently comprise an amino acid sequence selected from G, GG, GGG, S, SS, SSS, GS, GSGS (SEQ ID NO: 38), GSGSGS (SEQ ID NO: 39), GGS, GGSGGS (SEQ ID NO: 40), GGSGGSGGS (SEQ ID NO: 41), GGGS (SEQ ID NO: 42), GGGSGGGS (SEQ ID NO: 43), GGGSGGGSGGGS (SEQ ID NO: 44), GSS, GSSGSS (SEQ ID NO: 45), GSSGSSGSS (SEQ ID NO: 46), GGSS (SEQ ID NO: 47), GGSSGGSS (SEQ ID NO: 48), and GGSSGGSSGGSS (SEQ ID NO: 49).

11. A polypeptide described in any one of claims 4 to 10, wherein the polypeptide further comprises a fusion partner (FP), the FP being selected from Fc, albumin, and an albumin-binding fragment.

12. 12. The polypeptide of claim 11, wherein the Fc comprises: (a) a dog heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region; (b) a feline heavy chain constant region selected from an IgG1, IgG2a, and IgG2b constant region; or (c) an horse heavy chain constant region selected from an IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant region.

13. 13. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 12.

14. 14. An isolated or non-human host cell comprising the isolated nucleic acid of claim 13.

15. 15. A method for producing a polypeptide comprising culturing the host cell of claim 14 and isolating the polypeptide.

16. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

17. 13. Use of a polypeptide according to any one of claims 1 to 12 in the manufacture of a medicament for treating a companion animal species having a condition induced by IL13 and / or IL4, wherein the companion animal species is a dog, cat or horse.

18. 18. The use according to claim 17, wherein the condition induced by IL13 and / or IL4 is a pruritic or allergic condition selected from atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema.

19. 19. The use according to claim 17 or 18, wherein the polypeptide is adapted to be administrable parenterally.

20. 20. The use according to any one of claims 17 to 19, wherein the polypeptide is adapted to be administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal or inhalation route.

21. 21. The use of any one of claims 17 to 20, wherein a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor can be further administered.

22. The use according to any one of claims 17 to 21, wherein one or more antibodies can further be administered, and the one or more antibodies are selected from an anti-IL17 antibody, an anti-IL31 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, and an anti-BlyS antibody.

23. 13. Use of a polypeptide according to any one of claims 1 to 12 in the manufacture of a medicament for reducing IL13 and / or IL-4 signalling activity in a cell, wherein the cell is exposed to the polypeptide under permissive conditions for binding of the polypeptide to IL13 and / or IL4, thereby reducing binding of IL4 and / or IL13 to the native IL13 receptor and / or native IL-4 receptor and reducing IL13- and / or IL-4-mediated signalling, and wherein the cell is a canine, feline or equine cell.

24. 24. The use according to claim 23, wherein the cells are exposed to the polypeptide or pharmaceutical composition ex vivo.

25. A method for detecting IL13 or IL4 in a sample from a companion animal species, comprising contacting the sample with a polypeptide described in any one of claims 1 to 12 or a pharmaceutical composition described in claim 16 under conditions permissive for binding of the polypeptide to IL13 and / or IL4, and detecting whether a complex is formed between the polypeptide and IL13 and / or IL4 in the sample, wherein the sample is a biological sample obtained from a dog, cat, or horse.

26. A polypeptide comprising an extracellular domain of an IL13R polypeptide and an extracellular domain of an IL4R polypeptide, wherein the IL13R and IL4R polypeptides are derived from the same companion animal species, and the companion animal species is a dog, a cat, or a horse; the extracellular domain of the IL13R polypeptide comprises an amino acid sequence consisting of 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; and The polypeptide of claim 1, wherein the extracellular domain of the IL4R polypeptide comprises an amino acid sequence consisting of 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.

27. 13. A polypeptide according to any one of claims 1 to 12 for use in treating a companion animal species having a condition induced by IL13 and / or IL4, wherein the companion animal species is a dog, cat, or horse.

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