IL4 / IL13 receptor molecules for animals

Abstract

Provided are various embodiments related to IL13R / IL4R sequential polypeptides and IL13R / IL4R heterodimeric proteins from companion animal species that bind IL13 and / or IL4, including long-acting sequential polypeptides and heterodimeric proteins. Such heterodimeric proteins can be used in methods of treating IL13- and / or IL4-induced conditions in companion animals, such as dogs, cats, and horses.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Application No. 63 / 014090, filed April 22, 2020, and U.S. Provisional Application No. 63 / 014573, filed April 23, 2020, each of which is incorporated herein by reference in its entirety for all purposes.

[0002] [Sequence table] This application is filed with an electronic Sequence Listing, which is provided as a file entitled 2021-04-22_01157-0033-00PCT_ST25.txt, created on April 22, 2021, and is 614,620 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] [Field] The present disclosure relates to continuous polypeptides and heterodimeric proteins comprising interleukin-4 receptor and interleukin-13 receptor fragments from companion animal species that bind IL4 and / or IL13 of a companion animal species, e.g., canine IL4 and canine IL13, including long-acting molecules with extended serum half-lives. The present disclosure also relates to methods of using the continuous polypeptides and heterodimeric proteins to treat conditions induced by IL4 and / or IL13 in companion animals, e.g., dogs, cats, and horses, or to reduce IL4 and / or IL13 signaling activity in cells. [Background technology]

[0004] 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 bind to IL4 and / or IL13, reducing the free cytokine concentrations and thereby alleviating the clinical signs and symptoms associated with dermatitis, asthma, and other disorders. Companion species of 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 specifically bind to and be used to treat IL4 / IL13-induced conditions and reduce IL4 / IL13 signaling activity in companion animals. Summary of the Invention

[0005] Embodiment 1. A continuous polypeptide comprising the extracellular domain of an IL13R decoy polypeptide and the extracellular domain of an IL4R polypeptide, wherein the IL13R decoy and / or the IL4R polypeptide is derived from a companion animal species. Embodiment 2. A compound comprising the formula (I) IL13Rd-L1-IL4R-L2-FP, the formula (II) IL4R-L1-IL13Rd-L2-FP, the formula (III) IL13Rd-L1-FP-L2-IL4R, the formula (IV) IL4R-L1-FP-L2-IL13Rd, the formula (V) FP-L1-IL13Rd-L2-IL4R, or the formula (VI) FP-L1-IL4R-L2-IL13Rd, a) IL13Rd is the extracellular domain of an IL13R decoy polypeptide derived from a companion animal species; b) the 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) The contiguous polypeptide of embodiment 1, wherein the FP is a fusion partner, such as an IgG Fc polypeptide. Embodiment 3. A contiguous polypeptide comprising an extracellular domain of an IL13R polypeptide and an extracellular domain of an IL4R polypeptide, wherein the IL13R polypeptide and the IL4R polypeptide are derived from a companion animal species, and the contiguous polypeptide comprises formula (III) IL13R-L1-FP-L2-IL4R, formula (IV) IL4R-L1-FP-L2-IL13R, (V) FP-L1-IL13R-L2-IL4R, or formula (VI) FP-L1-IL4R-L2-IL13R; a) IL13R is the extracellular domain of an IL13R polypeptide from a companion animal species; b) the 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 contiguous polypeptide that is a fusion partner, such as an IgG Fc polypeptide. Embodiment 4. The continuous polypeptide of any one of embodiments 1 to 3, comprising a variant IgG Fc polypeptide derived from a companion animal species that is capable of binding to a neonatal Fc receptor (FcRn) with increased affinity compared to a wild-type Fc polypeptide, such as at low pH. Embodiment 5. A contiguous polypeptide comprising the extracellular domain of an IL13R polypeptide and the extracellular domain of an IL4R polypeptide, wherein the IL13R polypeptide and the IL4R polypeptide are derived from a companion animal species, and wherein the contiguous polypeptide comprises a variant IgG Fc polypeptide derived from the companion animal species that is capable of binding to a neonatal Fc receptor (FcRn) with increased affinity, e.g., at low pH, compared to a wild-type Fc polypeptide. Embodiment 6. A polypeptide comprising formula (I) IL13R-L1-IL4R-L2-Fc, formula (II) IL4R-L1-IL13R-L2-Fc, formula (III) IL13R-L1-Fc-L2-IL4R, formula (IV) IL4R-L1-Fc-L2-IL13R, (V) Fc-L1-IL13R-L2-IL4R, or formula (VI) Fc-L1-IL4R-L2-IL13R, a) IL13R is the extracellular domain of an IL13R polypeptide from a companion animal species; b) the 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) The contiguous polypeptide of embodiment 5, wherein Fc is an IgG Fc polypeptide. Embodiment 7. 5 x 10 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 10. The continuous polypeptide of any one of the preceding embodiments, which binds to IL13 of the companion animal species with a dissociation constant (Kd) of less than M. Embodiment 8. 5 x 10 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 10. The continuous polypeptide of any one of the preceding embodiments, which binds to IL4 of the companion animal species with a dissociation constant (Kd) of less than M. Embodiment 9. The continuous polypeptide of any one of the preceding embodiments, which reduces IL13 and / or IL4 signaling in a companion animal species. Embodiment 10. The continuous polypeptide of any one of the preceding embodiments, wherein the companion animal species is a dog, a cat, or a horse. Embodiment 11. The continuous polypeptide of any one of embodiments 3 to 10, wherein the extracellular domain of the 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. Embodiment 12. The continuous polypeptide of any one of embodiments 3 to 11, wherein the extracellular domain of the 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. Embodiment 13. The continuous polypeptide of any one of embodiments 3 to 12, wherein the extracellular domain of the 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. Embodiment 14. The continuous polypeptide of any one of embodiments 3 to 13, wherein the extracellular domain of the IL13R polypeptide is at least 98% identical or at least 99% 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 15. The consecutive polypeptide of any one of embodiments 3 to 14, 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. Embodiment 16. The consecutive polypeptide of any one of embodiments 3 to 15, 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. Embodiment 17. The contiguous polypeptide of any one of embodiments 3 to 16, wherein the extracellular domain of the IL13R polypeptide comprises an amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36. Embodiment 18. The contiguous polypeptide of any one of embodiments 3 to 17, wherein the extracellular domain of the IL13R polypeptide comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26. Embodiment 19. The continuous polypeptide of any one of embodiments 1, 2, 4, or 7 to 18, wherein the extracellular domain of the IL13R decoy polypeptide is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169. Embodiment 20. The contiguous polypeptide of any one of the preceding embodiments, wherein the extracellular domain of the IL4R polypeptide is at least 85% identical to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. Embodiment 21. The contiguous polypeptide of any one of the preceding embodiments, wherein the extracellular domain of the IL4R polypeptide is at least 90% identical to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. Embodiment 22. The contiguous polypeptide of any one of the preceding embodiments, wherein the extracellular domain of the IL4R polypeptide is at least 95% identical to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. Embodiment 23. The contiguous polypeptide of any one of the preceding embodiments, wherein the extracellular domain of the IL4R polypeptide is at least 98% identical or at least 99% identical to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. Embodiment 24. The contiguous polypeptide of any one of the preceding embodiments, wherein the extracellular domain of the IL4R polypeptide comprises an amino acid sequence selected from SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37. Embodiment 25. The contiguous polypeptide of any one of the preceding embodiments, wherein the extracellular domain of the IL4R polypeptide comprises an amino acid sequence selected from SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, and SEQ ID NO:27. Embodiment 26. The consecutive polypeptide of any one of embodiments 2 to 4, or 6 to 25, wherein 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: 151), GSGSGS (SEQ ID NO: 152), GGS, GGSGGS (SEQ ID NO: 153), GGSGGSGGS (SEQ ID NO: 154), GGGS (SEQ ID NO: 155), GGGSGGGS (SEQ ID NO: 156), GGGSGGGSGGGS (SEQ ID NO: 157), GSS, GSSGSS (SEQ ID NO: 158), GSSGSSGSS (SEQ ID NO: 159), GGSS (SEQ ID NO: 160), GGSSGGSS (SEQ ID NO: 161), and GGSSGGSSGGSS (SEQ ID NO: 162). Embodiment 27. The continuous polypeptide of any one of embodiments 2, 3, or 5 to 25, comprising an 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. Embodiment 28. a) a first contiguous polypeptide comprising at least one IL13R decoy extracellular domain (ECD) and a first Fc polypeptide; and b) a second sequential polypeptide comprising at least one IL4R ECD and a second Fc polypeptide. Including, A heterodimeric protein, wherein the IL13R decoy ECD and / or the IL4R ECD are derived from a companion animal species. Embodiment 29. The heterodimeric protein of embodiment 28, wherein the first Fc polypeptide and / or the second Fc polypeptide is a variant IgG Fc polypeptide derived from a companion animal species that can bind to a neonatal Fc receptor (FcRn) with increased affinity, such as at low pH, compared to a wild-type Fc polypeptide. Embodiment 30. a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide; and b) a second sequential polypeptide comprising at least one IL4R ECD and a second Fc polypeptide. Including, A heterodimeric protein, wherein the IL13R ECD and / or the IL4R ECD is derived from a companion animal species, and the first Fc polypeptide and / or the second Fc polypeptide is a variant IgG Fc polypeptide derived from the companion animal species that can bind to a neonatal Fc receptor (FcRn) with increased affinity, e.g., at low pH, compared to a wild-type Fc polypeptide. Embodiment 31. The heterodimeric protein of any one of embodiments 28 to 30, wherein the first consecutive polypeptide and / or the second consecutive polypeptide comprises one, two, three, or four IL4R ECDs and / or one, two, three, or four IL13R ECDs or IL13R decoy ECDs. Embodiment 32. The heterodimeric protein of any one of embodiments 28 to 31, wherein the first consecutive polypeptide and / or the second consecutive polypeptide further comprises at least one binding partner other than an IL4R ECD, an IL13R ECD, or an IL13R decoy ECD. Embodiment 33. The heterodimeric protein of embodiment 32, wherein at least one binding partner comprises IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-α, TSLP, and / or IgE. Embodiment 34. 5×10 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 34. The heterodimeric protein of any one of embodiments 28 to 33, which binds to IL13 and / or IL4 with a dissociation constant (Kd) of less than M. Embodiment 35. The heterodimeric protein of any one of embodiments 28 to 34, which reduces IL13 and / or IL4 signaling in a companion animal species. Embodiment 36. The heterodimeric protein of any one of embodiments 28 to 35, wherein the companion animal species is dog, cat, or horse. Embodiment 37. The heterodimeric protein of any one of embodiments 30 to 36, wherein the amino acid sequence of 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 38. The heterodimeric protein of any one of embodiments 30 to 37, wherein the amino acid sequence of at least one IL13R ECD contains 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 39. A heterodimeric protein of any one of embodiments 30 to 38, wherein the amino acid sequence of at least one IL13R ECD contains 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. Embodiment 40. The heterodimeric protein of any one of embodiments 30 to 39, wherein 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 41. The heterodimeric protein of any one of embodiments 28, 29, or 31 to 36, wherein the extracellular domain of the IL13R decoy polypeptide is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169. Embodiment 42. The heterodimeric protein of any one of embodiments 28 to 41, wherein the amino acid sequence of 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: 163, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37. Embodiment 43. The heterodimeric protein of any one of embodiments 28 to 42, wherein at least one IL4R ECD comprises an amino acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 163, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37. Embodiment 44. The heterodimeric protein of any one of embodiments 28 to 43, wherein the first Fc polypeptide or the second Fc polypeptide comprises a knob mutation. Embodiment 45. A heterodimeric protein of any one of embodiments 28 to 44, wherein the first Fc polypeptide or the second Fc polypeptide comprises a hole mutation. Embodiment 46. 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 46. ​​The heterodimeric protein of any one of embodiments 28 to 45, comprising: Embodiment 47. The first Fc polypeptide or the second 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, 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) 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. 47. The heterodimeric protein of any one of embodiments 28 to 46, comprising: Embodiment 48. 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 48. The heterodimeric protein of any one of embodiments 28 to 47, comprising: Embodiment 49. The first Fc polypeptide or the second 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, or position 138 of SEQ ID NO: 41; and / or b) 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) 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 49. The heterodimeric protein of any one of embodiments 28 to 48, comprising: Embodiment 50. The first Fc polypeptide or the second Fc polypeptide comprises: a) amino acid substitutions at positions corresponding to positions 138 and / or 140 and / or 181 of SEQ ID NO: 38, 137 and / or 139 and / or 180 of SEQ ID NO: 39, 137 and / or 139 and / or 180 of SEQ ID NO: 40, or 138 and / or 140 and / or 181 of SEQ ID NO: 41; and / or b) an amino acid substitution at a position corresponding to positions 154 and / or 156 and / or 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) amino acid substitutions at positions corresponding to positions 130 and / or 132 and / or 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 50. The heterodimeric protein of any one of embodiments 28 to 49, comprising: Embodiment 51. The first Fc polypeptide or the second Fc polypeptide comprises: a) serine at the position corresponding to position 138 and / or alanine at the position corresponding to position 140 and / or threonine at the position corresponding to position 181 of SEQ ID NO: 38, serine at the position corresponding to position 137, alanine at the position corresponding to position 139 and / or threonine at the position corresponding to position 180 of SEQ ID NO: 39, serine at the position corresponding to position 137 and / or alanine at the position corresponding to position 139 and / or threonine at the position corresponding to position 180 of SEQ ID NO: 40, or serine at the position corresponding to position 138 and / or alanine at the position corresponding to position 140 and / or threonine at the 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) Serine at position 130 and / or alanine at position 132 and / or 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. 51. The heterodimeric protein of any one of embodiments 28 to 50, comprising: Embodiment 52. The first Fc polypeptide or the second Fc polypeptide comprises: a) an amino acid substitution at position 138 and / or 140 and / or 181 of SEQ ID NO: 38, at position 137 and / or 139 and / or 180 of SEQ ID NO: 39, at position 137 and / or 139 and / or 180 of SEQ ID NO: 40, or at position 138 and / or 140 and / or 181 of SEQ ID NO: 41; and / or b) an amino acid substitution at position 154 and / or 156 and / or 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 132 and / or 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 52. The heterodimeric protein of any one of embodiments 28 to 51, comprising: Embodiment 53. The first Fc polypeptide or the second Fc polypeptide comprises: a) serine at position 138 and / or alanine at position 140 and / or threonine at position 181 of SEQ ID NO: 38, serine at position 137 and / or alanine at position 139 and / or threonine at position 180 of SEQ ID NO: 39, serine at position 137 and / or alanine at position 139 and / or threonine at position 180 of SEQ ID NO: 40, or serine at position 138 and / or alanine at position 140 and / or threonine at position 181 of SEQ ID NO: 41; and / or b) serine at position 154 and / or alanine at position 156 and / or 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) Serine at position 130 and / or alanine at position 132 and / or 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 53. The heterodimeric protein of any one of embodiments 28 to 52, comprising: Embodiment 54. The first Fc polypeptide or the second Fc polypeptide is selected from the group consisting 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, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO 54. The heterodimeric protein of any one of embodiments 28 to 53, comprising the amino acid sequence of 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 55. A heterodimeric protein of any one of embodiments 28 to 54, wherein the first consecutive 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 56. A heterodimeric protein of any one of embodiments 28 to 55, wherein the second consecutive 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 57. The continuous polypeptide or heterodimeric protein of any one of the preceding embodiments, wherein the variant IgG Fc polypeptide binds to FcRn with greater affinity than the wild-type IgG Fc polypeptide, as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool, at a pH in the range of about 5.0 to about 6.5, such as at a pH of about 5.0, about a pH of about 5.2, about a pH of about 5.5, about a pH of about 6.0, about a pH of about 6.2, or about a pH of about 6.5. Embodiment 58. The variant IgG Fc polypeptide has a pH of 5×10 or less as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool at a pH in the range of about 5.0 to about 6.5, such as about pH 5.0, about pH 5.5, about pH 6.0, or about pH 6.5. -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 10. The contiguous polypeptide or heterodimeric protein of any one of the preceding embodiments, which binds to FcRn with a dissociation constant (Kd) of less than M. Embodiment 59. A contiguous polypeptide or heterodimeric protein of any one of the preceding embodiments, having an increased serum half-life compared to a contiguous polypeptide or heterodimeric protein comprising a wild-type Fc polypeptide. Embodiment 60. The continuous polypeptide or heterodimeric protein of any one of the preceding embodiments, wherein the variant IgG Fc polypeptide binds to FcRn with increased affinity compared to the wild-type Fc polypeptide, and wherein the continuous polypeptide or heterodimeric protein has an increased serum half-life compared to a continuous polypeptide or heterodimeric protein comprising the wild-type Fc polypeptide. Embodiment 61. The variant IgG Fc polypeptide comprises: a) a tyrosine or phenylalanine at a position corresponding to position 23 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; b) a tyrosine at a position corresponding to position 82 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; c) a tyrosine at a position corresponding to position 82 and a histidine at a position corresponding to position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; d) a tyrosine at a position corresponding to position 82 and a tyrosine at a position corresponding to position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; e) a tyrosine at a position corresponding to position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; f) a tyrosine at a position corresponding to position 82 and a histidine at a position corresponding to position 208 of SEQ ID NO: 38 or SEQ ID NO: 41; g) a tyrosine at a position corresponding to position 82 and a tyrosine at a position corresponding to position 208 of SEQ ID NO: 38 or SEQ ID NO: 41; or h) Tyrosine at a position corresponding to position 208 of SEQ ID NO: or SEQ ID NO: 41 10. The contiguous polypeptide or heterodimeric protein of any one of the preceding embodiments, comprising: Embodiment 62. The variant IgG Fc polypeptide comprises: a) tyrosine or phenylalanine at position 23 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; b) a tyrosine at position 82 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; c) a tyrosine at position 82 and a histidine at position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; d) a tyrosine at position 82 and a tyrosine at position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; e) a tyrosine at position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; f) Tyrosine at position 82 and histidine at position 208 of SEQ ID NO: 38 or SEQ ID NO: 41 g) a tyrosine at position 82 and a tyrosine at position 208 of SEQ ID NO:38 or SEQ ID NO:41; or h) Tyrosine at position 208 of SEQ ID NO: 38 or SEQ ID NO: 41 10. The contiguous polypeptide or heterodimeric protein of any one of the preceding embodiments, comprising: Embodiment 63. The contiguous polypeptide or heterodimeric protein of any one of the preceding embodiments, wherein the variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129. Embodiment 64. The consecutive polypeptide or heterodimeric protein of any one of the preceding embodiments, comprising the amino acid sequence of SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, or SEQ ID NO:183. Embodiment 65. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, or SEQ ID NO:183. Embodiment 66. The contiguous polypeptide, heterodimeric protein, or polypeptide of any one of the preceding embodiments, wherein the contiguous polypeptide, heterodimeric protein, or polypeptide is sialylated. Embodiment 67. An isolated nucleic acid encoding the contiguous polypeptide, heterodimeric protein, or polypeptide of any one of the preceding embodiments. Embodiment 68. A host cell comprising the nucleic acid of embodiment 67. Embodiment 69. A host cell expressing the consecutive polypeptide, heterodimeric protein, or polypeptide of any one of embodiments 1 to 66. Embodiment 70. A method comprising culturing the host cell of embodiment 68 or 69 and isolating a polypeptide, a consecutive polypeptide, a first consecutive polypeptide, a second consecutive polypeptide, or a first consecutive polypeptide and a second consecutive polypeptide. Embodiment 71. A pharmaceutical composition comprising the consecutive polypeptide, heterodimeric protein, or polypeptide of any one of embodiments 1 to 66 and a pharmaceutically acceptable carrier. Embodiment 72. A method for treating a companion animal species having an IL13 and / or IL4-induced condition, comprising administering to the companion animal species a therapeutically effective amount of a consecutive polypeptide, heterodimeric protein, or polypeptide of any one of embodiments 1 to 66 or a pharmaceutical composition of embodiment 71. Embodiment 73. The method of embodiment 72, wherein the companion animal species is a dog, a cat, or a horse. Embodiment 74. The method of embodiment 72 or 73, 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 75. The method of any one of embodiments 72 to 74, wherein the continuous polypeptide, heterodimeric protein, polypeptide, or pharmaceutical composition is administered parenterally. Embodiment 76. The method of any one of embodiments 72 to 75, wherein the heterodimeric protein or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal, or inhalation route. Embodiment 77. The method of any one of embodiments 72 to 76, further comprising administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor. Embodiment 78. The method of any one of embodiments 62 to 77, further comprising 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-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, an anti-IL5 antibody, an anti-IL5R antibody, an anti-IL22 antibody, an anti-IL22R antibody, an anti-IL33 antibody, an anti-IL33R antibody, an anti-TSLP antibody, an anti-TSLPR antibody, and an anti-BlyS antibody. Embodiment 79. A method for reducing IL13 and / or IL4 signaling activity in a cell, comprising exposing the cell to a consecutive polypeptide, heterodimeric protein, or polypeptide of any one of embodiments 1 to 66, or the pharmaceutical composition of embodiment 71, under conditions that allow binding of the heterodimeric protein to IL13 and / or IL4, thereby (a) reducing binding of IL13 and / or IL-4 and / or IL-13 to the native IL13 receptor and / or the native IL-4 receptor and reducing IL13- and / or IL-4-mediated signaling. Embodiment 80. The method of embodiment 79, wherein the cells are exposed to the heterodimeric protein or pharmaceutical composition ex vivo. Embodiment 81. The method of embodiment 79, wherein the cell is exposed to the heterodimeric protein or pharmaceutical composition in vivo. Embodiment 82. The method of any one of embodiments 79 to 81, wherein the cell is a canine cell, a feline cell, or an equine cell. Embodiment 83. A method for detecting IL13 or IL4 in a sample from a companion animal species, comprising contacting the sample with a consecutive polypeptide, heterodimeric protein or polypeptide of any one of embodiments 1 to 66 or the pharmaceutical composition of embodiment 71 under conditions that allow 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 84. The method of embodiment 83, wherein the sample is a biological sample obtained from a dog, cat, or horse. [Brief explanation of the drawings]

[0006] [Figure 1] 1 is a graph of the sequential binding of canine IL4RECD-IL13RECD-Fc to canine IL4 and IL13 or canine IL13 and IL4 using IL4 and IL13 at a concentration of 30 μg / mL in PBS.

[0007] [Figure 2] 1 is a graph of the sequential binding of canine IL13RECD-IL4RECD-Fc to canine IL4 and IL13 or canine IL13 and IL4 using IL4 and IL13 at a concentration of 30 μg / mL in PBS.

[0008] [Figure 3]

[0033] Figure 1 is a graph of canine IL4RECD-IL13RECD-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.

[0009] [Figure 4] 1 shows a Biacore sensorgram of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) binding to wild-type canine IgG-B Fc polypeptide.

[0010] [Figure 5] 1 shows a Biacore sensorgram of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) binding to variant canine IgG-B Fc polypeptide L(23)Y.

[0011] [Figure 6] 1 shows a Biacore sensorgram of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) binding to the variant canine IgG-B Fc polypeptide L(23)F.

[0012] [Figure 7] 1 shows a Biacore sensorgram of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) binding to the variant canine IgG-B Fc polypeptide L(23)M.

[0013] [Figure 8] 1 shows a Biacore sensorgram of various concentrations of canine FcRn (12.5, 25, 50, 100, and 200 nM) binding to the variant canine IgG-B Fc polypeptide YTE.

[0014] [Figure 9] OctetRed sensorgrams of chimeric variant canine IgG-A Fc F00 antibody (A) and IgG-D Fc F00 antibody (B) binding to canine FcRn compared to chimeric variant canine IgG-A Fc without Phe mutation (C) and IgG-D Fc without Phe mutation (D).

[0015] [Figure 10] FIG. 1 shows the serum pharmacokinetic profiles of chimeric variant canine IgG-A F00 antibody (“IgG-A F00”; n=2) and chimeric variant canine IgG-A without the Phe mutation (“IgG-A”; n=2) following subcutaneous administration to rats at 2 mg / kg.

[0016] [Figure 11] 16 shows OctetRed sensorgrams of chimeric antibodies containing variant canine IgG-B Fc (0Y0, 0YH, 0YY, or 00Y) that bind to canine FcRn compared to chimeric antibodies containing wild-type canine IgG-B.

[0017] [Figure 12] 1 is a chart showing normalized antibody percent over time from the in vivo pharmacokinetic study in dogs described in Example 13. Description of a given sequence

[0018] Table 1 provides a list of the sequences referenced herein. JPEG0007821741000001.jpg255168JPEG0007821741000002.jpg255167JPEG0007821741000003.jpg255168JPEG0007821741000004.jpg255167JPEG0007821741000005.jpg255167JPEG0007821741000006.jpg255168JPEG0007821741000007.jpg255166JPEG0007821741000008.jpg255167JPEG0007821741000009.jpg255169JPEG0007821741000010.jpg255167JPEG0007821741000011.jpg255166JPEG0007821741000012.jpg255166JPEG0007821741000013.jpg255166JPEG0007821741000014.jpg255167JPEG0007821741000015.jpg255165JPEG0007821741000016.jpg255166JPEG0007821741000017.jpg255166JPEG0007821741000018.jpg255166JPEG0007821741000019.jpg255166JPEG0007821741000020.jpg255169JPEG0007821741000021.jpg255167JPEG0007821741000022.jpg255166JPEG0007821741000023.jpg255168JPEG0007821741000024.jpg255168JPEG0007821741000025.jpg255170JPEG0007821741000026.jpg255167JPEG0007821741000027.jpg255168JPEG0007821741000028.jpg255165JPEG0007821741000029.jpg255168JPEG0007821741000030.jpg255168JPEG0007821741000031.jpg248170JPEG0007821741000032.jpg255170JPEG0007821741000033.jpg255167JPEG0007821741000034.jpg255166JPEG0007821741000035.jpg255169JPEG0007821741000036.jpg255168JPEG0007821741000037.jpg25 5166JPEG0007821741000038.jpg255167JPEG0007821741000039.jpg255167JPEG0007821741000040.jpg255167JPEG0007821741000041.jpg230170. DETAILED DESCRIPTION OF THE INVENTION

[0019] Consecutive polypeptides, e.g., long-acting polypeptides, that bind to canine IL13 and / or IL4, feline IL13 and / or IL4, and / or equine IL13 and / or IL4 are provided. In some embodiments, the contiguous polypeptide comprises the extracellular domain of an IL13R polypeptide and the extracellular domain of an IL4R polypeptide. Methods for producing or purifying the contiguous polypeptides are also provided. Therapeutic methods using contiguous polypeptides that bind to 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.

[0020] Also provided are IL13R / IL4R heterodimeric proteins, e.g., long-acting proteins, that bind canine IL13 and / or IL4, feline IL13 and / or IL4, and / or equine IL13 and / or IL4. In some embodiments, the IL13R / IL4R heterodimeric proteins comprise a first contiguous polypeptide comprising the extracellular domain of an IL13R polypeptide and an Fc polypeptide, and a second contiguous polypeptide comprising the extracellular domain of an IL4R polypeptide and an Fc polypeptide. Methods for producing or purifying IL13R / IL4R heterodimeric proteins and contiguous polypeptides are also provided. Therapeutic methods using IL13R / IL4R heterodimeric proteins that bind to IL13 and / or IL4 and inhibit IL13- and / or IL-4-mediated signaling are also 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.

[0021] Also provided are variant IgG Fc polypeptides derived from companion animals with increased binding to Protein A, decreased binding to C1q, decreased binding to CD16, and increased binding to FcRn, which may be used in conjunction with the contiguous polypeptides or heterodimeric proteins provided herein.

[0022] For the convenience of the reader, the following definitions are provided for terms used herein.

[0023] As used herein, numerical terms such as Kd are calculated based on scientific measurements and, therefore, are subject to appropriate measurement errors. In some instances, the numerical terms may include values ​​that are rounded to the nearest significant figure.

[0024] 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 expressly stated otherwise. In the context of a claim that is multiple dependent, the use of "or" when referring to another claim refers only to that alternative claim.

[0025] Exemplary IL13R / IL4R Contiguous Polypeptides and Heterodimeric Proteins Novel IL13R / IL4R contiguous polypeptides and IL13R / IL4R heterodimeric proteins are provided, for example, heterodimeric proteins that bind to canine IL13 and / or IL4, feline IL13 and / or IL4, and / or equine IL13 and / or IL4.

[0026] 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 can 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 such as deletions, additions, and substitutions (generally conservative in nature) relative to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations in hosts producing the protein or errors during PCR amplification.

[0027] As used herein, "glycosylated" refers to a polypeptide to which one or more glycan moieties are covalently attached.

[0028] As used herein, "glycan" or "glycan moiety" refers to a glycosidically linked monosaccharide.

[0029] Glycans are attached to glycopeptides in several ways, of which N-linkage to asparagine and O-linkage to serine and threonine are most relevant for recombinant therapeutic glycoproteins. N-linked glycosylation occurs at the consensus sequence Asn-Xaa-Ser / Thr, where Xaa can be any amino acid except proline.

[0030] As used herein, "sialylated" refers to a polypeptide to which one or more sialic acid moieties are covalently attached.

[0031] Various approaches have been developed to produce glycosylated and sialylated proteins, see, for example, Savinova et al., Applied Biochem & Microbiol. 51(8):827-33 (2015).

[0032] As used herein, "PEGylated" refers to a polypeptide having one or more polyethylene glycol (PEG) moieties associated therewith or covalently or non-covalently attached thereto.

[0033] In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide comprises at least one glycan moiety attached to an N-linked glycosylation site. In some embodiments, the polypeptide is sialylated. In some embodiments, the polypeptide is PEGylated. In some embodiments, the polypeptide is PEGylated at a glycan. In some embodiments, the polypeptide is PEGylated at a primary amine. In some embodiments, the polypeptide is PEGylated at an N-terminal alpha amine. In some embodiments, the polypeptide is glycosylated, sialylated, and / or PEGylated.

[0034] The term "continuous polypeptide" herein refers to an uninterrupted amino acid sequence. A continuous polypeptide is typically translated from a single continuous DNA sequence. This can be created 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, resulting in the DNA sequences being expressed as a single protein. Typically, this is accomplished by cloning cDNA into an expression vector in frame with an existing gene.

[0035] As used herein, "IL4R" refers to a polypeptide comprising the entire or a fragment of the IL4 receptor subunit α that binds to IL-4.

[0036] 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 horses), unless otherwise specified. 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:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0037] As used herein, "IL13R" refers to a polypeptide comprising all or part of the IL13 receptor subunit alpha-1 that binds to IL-13.

[0038] 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 horses), unless otherwise specified. In some embodiments, IL13R is an extracellular domain fragment that binds to IL13. In some such embodiments, IL13R may be referred to as the 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.

[0039] As used herein, "IL13R decoy" or "IL13Rd" refers to a polypeptide comprising all or part of the IL13 receptor subunit α-2 that binds to IL-13.

[0040] For example, "IL13R decoy" or "IL13Rd" refers to an IL13 receptor subunit alpha-2 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 horses), unless otherwise specified. In some embodiments, the IL13R decoy is an extracellular domain fragment of the IL13 receptor subunit alpha-2 that binds to IL13. In some such embodiments, the IL13R decoy may be referred to as an IL13R decoy extracellular domain (ECD). In some embodiments, the IL13R decoy comprises the amino acid sequence of SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169.

[0041] The term "companion animal species" refers to animals suitable to be companions to humans. In some embodiments, companion animal species are small mammals such as dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, etc. In some embodiments, companion animal species are domestic animals such as horses, cows, pigs, etc.

[0042] An "extracellular domain" ("ECD") is the portion of a polypeptide that extends beyond the transmembrane domain into the extracellular space. As used herein, the term "extracellular domain" can include the entire extracellular domain or can include a truncated extracellular domain lacking one or more amino acids that bind to its ligand. The composition of an extracellular domain can depend on the algorithm used to determine which amino acids are membrane-resident. Different algorithms can predict, and different systems can express, different extracellular domains of a given protein.

[0043] The extracellular domain of an IL4R polypeptide can comprise the complete extracellular domain of an IL4R that binds IL4 or a truncated extracellular domain of an IL4R. 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 transition terms such as "comprising" or "comprises." In some embodiments, the extracellular domain of an IL4R polypeptide is the extracellular domain of an IL4R polypeptide derived from a companion animal species. 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:163, SEQ ID NO:25, or SEQ ID NO:27, or any fragment thereof. In some embodiments, the extracellular domain of the 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.

[0044] The extracellular domain of an IL13R polypeptide can comprise the 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 include the transmembrane or cytoplasmic domain, even when the terms are followed by open transition terms such as "comprising" or "comprises." In some embodiments, the extracellular domain of an IL13R polypeptide is the extracellular domain of an IL13R polypeptide derived from a companion 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.

[0045] The extracellular domain of an IL13Rd polypeptide can comprise the complete extracellular domain of IL13Rd that binds to IL13 or a truncated extracellular domain of IL13Rd. As used herein, the terms "extracellular domain of an IL13Rd polypeptide," "IL13Rd ECD," and similar terms refer to an IL13Rd polypeptide that does not include the transmembrane or cytoplasmic domain, even if the terms are followed by open transition terms such as "comprising" or "comprises." In some embodiments, the extracellular domain of an IL13Rd polypeptide is the extracellular domain of an IL13Rd polypeptide derived from a companion animal. For example, in some embodiments, the extracellular domain of an IL13Rd polypeptide is derived from canine IL13Rd, feline IL13Rd, or equine IL13Rd. In some embodiments, the extracellular domain of an IL13Rd polypeptide comprises the amino acid sequence of SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169, or any fragment thereof.

[0046] The terms "IL13R / IL4R sequential polypeptide" and "IL4R / IL13R sequential polypeptide" are used interchangeably to refer to a sequential polypeptide comprising an IL13R polypeptide and an IL4R polypeptide, and the terms do not refer to the order in which the IL13R and IL4R polypeptides appear in the sequential polypeptide, unless the order is otherwise indicated. For example, an IL13R / IL4R sequential polypeptide or an IL4R / IL13R sequential polypeptide can refer to an IL4R polypeptide that is preceded in sequence or followed in sequence by an IL13R polypeptide. In addition, an IL13R / IL4R sequential polypeptide or an IL4R / IL13R sequential polypeptide can refer to an IL13R polypeptide that is preceded in sequence or followed in sequence by an IL4R polypeptide.

[0047] The terms "IL13Rd / IL4R sequential polypeptide" and "IL4R / IL13Rd sequential polypeptide" are used interchangeably to refer to a sequential polypeptide comprising an IL13Rd polypeptide and an IL4R polypeptide, and the terms do not indicate the order in which the IL13Rd and IL4R polypeptides appear in the sequential polypeptide, unless the order is otherwise indicated. For example, an IL13Rd / IL4R sequential polypeptide or an IL4R / IL13Rd sequential polypeptide can refer to an IL4R polypeptide that is preceded in sequence or followed in sequence by an IL13Rd polypeptide. In addition, an IL13Rd / IL4R sequential polypeptide or an IL4R / IL13Rd sequential polypeptide can refer to an IL13Rd polypeptide that is preceded in sequence or followed in sequence by an IL4R polypeptide.

[0048] 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 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 the N-terminus of the IL4R polypeptide.

[0049] In some embodiments, an IL13Rd / IL4R continuous polypeptide comprises an IL13Rd polypeptide linked to an IL4R polypeptide at the C-terminus of the IL13Rd polypeptide or the N-terminus of the IL13R polypeptide. In some embodiments, an IL13Rd / IL4R continuous polypeptide comprises an IL4R polypeptide linked to an IL13Rd polypeptide at the C-terminus of the IL4R polypeptide or the N-terminus of the IL4R polypeptide.

[0050] 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, wherein the polypeptide is derived from a companion animal species. For example, the sequential polypeptide may comprise the extracellular domain of an IL4R polypeptide derived from a dog, a cat, or a horse, and / or may comprise the extracellular domain of an IL13R polypeptide derived from a dog, a cat, or a horse.

[0051] The IL13Rd / IL4R sequential polypeptides of the present invention may comprise the extracellular domain of an IL13Rd polypeptide and / or the extracellular domain of an IL4R polypeptide, wherein the polypeptides are derived from companion animal species. For example, the sequential polypeptides may comprise the extracellular domain of an IL4R polypeptide derived from a dog, a cat, or a horse, and / or the extracellular domain of an IL13Rd polypeptide derived from a dog, a cat, or a horse.

[0052] 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.

[0053] The terms "IL13Rd / IL4R heterodimeric protein" and "IL4R / IL13Rd heterodimeric protein" are used interchangeably to refer to a heterodimeric protein comprising a first consecutive polypeptide comprising an IL13Rd polypeptide and a second consecutive polypeptide comprising an IL4R polypeptide.

[0054] In some embodiments, the first contiguous polypeptide and / or the second contiguous polypeptide comprises an Fc polypeptide.

[0055] The IL13R / IL4R heterodimeric protein of the present invention may comprise the extracellular domain of an IL13R polypeptide and / or the extracellular domain of an IL4R polypeptide, where the polypeptide is derived from a companion animal species. For example, the heterodimeric protein may comprise the extracellular domain of an IL4R polypeptide derived from a dog, a cat, or a horse, and / or may comprise the extracellular domain of an IL13R polypeptide derived from a dog, a cat, or a horse.

[0056] The IL13Rd / IL4R heterodimeric protein of the present invention may comprise the extracellular domain of an IL13Rd polypeptide and / or the extracellular domain of an IL4R polypeptide, wherein the polypeptides are derived from companion animal species. For example, the heterodimeric protein may comprise the extracellular domain of an IL4R polypeptide derived from a dog, a cat, or a horse, and / or the extracellular domain of an IL13Rd polypeptide derived from a dog, a cat, or a horse.

[0057] "Wild-type" refers to a non-mutated form of a naturally occurring polypeptide, or a fragment thereof. A wild-type polypeptide may be recombinantly produced. "Wild-type IL13R ECD," "wild-type IL13Rd 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, IL13Rd, or IL4R.

[0058] A "variant" is a nucleic acid molecule or polypeptide that differs from a reference nucleic acid molecule or polypeptide by single or multiple amino acid substitutions, deletions, and / or additions, and that retains substantially at least one biological activity of the reference nucleic acid molecule or polypeptide.

[0059] A "biologically active" entity, or an entity possessing "biological activity," is an entity that has any function related to or associated with a metabolic or physiological process and / or possesses the structural, regulatory, or biochemical function of a naturally occurring molecule. Biologically active polynucleotide fragments are those that exhibit activity similar to, but not necessarily identical to, the activity of a polynucleotide of the invention. Biologically active polypeptides or fragments thereof include those that can participate in a biological reaction, including, but not limited to, ligand-receptor interactions or antigen-antibody binding. Biological activity can include an improved desired activity or a decreased undesirable activity. An entity can exhibit biological activity if it participates in a molecular interaction with another molecule, such as hybridization, if it has therapeutic value in alleviating a disease state, if it has preventative value in inducing an immune response, if it 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 if it can be used as a primer in a polymerase chain reaction (PCR).

[0060] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to nucleic acid molecule or polypeptide sequences are defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical with 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 performed using, for example, BLAST, BLAST-2, ALIGN, or MEGALINE. TM This can be achieved in a variety of ways within the skill of the art using publicly available computer software, such as (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 the sequences being compared.

[0061] In some embodiments, a variant has at least about 50% sequence identity with a reference nucleic acid molecule or polypeptide, 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. Such variants include, for example, polypeptides with one or more amino acid residues added or 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, or at least about 99% sequence identity to the sequence of the reference nucleic acid or polypeptide.

[0062] In some embodiments, the consecutive polypeptides comprise an extracellular domain of an IL13R polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, or at least about 99% 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 consecutive polypeptides comprise an extracellular domain of an IL13Rd polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169. In some embodiments, the contiguous polypeptide comprises an extracellular domain of an IL4R polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0063] As used herein, "position corresponding to position n" (where n is any number) refers to the 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 the purpose of determining whether a position of a subject polypeptide corresponds to position n of a reference polypeptide can be performed using, for example, BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN. TM This can be achieved in a variety of ways within the skill of the art using publicly available computer software, such as (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any parameters necessary to achieve maximum alignment over the entire length of the two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.

[0064] In some embodiments, the consecutive polypeptides comprise 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, the consecutive polypeptides comprise 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.

[0065] A "point mutation" is a mutation involving a single nucleotide or amino acid residue. The mutation can be the loss of a 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.

[0066] Amino acid substitutions can include, but are not limited to, substituting one amino acid in a polypeptide for another. Exemplary substitutions are shown in Table 2. Amino acid substitutions can be introduced into a molecule of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC, or enhanced pharmacokinetics.

[0067] JPEG0007821741000042.jpg121155

[0068] Amino acids can 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.

[0069] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0070] As used herein, a "fusion partner" refers to an additional component of an IL13R / IL4R continuous polypeptide, e.g., an additional polypeptide, such as albumin, an albumin-binding fragment, or a fragment of an immunoglobulin molecule. A fusion partner may include an oligomerization domain, such as the Fc domain of a heavy chain immunoglobulin.

[0071] The term "IgX Fc" or "IgX Fc polypeptide" refers to an Fc region 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" refers to the Fc region of the gamma chain, "IgA" or "IgA Fc" refers to the Fc region of the alpha chain, "IgD" or "IgD Fc" refers to the Fc region of the delta chain, "IgE" or "IgE Fc" refers to the Fc region of the epsilon chain, "IgM" or "IgM Fc" refers to the Fc region of the mu chain, etc.

[0072] A "fragment crystallizable polypeptide" or "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 comprises a fragment of an Fc domain that retains one or more biological activities of the entire Fc polypeptide. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind to FcRn. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind to C1q. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind to CD16. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind to Protein A. 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 and / or ADCC (antibody-dependent cellular cytotoxicity) induction.

[0073] "IgX Fc" or "IgX Fc polypeptide" refers to an Fc polypeptide derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" indicates the antibody isotype. Thus, "IgG Fc" indicates that the Fc polypeptide is derived from the gamma chain, "IgA Fc" indicates that the Fc polypeptide is derived from the alpha chain, "IgD Fc" indicates that the Fc polypeptide is derived from the delta chain, "IgE Fc" indicates that the Fc polypeptide is derived from the epsilon chain, "IgM Fc" indicates that the Fc polypeptide is derived from the mu chain, etc. In some embodiments, an IgG Fc polypeptide comprises a hinge, CH2, and CH3, but does not comprise a CH1 or CL. In some embodiments, an IgG Fc polypeptide comprises a CH2 and CH3, but does not comprise a CH1, hinge, or CL. In some embodiments, an IgG Fc polypeptide comprises a CH1, hinge, CH2, and CH3, with or without a CL. In some embodiments, an IgG Fc polypeptide comprises a CH1, hinge, CH2, and CH3, with or without a CL1. In some embodiments, an Fc polypeptide, such as an IgG Fc polypeptide, lacks one or more C-terminal amino acids, such as 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 2 amino acids, while retaining biological activity. In some embodiments, the biological activity is the ability to bind to FcRn. 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 and / or ADCC (antibody-dependent cellular cytotoxicity) induction. "IgX-N Fc" or "IgGXN Fc" indicates that the Fc polypeptide is derived from a particular subclass of antibody isotype (e.g., canine IgG subclasses IgG-A, IgG-B, IgG-C, or IgG-D; feline IgG subclasses IgG1a, IgG1b, or IgG2; or equine IgG subclasses IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where "N" indicates the subclass.

[0074] 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 IgG1a, IgG1b, or IgG2. In other examples, 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.

[0075] In some embodiments, the contiguous polypeptides comprise a first variant IgG Fc polypeptide comprising a "knob" mutation and a second variant IgG Fc polypeptide comprising a "hole" mutation. Non-limiting exemplary knob and hole mutations are described, for example, in Merchant, AM et al., An efficient route to human bispecific IgG. Nat Biotechnol, 16(7):677-81 (1998).

[0076] As used herein, a "knob" mutation refers to an interface mutation of a molecule (e.g., an Fc polypeptide) that contains bulky amino acids.

[0077] As used herein, a "hole mutation" refers to an interface mutation of a molecule (e.g., an Fc polypeptide) that comprises one or more smaller amino acids.

[0078] In some embodiments, the variant IgG Fc polypeptide comprises a knob mutation. In some embodiments, the 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.

[0079] In some embodiments, the 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.

[0080] In some embodiments, the 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.

[0081] In some embodiments, the 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.

[0082] In some embodiments, the 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.

[0083] In some embodiments, the variant IgG Fc polypeptide comprises a hole mutation. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to positions 138 and / or 140 and / or 181 of SEQ ID NO:38; 137 and / or 139 and / or 180 of SEQ ID NO:39; 137 and / or 139 and / or 180 of SEQ ID NO:40; 138 and / or 140 and / or 181 of SEQ ID NO:41; 154 and / or 156 and / or 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 130 and / or 132 and / or 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.

[0084] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at positions 138 and / or 140 and / or 181 of SEQ ID NO:38; 137 and / or 139 and / or 180 of SEQ ID NO:39; 137 and / or 139 and / or 180 of SEQ ID NO:40; 138 and / or 140 and / or 181 of SEQ ID NO:41; 154 and / or 156 and / or 197 of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46; or 130 and / or 132 and / or 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.

[0085] In some embodiments, the variant IgG Fc polypeptide comprises a serine at the position corresponding to position 138 and / or an alanine at the position corresponding to position 140 and / or a threonine at the position corresponding to position 181 of SEQ ID NO:38; a serine at the position corresponding to position 137 and / or an alanine at the position corresponding to position 139 and / or a threonine at the position corresponding to position 180 of SEQ ID NO:39; a serine at the position corresponding to position 137 and / or an alanine at the position corresponding to position 139 and / or a threonine at the position corresponding to position 180 of SEQ ID NO:40; a serine at the position corresponding to position 138 and / or alanine at the position corresponding to position 140 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.

[0086] In some embodiments, the 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 threonine at position 1; 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.

[0087] In some embodiments, the 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.

[0088] In some embodiments, the variant IgG Fc polypeptides have altered fetal receptor (FcRn) binding affinity, ie, the variant IgG Fc polypeptides have increased binding affinity for FcRn, such as at low pH.

[0089] In some embodiments, the variant IgG Fc polypeptide binds to FcRn with greater affinity than the wild-type IgG Fc polypeptide as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool at a pH ranging from about 5.0 to about 6.5, such as about pH 5.0, about pH 5.2, about pH 5.5, about pH 6.0, about pH 6.2, or about pH 6.5.

[0090] In some embodiments, the variant IgG Fc polypeptide has a pH of 5×10 or less as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool at a pH in the range of about 5.0 to about 6.5, such as a pH of about 5.0, a pH of about 5.5, a pH of about 6.0, or a pH of about 6.5. -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 -12Less than M or 1 x 10 -12 It binds to FcRn with a dissociation constant (Kd) less than M.

[0091] In some embodiments, long-acting IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13Rd / IL4R heterodimeric proteins, or IL13R / IL4R heterodimeric proteins are provided. In some embodiments, the IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13Rd / IL4R heterodimeric proteins, or IL13R / IL4R heterodimeric proteins have increased serum half-life. In some embodiments, the IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13Rd / IL4R heterodimeric proteins, or IL13R / IL4R heterodimeric proteins comprise a variant Fc polypeptide, and the sequential polypeptide or heterodimeric protein has increased serum half-life compared to a sequential polypeptide or heterodimeric protein comprising a wild-type Fc polypeptide.

[0092] In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13Rd / IL4R heterodimeric protein, or IL13R / IL4R heterodimeric protein comprises a variant IgG Fc polypeptide that can bind to FcRn with increased affinity compared to a wild-type Fc polypeptide, and the sequential polypeptide has an increased serum half-life compared to a sequential polypeptide comprising a wild-type Fc polypeptide.

[0093] In some embodiments, the contiguous polypeptide or heterodimeric protein comprises: a) a tyrosine or phenylalanine at a position corresponding to position 23 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; b) a tyrosine at a position corresponding to position 82 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; c) a tyrosine at a position corresponding to position 82 and a histidine at a position corresponding to position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; d) a tyrosine at a position corresponding to position 82 and a tyrosine at a position corresponding to position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; e) a tyrosine at a position corresponding to position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; f) a tyrosine at a position corresponding to position 82 and a histidine at a position corresponding to position 208 of SEQ ID NO: 38 or SEQ ID NO: 41; g) a tyrosine at a position corresponding to position 82 and a tyrosine at a position corresponding to position 208 of SEQ ID NO: 38 or SEQ ID NO: 41; or h) Tyrosine at a position corresponding to position 208 of SEQ ID NO: 38 or SEQ ID NO: 41 and a variant IgG Fc polypeptide comprising:

[0094] In some embodiments, the contiguous polypeptide or heterodimeric protein comprises: a) tyrosine or phenylalanine at position 23 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; b) a tyrosine at position 82 of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; c) a tyrosine at position 82 and a histidine at position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; d) a tyrosine at position 82 and a tyrosine at position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; e) a tyrosine at position 207 of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, 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; f) a tyrosine at position 82 and a histidine at position 208 of SEQ ID NO:38 or SEQ ID NO:41; g) a tyrosine at position 82 and a tyrosine at position 208 of SEQ ID NO:38 or SEQ ID NO:41; or h) Tyrosine at position 208 of SEQ ID NO: 38 or SEQ ID NO: 41 and a variant IgG Fc polypeptide comprising:

[0095] "Signal sequence" refers to a sequence of amino acid residues or a polynucleotide encoding the same that facilitates secretion of a polypeptide of interest and is typically cleaved upon transport of the polypeptide across the cell surface membrane.

[0096] "Linker" refers to one or more amino acid residues that connect a first polypeptide to a second polypeptide.

[0097] In some embodiments, the linker is a glycine-rich and / or serine-rich flexible unstructured linker. In some embodiments, the linker comprises the amino acids G (Gly) and / or S (Ser). For example, the linker may include G or repeats of G (e.g., GG, GGG, etc.); GS or repeats of GS (e.g., GSGS (SEQ ID NO: 151), GSGSGS (SEQ ID NO: 152), etc.); GGS or repeats thereof (e.g., GGSGGS (SEQ ID NO: 153), GGSGGSGGS (SEQ ID NO: 154), etc.); GGGS (SEQ ID NO: 155) or repeats thereof (e.g., GGGSGGGS (SEQ ID NO: 156), GGGSGGGSGGGS (SEQ ID NO: 157), etc.); GSS or repeats thereof (e.g., GSSGSS (SEQ ID NO: 158), GSSGSSGSS (SEQ ID NO: 159), etc.); or GGSS (SEQ ID NO: 160) or repeats thereof (e.g., GGSSGGSS (SEQ ID NO: 161), GGSSGGSSGGSS (SEQ ID NO: 162), etc.).

[0098] In some embodiments, the continuous polypeptide comprises at least one linker. In some embodiments, the continuous polypeptide comprises an optional signal sequence and at least one optional linker. In some embodiments, the continuous polypeptide does not comprise a signal sequence or a linker. In some embodiments, the continuous polypeptide is translated with a signal sequence, but the signal sequence is cleaved from the continuous polypeptide.

[0099] In some embodiments, the IL13R / IL4R translocator polypeptide comprises: Formula (I): IL13R-L1-IL4R-L2-FP, Formula (II): IL4R-L1-IL13R-L2-FP, Formula (III): IL13R-L1-FP-L2-IL4R, Formula (IV): IL4R-L1-FP-L2-IL13R, Formula (V): FP-L1-IL13R-L2-IL4R, or Formula (VI): FP-L1-IL4R-L2-IL13R wherein IL13R is an IL13R extracellular domain (ECD) polypeptide from a companion animal species, IL4R is an IL4R ECD polypeptide from a companion animal species, L1 is a first optional linker, L2 is a second optional linker, and FP is an optional fusion partner such as an Fc polypeptide.

[0100] In some embodiments, the IL13Rd / IL4R sequential polypeptide comprises: Formula (I): IL13Rd-L1-IL4R-L2-FP, Formula (II): IL4R-L1-IL13Rd-L2-FP, Formula (III): IL13Rd-L1-FP-L2-IL4R, Formula (IV): IL4R-L1-FP-L2-IL13Rd, Formula (V): FP-L1-IL13Rd-L2-IL4R, or Formula (VI): FP-L1-IL4R-L2-IL13Rd wherein IL13Rd is an IL13Rd extracellular domain (ECD) polypeptide from a companion animal species, IL4R is an IL4R ECD polypeptide from a companion animal species, L1 is a first optional linker, L2 is a second optional linker, and FP is an optional fusion partner such as an Fc polypeptide.

[0101] 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, and SEQ ID NO:31.

[0102] In some embodiments, the IL13Rd / IL4R continuous polypeptide comprises an amino acid sequence selected from SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, and SEQ ID NO:183.

[0103] In some embodiments, the 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 IL4R ECD is derived from a companion animal species.

[0104] In some embodiments, the first contiguous polypeptide or the second contiguous polypeptide has the formula: IL13R(n)-L-Fc or IL4R(n)-L-Fc wherein IL13R(n) is at least one IL13R extracellular domain (ECD) polypeptide from a companion animal species; IL4R(n) is at least one IL4R ECD polypeptide from a companion animal species; (n) is one, two, three, four or more ECD polypeptides; L is an optional linker; and Fc is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a knob or hole mutation.

[0105] In some embodiments, the heterodimeric protein comprises a) a first contiguous polypeptide comprising at least one IL13Rd 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 IL13Rd ECD and / or IL4R ECD is derived from a companion animal species.

[0106] In some embodiments, the first contiguous polypeptide or the second contiguous polypeptide has the formula: IL13Rd(n)-L-Fc or IL4R(n)-L-Fc wherein IL13Rd(n) is at least one IL13Rd extracellular domain (ECD) polypeptide from a companion animal species; IL4R(n) is at least one IL4R ECD polypeptide from a companion animal species; (n) is one, two, three, four or more ECD polypeptides; L is an optional linker; and Fc is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a knob or hole mutation.

[0107] Additionally, other binding partners can be included in the contiguous polypeptide before, after, and / or between any one or more of the IL13R, IL13Rd, or IL4R ECD polypeptides. Other potential binding partners include IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-α, TSLP, and / or IgE.

[0108] 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.

[0109] 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.

[0110] In some embodiments, the contiguous polypeptide or heterodimeric protein comprises the amino acid sequence of SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, or SEQ ID NO:183.

[0111] Exemplary Expression and Production Polynucleotide sequences encoding all or a portion (e.g., the extracellular domain) of a contiguous polypeptide, with or without a signal sequence, are provided. If a homologous signal sequence (i.e., the signal sequence of a native IL4R, IL13R, or IL13Rd) is not used in constructing the nucleic acid molecule, another signal sequence can be used, such as any one of the signal sequences described in PCT / US06 / 02951.

[0112] Typically, a nucleotide sequence encoding a polypeptide of interest, such as a continuous polypeptide, is inserted into an expression vector suitable for expression in a selected host cell.

[0113] 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 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, such as an antibiotic resistance gene. A vector is appropriate for the host cell in which it is expressed. When a gene of interest is present in the vector, the vector may be called a "recombinant vector."

[0114] A "host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate animal 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; progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. A host cell includes cells transfected in vivo with a polynucleotide encoding the amino acid sequences provided herein.

[0115] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the 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 of the components of the cell in which it is produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide in which it is typically found in nature (e.g., genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) or, for example, if it is an RNA polynucleotide, if it has been separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated."

[0116] In some embodiments, the heterodimeric protein or sequential polypeptide is isolated using chromatography, such as size exclusion chromatography, ion exchange chromatography, Protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0117] The terms "label" and "detectable label" refer to a moiety attached to an IL13R / IL4R continuous polypeptide to render it detectable. In some embodiments, the label is a detectable marker that can generate a signal that is detectable by visual or instrumental means, e.g., by incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined 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 such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, a moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety.

[0118] Exemplary IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, and IL13Rd / IL4R heterodimeric proteins as decoy receptor traps The IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, and IL13Rd / IL4R heterodimeric proteins of the present invention can function as decoy receptors to capture IL13 and / or IL4 and inhibit their interaction with IL13R and / or IL4R on the cell surface. Decoy receptors such as those of the present invention recognize their ligands with high affinity and specificity but are constitutively unable to signal. They compete with wild-type receptors for ligand binding and participate in ligand / receptor interactions, thereby regulating the activity or number of functional receptors and / or downstream cellular activities from the receptor. Decoy receptors can act as molecular traps for agonist ligands, thereby inhibiting ligand-induced receptor activation.

[0119] As used herein, "IL13" refers to any native IL13 resulting from expression and processing of IL13 in cells. Unless otherwise indicated, 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 horses). The term also includes naturally occurring variants of IL13, such as splice variants or allelic variants.

[0120] 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.

[0121] As used herein, "IL4" refers to any native IL4 resulting from expression and processing of IL4 in a cell. Unless otherwise indicated, 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 horses). The term also includes naturally occurring variants of IL4, such as splice variants or allelic variants.

[0122] 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.

[0123] The present invention provides IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, and IL13Rd / IL4R heterodimeric proteins as therapeutic agents. The sequential and heterodimeric proteins of the present invention bind to IL13 and / or IL4, as described in more detail herein, which have been demonstrated to be associated with allergic diseases. In various embodiments, the sequential and heterodimeric proteins of the present invention are capable of binding to IL13 and / or IL4 with very high affinity. In various embodiments, the sequential and heterodimeric proteins of the present invention are capable of disrupting IL13 and / or IL4 signaling.

[0124] The term "affinity" refers to the overall strength 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 is generally determined by the dissociation constant (K D The 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.

[0125] "K D "," "K d The terms "," "Kd" or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of a receptor fusion-ligand interaction. In some embodiments, the K d is measured by using a biolayer interference assay using a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont, CA) according to the supplier's instructions. Briefly, biotinylated antigen is attached to the sensor tip, and the binding of the fusion molecule is monitored for 90 seconds, followed by dissociation 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 subtracted to correct for drift. Data are fitted to a 2:1 binding model using ForteBio data analysis software to determine the binding rate constant (k on ), dissociation rate constant (k off ), and K d Determine the equilibrium dissociation constant (K d ) is k off / k on The term "k" refers to the rate constant for binding of molecule X to its partner Y, and the term "k" refers to the rate constant for dissociation of molecule X or partner Y from the molecule X / partner Y complex.

[0126] 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, and the reaction, association, or affinity is detectable, for example, by one or more methods known in the art, such as immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance, etc.

[0127] "Surface plasmon resonance" can be used, for example, in BIAcore TMThe system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ) demonstrates an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix. For further description, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

[0128] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from a layer of immobilized proteins 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 non-limiting exemplary device for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0129] In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein has a concentration of 5×10 or more 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 -12and 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. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein ... -6 M and 1×10 -6 Between M, 5 x 10 -6 M and 5x10 -7 Between M, 5 x 10 -6 M and 1×10 -7 Between M, 5 x 10 -6 M and 5x10 -8 Between M, 5 x 10 -6 M and 1×10 -8 Between M, 5 x 10 -6 M and 5x10 -9 Between M, 5 x 10 -6 M and 1×10 -9 Between M, 5 x 10 -6 M and 5x10 -10 Between M, 5 x 10 -6 M and 1×10 -10 Between M, 5 x 10 -6 M and 5x10 -11 Between M, 5 x 10 -6 M and 1×10 -11 Between M, 5 x 10 -6 M and 5x10 -12 Between M, 5 x 10 -6 M and 1×10 -12 Between M, 1 x 10 -6 M and 5x10 -7 Between M, 1 x 10 -6 M and 1×10 -7 Between M, 1 x 10 -6 M and 5x10 -8 Between M, 1 x 10 -6 M and 1×10 -8 Between M, 1 x 10 -6 M and 5x10 -9 Between M, 1 x 10 -6 M and 1×10 -9 Between M, 1 x 10 -6 M and 5x10 -10 Between M, 1 x 10 -6M and 1×10 -10 Between M, 1 x 10 -6 M and 5x10 -11 Between M, 1 x 10 -6 M and 1×10 -11 Between M, 1 x 10 -6 M and 5x10 -12 Between M, 1 x 10 -6 M and 1×10 -12 Between M, 5 x 10 -7 M and 1×10 -7 Between M, 5 x 10 -7 M and 5x10 -8 Between M, 5 x 10 -7 M and 1×10 -8 Between M, 5 x 10 -7 M and 5x10 -9 Between M, 5 x 10 -7 M and 1×10 -9 Between M, 5 x 10 -7 M and 5x10 -10 Between M, 5 x 10 -7 M and 1×10 -10 Between M, 5 x 10 -7 M and 5x10 -11 Between M, 5 x 10 -7 M and 1×10 -11 Between M, 5 x 10 -7 M and 5x10 -12 Between M, 5 x 10 -7 M and 1×10 -12 Between M, 1 x 10 -7 M and 5x10 -8 Between M, 1 x 10 -7 M and 1×10 -8 Between M, 1 x 10 -7 M and 5x10 -9 Between M, 1 x 10 -7 M and 1×10 -9 Between M, 1 x 10 -7 M and 5x10 -10 Between M, 1 x 10 -7 M and 1×10 -10 Between M, 1 x 10 -7 M and 5x10 -11 Between M, 1 x 10 -7 M and 1×10 -11 Between M, 1 x 10 -7 M and 5x10 -12 Between M, 1 x 10 -7M and 1×10 -12 Between M, 5 x 10 -8 M and 1×10 -8 Between M, 5 x 10 -8 M and 5x10 -9 Between M, 5 x 10 -8 M and 1×10 -9 Between M, 5 x 10 -8 M and 5x10 -10 Between M, 5 x 10 -8 M and 1×10 -10 Between M, 5 x 10 -8 M and 5x10 -11 Between M, 5 x 10 -8 M and 1×10 -11 Between M, 5 x 10 -8 M and 5x10 -12 Between M, 5 x 10 -8 M and 1×10 -12 Between M, 1 x 10 -8 M and 5x10 -9 Between M, 1 x 10 -8 M and 1×10 -9 Between M, 1 x 10 -8 M and 5x10 -10 Between M, 1 x 10 -8 M and 1×10 -10 Between M, 1 x 10 -8 M and 5x10 -11 Between M, 1 x 10 -8 M and 1×10 -11 Between M, 1 x 10 -8 M and 5x10 -12 Between M, 1 x 10 -8 M and 1×10 -12 Between M, 5 x 10 -9 M and 1×10 -9 Between M, 5 x 10 -9 M and 5x10 -10 Between M, 5 x 10 -9 M and 1×10 -10 Between M, 5 x 10 -9 M and 5x10 -11 Between M, 5 x 10 -9 M and 1×10 -11 Between M, 5 x 10 -9 M and 5x10 -12 Between M, 5 x 10 -9 M and 1×10 -12 Between M, 1 x 10 -9M and 5x10 -10 Between M, 1 x 10 -9 M and 1×10 -10 Between M, 1 x 10 -9 M and 5x10 -11 Between M, 1 x 10 -9 M and 1×10 -11 Between M, 1 x 10 -9 M and 5x10 -12 Between M, 1 x 10 -9 M and 1×10 -12 Between M, 5 x 10 -10 M and 1×10 -10 Between M, 5 x 10 -10 M and 5x10 -11 Between M, 1 x 10 -10 M and 5x10 -11 Between M, 1 x 10 -10 M and 1×10 -11 Between M, 1 x 10 -10 M and 5x10 -12 Between M, 1 x 10 -10 M and 1×10 -12 Between M, 5 x 10 -11 M and 1×10 -12 Between M, 5 x 10 -11 M and 5x10 -12 Between M, 5 x 10 -11 M and 1×10 -12 Between M, 1 x 10 -11 M and 5x10 -12 Between M or 1 x 10 -11 M and 1×10 -12 In some embodiments, the IL13R / IL4R consecutive polypeptide, the IL13Rd / IL4R consecutive polypeptide, the IL13R / IL4R heterodimeric protein, or the IL13Rd / IL4R heterodimeric protein binds to canine IL13 and / or IL4, feline IL13 and / or IL4, or equine IL13 and / or IL4 with a Kd between 0.01 and 0.01 M. In some embodiments, the IL13R / IL4R consecutive polypeptide, the IL13Rd / IL4R consecutive polypeptide, the IL13R / IL4R heterodimeric protein, or the IL13Rd / IL4R heterodimeric protein binds to canine IL13 and / or IL4, feline IL13 and / or IL4, or equine IL13 and / or IL4.

[0130] "Decrease" or "inhibit" means to decrease, reduce, or stop an activity, function, or amount compared to a reference. In some embodiments, "decrease" or "inhibit" refers to the ability to cause an overall decrease of 20% or more. In some embodiments, "decrease" or "inhibit" refers to the ability to cause an overall decrease of 50% or more. In some embodiments, "decrease" or "inhibit" refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control dose (e.g., 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 can be obtained from a healthy or non-diseased sample. In some instances, a reference is obtained from a non-diseased or untreated sample of a companion animal. In some instances, a reference is obtained from one or more healthy animals of a particular species that are not the animal being tested or treated.

[0131] As used herein, the term "substantially reduced" refers to a sufficiently high degree of reduction between a numerical value and a reference numerical value such that one of skill in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the values. In some embodiments, a substantially reduced numerical value is reduced by more than approximately any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

[0132] In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein can 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 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%.

[0133] "Increased" or "greater" refers to an increase relative to a reference. In some embodiments, "increased" or "greater" refers to the ability to cause an overall increase of about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 125% or more, about 150% or more, about 200% or more, or about 300% or more compared to a reference value. In some embodiments, "increase" or "greater" refers to the ability to cause an overall increase of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% compared to a reference value.

[0134] In some embodiments, a variant Fc polypeptide, such as a variant IgG Fc polypeptide, can bind to FcRn or FcRn / B2M with an affinity that is increased by about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 125% or more, about 150% or more, about 200% or more, or about 300% or more compared to a reference Fc polypeptide. In some embodiments, a variant Fc polypeptide can bind to FcRn or FcRn / B2M with an affinity that is increased by about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% compared to a reference Fc polypeptide. In some embodiments, the reference Fc polypeptide is a wild-type Fc polypeptide. In some embodiments, the Fc polypeptide is a different variant Fc polypeptide. In some embodiments, the affinity is measured by biolayer interferometry at a pH ranging from about 5.0 to about 6.5.

[0135] In some embodiments, pharmacokinetic analysis is performed to determine any number of pharmacokinetic parameters, including half-life, T, C, and area under the curve (AUC). For example, an IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein described herein is administered to an animal, and serum samples are collected at different time intervals (e.g., pre-injection and / or 0.5, 1, 6, 24, 48, 72, 168, 216, and / or 336 hours post-administration). The sequential polypeptide or heterodimeric protein concentration in the serum samples can be determined, for example, by ELISA.

[0136] In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein has a serum half-life that is about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 125% or more, about 150% or more, about 200% or more, about 250% or more, or about 300% or more relative to a reference sequential polypeptide or heterodimeric protein. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein has a serum half-life that is about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% of that of the reference sequential polypeptide or heterodimeric protein. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein has a serum half-life that is about 1.5-fold or more, about 2-fold or more, or about 3-fold or more of that of the reference sequential polypeptide or heterodimeric protein. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein has a serum half-life of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, about 25% to about 70%, or about 200% to about 300% of that of a reference sequential polypeptide or heterodimeric protein. In some embodiments, the reference sequential polypeptide or heterodimeric protein comprises a wild-type Fc polypeptide. In some embodiments, the Fc polypeptide is a different variant Fc polypeptide.

[0137] Exemplary Pharmaceutical Compositions The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0138] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier common 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, 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; polyvinylpyrrolidone, cellulose-based materials; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.

[0139] The pharmaceutical composition can be stored in lyophilized form. Thus, in some embodiments, the preparation method includes a lyophilization step. The lyophilized composition can then be re-formulated prior to administration to dogs, cats, or horses, typically as an aqueous composition suitable for parenteral administration. In other embodiments, particularly when the antibody is highly stable to thermal 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 be included. Thus, the present invention provides pharmaceutical compositions in solid or liquid form.

[0140] The pH of the pharmaceutical composition when administered can range from about pH 5 to about pH 8. The compositions of the present invention are sterile 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 antibacterial agents.

[0141] Exemplary Uses of IL13R / IL4R Sequential Polypeptides, IL13Rd / IL4R Sequential Polypeptides, IL13R / IL4R Heterodimeric Proteins, and IL13Rd / IL4R Heterodimeric Proteins and Pharmaceutical Compositions The IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, and IL13Rd / IL4R heterodimeric proteins of the present invention, or pharmaceutical compositions comprising the sequential polypeptides or heterodimeric proteins thereof, may be useful for treating IL13- 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- or IL4-induced conditions include, but are not limited to, pruritic or allergic diseases. In some embodiments, the IL13- 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.

[0142] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" includes any administration or application of a therapeutic agent for a 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 the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or slowing of disease spread, prevention or slowing of disease recurrence, slowing 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 (partial or complete). Also encompassed by "treatment" is the reduction of the pathological consequences of a proliferative disease. 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.

[0143] In some embodiments, an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition comprising a sequential polypeptide or heterodimeric protein thereof, can be utilized in accordance with the methods herein to treat an IL13- or IL4-induced condition. In some embodiments, an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition comprising a sequential polypeptide or heterodimeric protein thereof, is administered to a companion animal, such as a dog, cat, or horse, to treat an IL13- or IL4-induced condition.

[0144] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the type of disease being treated, the disease state, the severity and course of the disease, the type of therapeutic objective, previous treatments, clinical history, response to previous treatments, the discretion of the attending physician, 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 is also an amount 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 can 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.

[0145] In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein, or pharmaceutical composition comprising the sequential polypeptide or heterodimeric protein thereof, is administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein, or pharmaceutical composition comprising the sequential polypeptide or heterodimeric protein thereof, is administered as a bolus injection or by continuous infusion over a predetermined period of time. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition comprising the sequential polypeptide or heterodimeric protein thereof, is administered intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation.

[0146] The IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins 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 IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins may be administered in an amount ranging from 0.1 mg / kg to 50 mg / kg of body weight per dose. In some embodiments, the IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins may be administered in an amount ranging from 1 mg / kg to 10 mg / kg of body weight per dose. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein 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.

[0147] The IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein, or pharmaceutical composition comprising the sequential polypeptide or heterodimeric protein thereof, can be administered to the companion animal once or over a series of treatments. For example, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein, or pharmaceutical composition comprising the sequential polypeptide or heterodimeric protein thereof can be administered at least once, more than once, at least two times, at least three times, at least four times, or at least five times.

[0148] In some embodiments, the dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more week-long treatment-free periods. In other embodiments, the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more day- or week-long treatment-free periods.

[0149] In some embodiments, the long-acting IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein is administered at a reduced dose and / or with increased intervals between administrations compared to a reference sequential polypeptide or heterodimeric protein.

[0150] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential or sequential administration in any order. The term "in combination" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent is within a short time period relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered at a time interval of about a specified number of minutes or less. 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 continues after the administration of one or more other agents is discontinued, or where the administration of one or more agents is initiated before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is separated in time by more than about a specified number of minutes. As used herein, "in conjunction with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an animal.

[0151] In some embodiments, the method comprises administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor in combination with an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition comprising the sequential polypeptide or heterodimeric protein thereof. In some embodiments, the method comprises administering an anti-IL31 antibody, an anti-IL4R antibody, 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-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, or an anti-BlyS antibody in combination with an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition comprising the sequential polypeptide or sequential polypeptides thereof.

[0152] Provided herein are methods of exposing cells to an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition comprising a sequential polypeptide or heterodimeric protein, under conditions that allow binding to IL13 and / or IL4. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein or pharmaceutical composition ex vivo. In some embodiments, the cells are exposed to the IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or pharmaceutical composition in vivo. In some embodiments, cells are exposed to an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition under conditions that allow binding of the sequential polypeptide or heterodimeric protein to extracellular IL13 and / or IL4. In some embodiments, cells may be exposed to an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition in vivo by any one or more of the 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 an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, or a pharmaceutical composition ex vivo by exposing the cells to culture medium containing the heterodimeric protein or pharmaceutical composition.In some embodiments, the permeability of the cell membrane can be affected by using any number of methods understood by one of skill in the art (e.g., electroporation of the cells or exposure of the cells to a solution containing calcium chloride) prior to exposing the cells to culture medium containing the fusogenic molecule or pharmaceutical composition.

[0153] In some embodiments, exposure results in a reduction in IL13 and / or IL4 signaling function by the cell. In some embodiments, the IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein can reduce IL13 and / 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 and / or IL4 signaling function in the absence of the sequential polypeptide or heterodimeric protein.In some embodiments, the reduction in IL13 and / 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%.

[0154] Provided herein are methods of using IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins for the detection, diagnosis, and monitoring of anti-IL13 or IL4-induced conditions. Provided herein are methods of determining whether a companion animal will respond to IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric protein therapy. In some embodiments, the methods comprise using IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins to detect whether the animal has cells that express IL13 or IL4. 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 (e.g., a control). In some embodiments, the method may be useful for determining whether an IL13R / IL4R sequential polypeptide, IL13Rd / IL4R sequential polypeptide, IL13R / IL4R heterodimeric protein, or IL13Rd / IL4R heterodimeric protein described herein is an appropriate therapeutic agent for a subject.

[0155] In some embodiments, the sample is a biological sample. The term "biological sample" refers to a quantity of material from a living organism or formerly living organism. 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.

[0156] In some embodiments, cells or cell / tissue lysates are contacted with an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein, and binding between the sequential polypeptide or heterodimeric protein and the cells is determined. If the test cells exhibit binding activity compared to reference cells of the same tissue type, it may indicate that the subject would benefit from treatment with an IL13R / IL4R sequential polypeptide, an IL13Rd / IL4R sequential polypeptide, an IL13R / IL4R heterodimeric protein, or an IL13Rd / IL4R heterodimeric protein. In some embodiments, the test cells are from a tissue of a companion animal.

[0157] 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), turbidimetric 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 the various methods used, which are often determined by the availability of assay equipment and compatible immunoassay procedures. Suitable labels include, but are not limited to, radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or p-lactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot supplied by Quantum Dot Corporation, Palo Alto, Calif.). TM General techniques to be used in performing the various immunoassays noted above are known to those of skill in the art.

[0158] For diagnostic purposes, IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins 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, the sequential polypeptide or heterodimeric protein need not be labeled, and its presence can be detected, for example, using an antibody that binds to the sequential polypeptide or heterodimeric protein. In some embodiments, the IL13R / IL4R sequential polypeptides, IL13Rd / IL4R sequential polypeptides, IL13R / IL4R heterodimeric proteins, or IL13Rd / IL4R heterodimeric proteins can be used 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). Anti-IL13 and IL4 antibodies and polypeptides can also be used in in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody or polypeptide is coupled to a radionuclide (e.g., 111 In, 99 Tc, 14 C. 131 I, 125 I, 3 H, or any other radionuclide label, including those outlined herein, can be used to localize cells or tissues of interest using immunoscintigraphy. Consecutive polypeptides or heterodimeric proteins can also be used as staining reagents in pathology, using techniques well known in the art.

[0159] In some embodiments, the IL13R / IL4R sequential polypeptide, the IL13Rd / IL4R sequential polypeptide, the IL13R / IL4R heterodimeric protein, or the IL13Rd / IL4R heterodimeric protein is used for diagnostic purposes, and the IL13R / IL4R sequential polypeptide, the IL13Rd / IL4R sequential polypeptide, the IL13R / IL4R heterodimeric protein, or the IL13Rd / IL4R heterodimeric protein is used as a therapeutic agent. In some embodiments, the diagnostic protein and the therapeutic protein are different. In some embodiments, the diagnostic protein and the therapeutic protein are the same.

[0160] The following examples illustrate certain aspects of the disclosure and are not intended to limit the disclosure in any way. [Example]

[0161] Example 1 Expression and purification of canine IL-4 and L-13 The 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 the nucleotide sequence encoding the canine IL4 protein (SEQ ID NO: 1) with a C-terminal poly-His tag.

[0162] 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.

[0163] Example 2 Extracellular domains of IL13R and IL4R The extracellular domains of canine, feline, and equine IL4R involved in binding canine, feline, and equine IL4 and / or IL13 have been identified and delimited. Exemplary full-length extracellular domains of canine IL4R, feline IL4R, and equine IL4 have been identified as SEQ ID NO: 23 and SEQ ID NO: 163 (canine), SEQ ID NO: 25 (feline), and SEQ ID NO: 27 (equine). Exemplary extracellular domain fragments of canine IL4R, feline IL4R, and equine IL4R that are hypothesized to retain biological activity have been identified as SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, respectively.

[0164] The extracellular domains of canine, feline, and equine IL13R involved in binding canine, feline, and equine IL4 and / or IL13 have been identified and delimited. Exemplary full-length extracellular domains of canine IL13R, feline IL13R, and equine IL13R have been identified as SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively. Exemplary extracellular domain fragments of canine IL13R, feline IL13R, and equine IL13R that are hypothesized to retain biological activity have been identified as SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively.

[0165] The unpaired cysteine ​​(Cys) in canine IL13R (position 18 of SEQ ID NO: 22), feline IL13R (position 18 of SEQ ID NO: 24), and equine IL13R (position 18 of SEQ ID NO: 26) was informatively identified and determined to be buried (not exposed) based on 3D 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.

[0166] Example 3 Expression and purification of canine IL13R / IL4R contiguous polypeptides from CHO cells A nucleotide sequence encoding a canine IL13R ECD / IL4R ECD continuous polypeptide linked to an IgGB Fc polypeptide was designed along with a signal sequence. For the continuous polypeptide "IL13RECD-IL4RECD-IgGB Fc" (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 continuous polypeptide "IL4RECD-IL13RECD-IgGB Fc" (SEQ ID NO: 21), the extracellular domain of IL13R precedes the extracellular domain of IL4R.

[0167] 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 one-step protein A column chromatography.

[0168] Each of IL13RECD-IL4RECD-IgGB Fc and IL4RECD-IL13RECD-IgGB Fc can be expressed and purified in a single step using a Protein A column or other chromatography 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 removal steps can be applied. The purified protein can be mixed with additives and sterilized by filtration to prepare a pharmaceutical composition of the present invention. The pharmaceutical composition can be administered to dogs with atopic dermatitis or asthma in an amount sufficient to bind to and / or inhibit either IL13 and / or IL4.

[0169] Then, using Vector, you can TMPilot-scale transfections were performed in CHO-S cells using a transfection reagent (Life Technologies). The supernatant was collected by clarifying the conditioned medium. The protein was purified by a single-pass Protein A chromatography step and used for further studies.

[0170] Example 4 Demonstration of IL13 and IL4 binding activity This example demonstrates that both IL13RECD-IL4RECD-IgGB Fc (SEQ ID NO: 20) and IL4RECD-IL13RECD-IgGB Fc (SEQ ID NO: 21) bind to canine IL4 and IL13 with kinetics required for therapeutic activity.

[0171] 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 tip. Association of IL4 with various concentrations (12, 16, and 44 nM) of IL13RECD-IL4RECD-IgGB Fc (SEQ ID NO: 20) was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was subtracted to correct for drift. ForteBio TM Using data analysis software, the data were fitted to a 1:1 binding model, and k on , k off The Kd of IL13RECD-IL4RECD-IgGB Fc 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 of IL13RECD-IL4RECD-IgGB Fc and the ligand IL4 was 8 x 10 -11 It was.

[0172] Association of canine IL4 with various concentrations (40.7 and 140 nM) of IL4RECD-IL13RECD-IgGB Fc (SEQ ID NO: 21) was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was subtracted for drift correction. ForteBio TM Data analysis software was used to fit the data to a 1:1 binding model, k on , k off The Kd of IL4RECD-IL13RECD-IgGB Fc 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 of IL4RECD-IL13RECD-IgGB Fc and the ligand IL4 was 1.1 x 10 -11 It was.

[0173] 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).

[0174] A sequential binding experiment of IL4 and IL13 was performed using IL13RECD-IL4RECD-IgGB Fc (SEQ ID NO: 20). Biotinylated canine IL13RECD-IL4RECD-IgGB Fc was captured on a streptavidin sensor tip. Canine IL13RECD-IL4RECD-IgGB Fc was exposed to (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 (Figure 2). This experiment demonstrated that once IL13RECD-IL4RECD-IgGB Fc bound to IL13, it may not bind to IL4, and once it bound to IL4, its ability to bind to IL13 was reduced.

[0175] Sequential binding experiments of IL4 and IL13 were performed using IL4RECD-IL13RECD-IgGB Fc (SEQ ID NO: 21). Biotinylated canine IL4RECD-IL13RECD-IgGB Fc was captured on a streptavidin sensor tip. Canine IL4RECD-IL13RECD-IgGB Fc was exposed to (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 (Figure 1). These experiments demonstrated that once IL4RECD-IL13RECD-IgGB Fc bound to IL13, it may not bind to IL4, and once bound to IL4, its ability to bind IL13 was reduced.

[0176] The tight binding of IL13RECD-IL4RECD-IgGB Fc and IL4RECD-IL13RECD-IgGB Fc to IL4 or IL13 is thought to be due to the contribution of simultaneous binding of both IL4RECD and IL13RECD.

[0177] Example 5 Cellular functional activity of canine IL4RECD-IL13RECD-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 in the proliferation assay. Cells were grown in exponential growth phase in RPMI 1640 medium (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 IL4RECD-IL13RECD-IgGB Fc (SINK) was added at a dilution series, followed by canine IL4 (Sino Biological Inc, Cat. No. 70021-DNAE-5) at 50 ng / ml. Cells were incubated in a total volume of 100 μl for 48 hours at 37°C, 5% CO2. 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, Cat. No. G7570).

[0178] In this assay, 100 μl of premixed reagents A and B was added to each well. After 2 minutes of shaking on an orbital shaker, cells were lysed. Monooxygenation of luciferin was catalyzed by luciferase in the presence of intracellular Mg2+ and ATP, generating a luminescent signal proportional to the amount of ATP present in the cells. The amount of ATP is directly proportional to the number of cells present in the 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).

[0179] Data was analyzed using a four parameter logistic fit. IC50 is 2.0 nM. See Figure 3.

[0180] Example 6 Canine, feline, and equine IgG Fc polypeptides for IL13R and IL4R heterodimeric proteins Pairs of variant canine IgG Fc polypeptides, variant feline IgG Fc polypeptides, and variant equine IgG Fc polypeptides were designed to allow for the preparation of heterodimeric proteins containing at least one IL13R ECD and at least one IL4R ECD using a knob-in-hole heterodimerization approach. First, pairings of two Fc polypeptides were designed by introducing CH3 interface mutations such that the first Fc polypeptide contains a bulky amino acid (knob) and the second Fc polypeptide contains a smaller amino acid in the same general position (hole).

[0181] A threonine to tryptophan amino acid substitution (T138W or T137W) can be introduced as a knob at a position corresponding to position 138 of canine IgG-A (SEQ ID NO: 38), position 137 of canine IgG-B Fc (SEQ ID NO: 39), position 137 of canine IgG-C Fc (SEQ ID NO: 40), or position 138 of canine IgG-D Fc (SEQ ID NO: 41). Examples of amino acid sequences of first variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptides containing the knob mutation are SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively.

[0182] Hole amino acid substitutions can be introduced: threonine to serine at the position corresponding to position 138 of canine IgG-A (SEQ ID NO: 38) or IgG-D (SEQ ID NO: 41), and / or leucine to alanine at the position corresponding to position 140, and / or tyrosine to threonine at the position corresponding to position 180 (T138S, L140A, and / or Y180T); or threonine to serine at the position corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 39) or IgG-C (SEQ ID NO: 40), and / or leucine to alanine at the position corresponding to position 139, and / or tyrosine to threonine at the position corresponding to position 179 (T137S, L139A, and / or Y179T). Examples of amino acid sequences of second variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptides containing the 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.

[0183] A threonine to tryptophan amino acid substitution (T154W) at the position corresponding to position 154 of feline IgG1a Fc (SEQ ID NO: 42 or SEQ ID NO: 43), feline IgG1b Fc (SEQ ID NO: 44 or SEQ ID NO: 45), or feline IgG2 (SEQ ID NO: 46) can be introduced as a knob. Examples of amino acid sequences of first variant feline IgG1a, and IgG1b, and IgG2 Fc polypeptides containing the knob mutation are SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.

[0184] Amino acid substitutions of threonine to serine at position corresponding to position 154 and / or leucine to alanine at position corresponding to position 156 and / or tyrosine to threonine at position corresponding to position 197 (T154S, L156A, and / or Y(197)T) of feline IgG1a (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) can be introduced as holes. Examples of amino acid sequences of second variant feline IgG1a, IgG1b, IgG2 Fc polypeptides containing hole mutations 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.

[0185] A threonine to tryptophan amino acid substitution (T130W) at the position corresponding to position 130 of equine IgG1 Fc (SEQ ID NO: 47), equine IgG2 Fc (SEQ ID NO: 48), equine IgG3 Fc (SEQ ID NO: 49), equine IgG4 Fc (SEQ ID NO: 50), equine IgG5 Fc (SEQ ID NO: 51), equine IgG6 Fc (SEQ ID NO: 52), or equine IgG7 Fc (SEQ ID NO: 53) can be introduced as a knob. Examples of amino acid sequences of first variant equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides containing the 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.

[0186] Amino acid substitutions of threonine to serine at the position corresponding to position 130, leucine to alanine at the position corresponding to position 132, and / or tyrosine to threonine at the position corresponding to position 173 (T130W, L(132)A, and / or Y(173)T) can be introduced as holes in equine IgG1 Fc (SEQ ID NO: 47), equine IgG2 Fc (SEQ ID NO: 48), equine IgG3 Fc (SEQ ID NO: 49), equine IgG4 Fc (SEQ ID NO: 50), equine IgG5 Fc (SEQ ID NO: 51), equine IgG6 Fc (SEQ ID NO: 52), or equine IgG7 Fc (SEQ ID NO: 53). Exemplary amino acid sequences of second variant equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides containing the 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.

[0187] Example 7 IL13R / IL4R ECD heterodimer protein In addition to the contiguous IL13R / IL4R ECD polypeptide format, heterodimeric protein pairs can have the following formats: Heterodimeric Protein A: Polypeptide 1: IL13R(n)-L-Fc1 and Polypeptide 2: IL4R(n)-L-Fc2; or Heterodimeric Protein B: Polypeptide 1: IL4R(n)-L-Fc1 and Polypeptide 2: IL13R(n)-L-Fc2 wherein IL13R(n) is at least one IL13R extracellular domain (ECD) polypeptide from a companion animal species, IL4R(n) is at least one IL4R ECD polypeptide from a companion animal species, (n) is one, two, three, four, or more ECD polypeptides, L is an optional linker, Fc1 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a knob mutation, and Fc2 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a hole mutation. Optional linkers can also be used between multiple ECD polypeptides. In addition, other binding partners can be included before, after, and / or between any one or more ECD polypeptides. Other potential binding partners include IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-α, TSLP, and / or IgE.

[0188] Examples of pairs of consecutive polypeptides 1 and 2 that can form heterodimeric proteins 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. Host cells can be co-transfected with vectors expressing these consecutive polypeptide pairs to produce the described heterodimeric proteins.

[0189] Example 8 Screening of variant canine IgG-B polypeptides with enhanced canine FcRn / B2M binding A poly-His tagged canine FcRn (SEQ ID NO: 114) and canine B2M (SEQ ID NO: 115) heterodimeric complex was transiently expressed in HEK cells and purified using Ni-NTA chromatography.

[0190] A rapid screening (FASEBA) study was performed on the expression, biophysical properties, and affinity of a canine IgG-B Fc phage library. Briefly, the open reading frame of the canine IgG-B Fc polypeptide was subcloned into the plasmid pFASEBA. Based on three-dimensional protein modeling of the canine IgG-B / canine FcRn / canine B2M complex, 12 amino acid positions in canine IgG-B were identified as potentially involved in the binding between IgG-B and FcRn / B2M. The 12 identified positions in canine IgG-B were Thr(21), Leu(22), Leu(23), Ile(24), Ala(25), Thr(27), Gly(80), His(81), Gln(82), Leu(85), Met(201), and Asn(207) of SEQ ID NO: 39.

[0191] Twelve single-site NNK mutation libraries of canine IgG-B Fc were prepared, each containing variant IgG-B Fc polypeptides with each of the 20 possible amino acid substitutions at each of the 12 sites. Each phage library was panned against the canine FcRn / B2M complex at pH 6.0. After three rounds of panning, a total of 53 Fc phage clones were identified as potentially having enhanced FcRn / B2M binding, and the mutations were identified by sequencing.

[0192] Single E. coli colonies expressing each of the 53 variant canine IgG-B Fc polypeptides with a SASA tag were cultured and induced to express the Fc polypeptides. Cell culture medium containing the variant canine IgG-B Fc polypeptides was exposed to immobilized BSA on a plate or Biacore chip. The plate or chip with the bound variant canine IgG-B Fc polypeptides was exposed to soluble canine FcRn / B2M complexes and screened for slower off-rates (koff) at pH 6. Each variant IgG-B Fc polypeptide was identified to exhibit slower koff in the canine FcRn / B2M complex compared to the wild-type IgG-B Fc polypeptide. Four lead variant canine IgG-B polypeptides were identified: L(23)Y (SEQ ID NO: 117; "Y00"); L(23)F (SEQ ID NO: 116; "F00"); L(23)M; and L(23)S.

[0193] The koff of each of the lead variant canine IgG-B polypeptides was further investigated. Biotinylated canine FcRn / B2M complexes were immobilized on a Biacore chip and exposed to each variant canine IgG B polypeptide as an analyte using a Biacore T200 at pH 6.0. The koff (1 / s) of the wild-type canine IgG-B Fc polypeptide was 1.22 × 10 -1 and the k(1 / s) of the variant canine IgG-B Fc polypeptide L(23)Y ("Y00") is 1.38 x 10 -2 and the koff (1 / s) of the variant IgG-B Fc polypeptide L(23)F ("F00") is 6.31 x 10 -2 and 8.47 x 10 -2 and the koff(1 / s) of the variant canine IgG-B polypeptide L(23)M is 1.26 x 10 -1 and the koff(1 / s) of the variant canine IgG-B polypeptide L(23)S is 2.41 × 10 -1 It was.

[0194] Binding analysis was performed using a Biacore T200. Briefly, each lead variant canine IgG-B Fc polypeptide with a SASA tag was immobilized on a Series S SensorChip CM5. The association of each variant IgG-B Fc polypeptide with various concentrations of the canine FcRn / B2M complex (12.5, 25, 50, 100, and 200 nM) was monitored at 25°C until steady-state binding was achieved. A running buffer of 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 6.0 was used. A buffer-only blank curve was used as a control. The results are presented in Figures 10-14. The steady-state Kd of the wild-type canine IgG-B Fc polypeptide was 1.25 x 10 -6 (Figure 4); the steady-state Kd of the variant canine IgG-B Fc polypeptide L(23)Y ("Y00") is 1.13 x 10 -7 (Figure 5); the steady-state Kd of the variant canine IgG-B Fc polypeptide L(23)F ("F00") is 3.67 x 10 -7 (Figure 6); the steady-state Kd of the variant canine IgG-B Fc polypeptide L(23)M is 4.06 x 10 -7 (Figure 7); the steady-state Kd of the variant canine IgG-B Fc polypeptide YTE is 8.62 x 10 -8 (Figure 8).

[0195] Example 9 Phe mutations in canine IgG enhance canine FcRn interaction The affinity of variant canine Fc polypeptides for FcRn was evaluated in the context of chimeric antibodies. An antibody variable light chain fused to a canine kappa light chain and a variant canine IgG-D Fc polypeptide comprising SEQ ID NO: 118 (F00; Protein A+; C1q-; CD16-) or SEQ ID NO: 119 (Protein A+; C1q+; CD16+), and a variable heavy chain fused to a variant canine IgG-D Fc polypeptide comprising SEQ ID NO: 120 (F00; Protein A+; C1q-; CD16-) or SEQ ID NO: 121 (Protein A+; C1q+; CD16+) were expressed.

[0196] Binding analysis was performed using the biosensor OctetRed as follows. Briefly, biotinylated TNFα was captured on a streptavidin sensor tip. Antibody conjugates at 20 μg / mL bound to TNFα. The conjugate was then used to bind canine FcRn (50 μg / mL) at pH 6.0. Dissociation was performed at pH 7.2.

[0197] The Phe mutation enhanced canine FcRn binding at low pH (pH 6.0, 20 mM Na citrate, 140 mM NaCl), as shown by the binding profiles of the chimeric variant canine IgG-A "F00" antibody (Figure 9, A) and IgG-D "F00" antibody (Figure 9, B) compared with chimeric variant canine IgG-A without the Phe mutation (Figure 9, C) and IgG-D without the Phe mutation (Figure 9, B). The chimeric variant canine IgG-A and IgG-D antibodies with the Phe mutation (Figure 9, A and B) showed enhanced association with canine FcRn at low pH (pH 6.0) and rapid dissociation at neutral pH (PBS pH 7.2). A similar enhanced binding profile was also observed with the chimeric variant canine IgG-B "F00" antibody.

[0198] Example 10 Pharmacokinetics of Phe mutations in canine IgG Pharmacokinetic analysis was performed using Sprague-Dawley rats. The rats were subcutaneously administered 2 mg / kg of the chimeric variant canine IgG-A "F00" antibody and the chimeric variant canine IgG-A without the Phe mutation (two rats per group). Serum samples were collected from the rats before injection and at 0.5, 1, 6, 24, 48, 72, 168, 216, and 336 hours after injection. The canine chimeric antibody concentrations in the serum samples were determined by ELISA as follows.

[0199] Capture antibody (1 μg / mL in PBS) was coated onto a 96-well Maxisorp plate at 100 μl per well. The plate was incubated overnight at 4°C and washed five times with PBST (PBS containing 0.05% Tween-20). Each well was blocked with 200 μl of 5% BSA in PBST, and the plate was incubated for 1 hour at room temperature. The plate was washed five times with PBST. Dilutions of control antibody (1000 ng / mL to 0.1 ng / mL) were added to the plate in duplicate and used with blank wells containing no control antibody to generate a standard curve. Serum samples were prepared at 10-, 20-, and 40-fold dilutions in 5% BSA-PBST and added to the plate. The plate was incubated for 1 hour at room temperature and washed five times with PBST. 100 μl of HRP-conjugated antibody (Bio-Rad, catalog no. HCA204P) was added to each well at 0.25 μg / mL in 5% BSA-PBST. Plates were incubated for 1 hour at room temperature and washed five times with PBST. 100 μl of QuantaBlu (Thermo Scientific, catalog no. 15169) was added to each well. Fluorescence was measured at 325 nm / 420 nm (emission / excitation) after 10-15 minutes of incubation. Anti-TNFα titers in serum samples were calculated against a standard curve.

[0200] AUC of IgG-A 0-336h is 150970, and IgG-A "F00" is 848924ng / mL * The terminal half-lives were estimated to be 33 and 152 hours, respectively (Figure 10). Thus, the single Phe mutation significantly improved the pharmacokinetic profile of the antibody in rats.

[0201] Example 11 Phe mutations in dog, cat, and horse IgG Fc The interaction between Phe mutations and FcRn in canine IgG-A, IgG-B, IgG-C, and IgG-D Fc was modeled using three-dimensional protein structure analysis. The aromatic side chain of Phe appears to make hydrophobic interactions with canine FcRn at the hydrophobic ring (π-CH) of the Pro in the "WPE" motif. In addition, the hydrophobic side chain of Phe may be in direct contact with the Glu side chain adjacent to the Pro in the same "WPE" motif. This interaction may result in energy loss if the Glu side chain is deprotonated and negatively charged, such as at neutral pH. Therefore, some protonation of the Glu residue may be necessary to minimize aromaticity to the Glu-H interaction. This may explain why the interaction between variant IgGs with Phe mutations and FcRn is reduced at neutral pH. Based on the protein structure analysis, the interaction appears to be conserved among canine IgG-A, IgG-B, IgG-C, and IgG-D Fc.

[0202] Furthermore, the interaction between the Phe mutations in feline IgG1a and IgG2 Fc was modeled when complexed with feline FcRn, and the same interactions observed with canine IgG Fc appeared to be conserved in feline IgG Fc.

[0203] The interactions between Phe mutations in equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc in complex with equine FcRn were also modeled, and the same interactions appeared to be maintained in equine IgG Fc.

[0204] Example 12 Other exemplary variant canine IgG Fc enhances canine FcRn interaction The affinity of additional variant canine Fc polypeptides for FcRn was evaluated in the context of chimeric antibodies. Antibody variable light chains fused to canine kappa light chains and variable heavy chain sequences fused to wild-type IgG-B Fc polypeptide (comprising SEQ ID NO: 39), variant canine IgG-B Fc polypeptide 0Y0 (comprising SEQ ID NO: 122), variant canine IgG-B Fc polypeptide 0YH (comprising SEQ ID NO: 123), variant canine IgG-B Fc polypeptide 0YY (comprising SEQ ID NO: 124), and variant canine IgG-B Fc polypeptide 00Y (SEQ ID NO: 125) were expressed.

[0205] Binding analysis was performed using the biosensor OctetRed as follows. Briefly, biotinylated targets were captured on a streptavidin sensor tip. Antibody conjugates at 20 μg / mL bound to the biotinylated targets. The conjugates were then used to bind canine FcRn (50 μg / mL) at pH 6.0. Dissociation was performed at pH 7.2.

[0206] Each of the chimeric variant canine IgG-B antibodies exhibited enhanced binding to canine FcRn at pH 6.0 compared to the chimeric wild-type canine IgG-B antibody, and each had a significant dissociation rate at neutral pH (Figure 11).

[0207] Example 13 Variant canine IgG Fc extends antibody half-life in vivo in dogs The in vivo half-life of variant canine Fc polypeptides relative to FcRn was evaluated in the context of chimeric antibodies. Antibody variable light chains fused to canine kappa light chains and variable heavy chains fused to wild-type IgG-B Fc polypeptide YTE (comprising SEQ ID NO: 39), variant canine IgG-B Fc polypeptide YTE (comprising SEQ ID NO: 126), variant canine IgG-B Fc polypeptide 0Y0 (comprising SEQ ID NO: 122), variant canine IgG-B Fc polypeptide F00 (comprising SEQ ID NO: 116), variant canine IgG-B Fc polypeptide 0YH (comprising SEQ ID NO: 123), and variant canine IgG-B Fc polypeptide Y00 (comprising SEQ ID NO: 117) were expressed and purified to 40 mg / mL in PBS, pH 7.2.

[0208] Canine pharmacokinetics was performed at Absorption Systems California, LLC. Male beagle dogs (approximately 8-14 kg) were obtained from Marshall Bioresources, North Roads, NY. A total of 12 dogs were used in the study, with n = 2 dogs per group. Six antibodies were administered subcutaneously to dogs at 4 mg / kg. Serum samples were collected pre-injection and at 6, 24, 48, 72, 96, 120, 144, 168, 216, 264, 336, 504, and 672 hours post-injection. Canine chimeric antibody concentrations were determined by ELISA as described. Cp between 144 and 336 hours was converted to Ln[Cp], and Ln[Cp] t =-k * t+Ln[Cp] 144h The curves were fitted to a linear equation of the form: Terminal half-life was then calculated from the slope k, as listed in Table 3 below. The 0Y0, F00, 0YH, and Y00 mutations in the canine IgG-B Fc significantly improved in vivo antibody half-life in dogs. The normalized antibody percentages over time from the study are shown in Figure 12.

[0209] JPEG0007821741000043.jpg86170

[0210] Example 14 Extracellular domain of IL13 receptor decoy The extracellular domains of mammalian IL13 receptor decoys (IL13Rd), such as canine, feline, and equine IL13Rd, can bind to IL13. The IL13-binding domains of canine, feline, and equine IL13Rd have been identified and delimited. The full-length precursor sequences of canine, feline, and equine IL13Rd correspond to SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166, respectively. Exemplary extracellular domains of canine, feline, and equine IL13Rd have been identified as SEQ ID NO: 167, SEQ ID NO: 168, and SEQ ID NO: 169, respectively.

[0211] Nucleotide sequences encoding canine IL13Rd ECD / IL4R ECD contiguous polypeptides linked to an IgGB Fc polypeptide with a signal sequence were designed, and the contiguous polypeptides were expressed and purified. Binding assays to canine IL13 were performed. All three contiguous polypeptides bound to IL13 with kinetics required for therapeutic activity. In all cases, the Kd was in the nM range.

[0212] Example 15 IL13Rd ECD / IL4R ECD continuous polypeptide and heterodimeric protein The contiguous IL13Rd ECD / IL4R ECD polypeptide can have the following format: Formula (I): IL13Rd-L1-IL4R-L2-FP, Formula (II): IL4R-L1-IL13Rd-L2-FP, Formula (III): IL13Rd-L1-FP-L2-IL4R, Formula (IV): IL4R-L1-FP-L2-IL13Rd, Formula (V): FP-L1-IL13Rd-L2-IL4R, or Formula (VI): FP-L1-IL4R-L2-IL13Rd wherein IL13Rd is an IL13Rd extracellular domain (ECD) polypeptide from a companion animal species, IL4R is an IL4R ECD polypeptide from a companion animal species, L1 is a first optional linker, L2 is a second optional linker, and FP is an optional fusion partner such as an Fc polypeptide.

[0213] In addition to the contiguous IL13Rd / IL4R ECD polypeptide format, heterodimeric protein pairs can have the following formats: Heterodimeric Protein A: Polypeptide 1: IL13Rd(n)-L-Fc1 and Polypeptide 2: IL4R(n)-L-Fc2; or Heterodimeric Protein B: Polypeptide 1: IL4R(n)-L-Fc1 and Polypeptide 2: IL13Rd(n)-L-Fc2 wherein IL13Rd(n) is at least one IL13Rd extracellular domain (ECD) polypeptide from a companion animal species, IL4R(n) is at least one IL4R ECD polypeptide from a companion animal species, (n) is one, two, three, four, or more ECD polypeptides, L is an optional linker, Fc1 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a knob mutation, and Fc2 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a hole mutation. Optional linkers can also be used between multiple ECD polypeptides. In addition, other binding partners can be included before, after, and / or between any one or more ECD polypeptides. Other potential binding partners include IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-α, TSLP, and / or IgE. Host cells can be co-transfected with vectors expressing these consecutive polypeptide pairs to produce the described heterodimeric proteins.

Claims

1. a polypeptide comprising an extracellular domain of an IL13R polypeptide and an extracellular domain of an IL4R polypeptide, wherein the IL13R and / or IL4R polypeptide is derived from a companion animal species; the polypeptide comprises formula (I) IL13R-L1-IL4R-L2-FP, formula (II) IL4R-L1-IL13R-L2-FP, formula (III) IL13R-L1-FP-L2-IL4R, formula (IV) IL4R-L1-FP-L2-IL13R, (V) FP-L1-IL13R-L2-IL4R, or formula (VI) FP-L1-IL4R-L2-IL13R; a) the IL13R is the extracellular domain of an IL13R polypeptide from a companion animal species; b) the 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 i) SEQ ID NO: 117, or ii) SEQ ID NO: 122 A polypeptide which is a fusion partner comprising an IgG Fc polypeptide comprising:

2. The polypeptide according to claim 1, wherein the extracellular domain of the IL13R polypeptide is the extracellular domain of an IL13R decoy polypeptide.

3. 3. The polypeptide of claim 1 or 2, wherein the IgG Fc polypeptide is capable of binding to neonatal Fc receptor (FcRn) with increased affinity compared to the wild-type Fc polypeptide, such as at low pH.

4. Polypeptides are measured by biolayer interferometry, 5 x 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 binds to IL13 of a companion animal species with a dissociation constant (Kd) of less than M; and / or Polypeptides are measured by biolayer interferometry, 5 x 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 binds to IL4 of companion animal species with a dissociation constant (Kd) less than M; A polypeptide described in any one of claims 1 to 3.

5. 5. The polypeptide of claim 1, wherein the polypeptide reduces IL13 and / or IL4 signaling in a companion animal species; the companion animal species may be a dog, a cat, or a horse.

6. 6. The polypeptide of claim 3, wherein the extracellular domain of the IL13R polypeptide is at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% 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.

7. 7. The polypeptide of claim 3, 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.

8. the extracellular domain of the IL13R polypeptide comprising an amino acid sequence selected from SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36; and / or comprising an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26; A polypeptide according to any one of claims 3 to 7.

9. the extracellular domain of the IL13R polypeptide is at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169; and / or the extracellular domain of the IL4R polypeptide is at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, and the extracellular domain of the IL4R polypeptide may comprise an amino acid sequence selected from SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:23, SEQ ID NO:163, SEQ ID NO:25, and SEQ ID NO:27; A polypeptide described in any one of claims 1 to 8.

10. 10. The polypeptide of claim 1, wherein 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: 151), GSGSGS (SEQ ID NO: 152), GGS, GGSGGS (SEQ ID NO: 153), GGSGGSGGS (SEQ ID NO: 154), GGGS (SEQ ID NO: 155), GGGSGGGS (SEQ ID NO: 156), GGGSGGGSGGGS (SEQ ID NO: 157), GSS, GSSGSS (SEQ ID NO: 158), GSSGSSGSS (SEQ ID NO: 159), GGSS (SEQ ID NO: 160), GGSSGGSS (SEQ ID NO: 161), and GGSSGGSSGGSS (SEQ ID NO: 162).

11. 11. The polypeptide of any one of claims 1 to 10, wherein the variant IgG Fc polypeptide binds to FcRn with greater affinity than the wild-type IgG Fc polypeptide as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool at a pH ranging from about 5.0 to about 6.5, such as about pH 5.0, about pH 5.2, about pH 5.5, about pH 6.0, about pH 6.2, or about pH 6.

5.

12. The variant IgG Fc polypeptide has a 5×10 affinity to at least 500 ng / mL of IgG Fc polypeptide, as measured by biolayer interferometry, surface plasmon resonance, or any protein-protein interaction tool at a pH in the range of about 5.0 to about 6.5, such as a pH of about 5.0, a pH of about 5.5, a pH of about 6.0, or a pH of about 6.

5. －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 12. The polypeptide of claim 1, which binds to FcRn with a dissociation constant (Kd) of less than M.

13. 13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, or SEQ ID NO:

183.

14. 14. The polypeptide of any one of claims 1 to 13, wherein the polypeptide is sialylated.

15. 15. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 14.

16. A host cell comprising the nucleic acid of claim 15.

17. 17. A method for producing a polypeptide, comprising culturing the host cell of claim 16 and isolating the polypeptide.

18. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

19. A method for treating a companion animal species having an IL13 and / or IL4 induced condition, comprising administering to the companion animal species a therapeutically effective amount of a polypeptide described in any one of claims 1 to 14 or a pharmaceutical composition described in claim 18, wherein the companion animal species may be a dog, cat, or horse.

20. 20. The method of claim 19, wherein the IL13 and / or IL4 induced condition is a pruritic or allergic condition, such as atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema.

21. 21. The method of claim 19 or 20, wherein the polypeptide or pharmaceutical composition is administered parenterally.

22. 22. The method of any one of claims 19 to 21, wherein the polypeptide or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal, or inhalation route.

23. 23. The method of any one of claims 19 to 22, further comprising administering a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor, and / or 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-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, and an anti-BlyS antibody.

24. A method for reducing IL13 and / or IL4 signaling activity in cells of a companion animal species, the method comprising exposing the cells to a polypeptide of any one of claims 1 to 14 or a pharmaceutical composition of claim 18 under conditions that permit binding of the polypeptide to IL13 and / or IL4, thereby reducing binding of IL4 and / or IL13 to the natural IL13 receptor and / or the natural IL4 receptor and reducing IL13 and / or IL4 mediated signaling, the cells may be canine, feline or equine cells.

25. 25. The method of claim 24, wherein the cells are exposed to the polypeptide or pharmaceutical composition ex vivo.

26. 25. The method of claim 24, wherein the cell is exposed to the polypeptide or pharmaceutical composition in vivo.

27. 19. A method for detecting IL13 or IL4 in a sample from a companion animal species, the method comprising contacting the sample with a polypeptide described in any one of claims 1 to 14 or a pharmaceutical composition described in claim 18 under conditions that allow 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 biological sample may be obtained from a dog, cat, or horse.

28. 14. The polypeptide of claim 13, wherein the amino acid sequence is selected from SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, and SEQ ID NO:183, and optionally contains 1, 2, 3, 4, or 5 amino acid substitutions.

29. 29. The polypeptide of claim 28, wherein the amino acid substitutions are conservative substitutions.

30. 2. The polypeptide of claim 1, wherein the IgG Fc polypeptide is capable of binding to neonatal Fc receptor (FcRn) with increased affinity compared to a wild-type Fc polypeptide.

31. A pharmaceutical for treating a companion animal species having an IL13 and / or IL4 induced state, comprising a polypeptide described in any one of claims 1 to 14 or a pharmaceutical composition described in claim 18, wherein the companion animal species may be a dog, cat, or horse.

32. A drug for reducing IL13 and / or IL4 signaling activity in a cell, comprising a polypeptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 18, wherein the cell is exposed to the polypeptide or pharmaceutical composition under conditions that allow binding of the polypeptide to IL13 and / or IL4, thereby reducing binding of IL4 and / or IL13 to the natural IL13 receptor and / or the natural IL4 receptor and reducing IL13 and / or IL4 mediated signaling, and the cell may be a canine cell, feline cell, or equine cell.

33. An agent for detecting IL13 or IL4 in a sample from a companion animal species, the agent comprising a polypeptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 18, wherein the sample is contacted with the polypeptide or pharmaceutical composition under conditions which allow binding of the polypeptide to IL13 and / or IL4, and whether a complex is formed between the polypeptide and IL13 and / or IL4 in the sample is detected, and the biological sample may be obtained from a dog, cat, or horse.

Images