IgG Fc variant for animals

Abstract

Various embodiments are provided for variant IgG Fc polypeptides for companion animals that exhibit increased Protein A binding for ease of purification, decreased C1q binding for reduced complement-mediated immune responses, decreased CD16 binding (e.g., decreased antibody-dependent cellular cytotoxicity (ADCC) induction), improved stability, and / or the ability to form multimeric proteins. Additionally, various embodiments are provided for antibodies and fusion proteins comprising such variant IgG Fc polypeptides. In various embodiments, such polypeptides may be used to treat companion animals, such as dogs, cats, and horses.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 785,680, filed Dec. 27, 2018, which is hereby incorporated by reference in its entirety for all purposes.

[0002] [Field] The present disclosure relates to variant IgG Fc polypeptides of companion animals having enhanced characteristics including increased Protein A binding (e.g., for ease of purification), decreased C1q binding (e.g., for reduced complement - mediated immune response), decreased CD16 binding (e.g., for reduced antibody - dependent cell cytotoxicity (ADCC) induction), improved stability, and / or the ability to form heterodimeric proteins. The variant IgG Fc polypeptides of the present disclosure may have broad utility in therapeutic agents for companion animals. For example, the variant IgG Fc polypeptides may be used in the design and manufacture of long - acting GLP1 polypeptides for treating, e.g., diabetes, obesity, or related indications in companion animals such as dogs, cats, and horses. In addition, the variant IgG Fc polypeptides may be used in the design and manufacture of antibodies or fusion proteins for treating various disorders in companion animals.

Background Art

[0003] IgG Fc plays an important role in Fc - mediated functions through its interactions with FcRn, Fc receptors, and C1q. In companion animals, various IgG subtypes differ in these functions, which is often considered when selecting a particular IgG antibody or IgG Fc fusion protein for therapeutic or diagnostic use. For example, the ability of an IgG subtype having a weak or non - measurable binding affinity for C1q or CD16 may be advantageous. In addition, the ability of IgG Fc to bind to Protein A can be useful for purification using Protein A affinity purification platforms.

[0004] However, most IgG Fc subtypes of dogs, cats, and horses do not have protein A binding properties, weak or non-measurable binding affinity to CD16, and weak or non-measurable binding affinity to C1q. For example, among the four canine IgG Fc subtypes (IgG-A, IgG-B, IgG-C, and IgG-D), only canine IgG-B Fc has an apparent affinity for protein A. On the other hand, only canine IgG-A Fc and IgG-D Fc have no or weak C1q binding or CD16 binding. Antibodies and Fc fusion proteins containing variant IgG Fc polypeptides with reduced binding to C1q and / or CD16 and / or capable of binding to protein A are desirable.

Summary of the Invention

[0005] Embodiment 1. A polypeptide comprising at least one therapeutic polypeptide and / or at least one antibody and a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide a) comprises at least one amino acid modification relative to the wild-type IgG Fc polypeptide of a companion animal species, and the variant IgG Fc polypeptide has an increased binding affinity for protein A relative to the wild-type IgG Fc polypeptide; b) comprises at least one amino acid modification relative to the wild-type IgG Fc polypeptide of a companion animal species, and the variant IgG Fc polypeptide has a decreased binding affinity for C1q relative to the wild-type IgG Fc polypeptide; c) comprises at least one amino acid modification relative to the wild-type IgG Fc polypeptide of a companion animal species, and the variant IgG Fc polypeptide has a decreased binding affinity for CD16 relative to the wild-type IgG Fc polypeptide; d) It comprises a hinge region containing at least one amino acid modification relative to a wild-type feline or equine IgG Fc polypeptide, and the variant IgG Fc polypeptide is determined by SDS-PAGE analysis under reducing and / or non-reducing conditions to have increased recombinant production relative to the wild-type IgG Fc polypeptide and / or increased hinge disulfide formation; e) It comprises at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, and the at least one amino acid substitution is cysteine, and the variant IgG Fc polypeptide is capable of forming at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide; f) It comprises at least one amino acid substitution relative to a wild-type canine IgG-A or IgG-D Fc polypeptide, and the variant IgG Fc polypeptide has increased binding affinity for C1q and / or CD16 relative to the wild-type canine IgG-A or IgG-D Fc polypeptide; and / or g) It comprises a CH1 region containing at least one amino acid modification relative to a wild-type canine or feline IgG CH1 region, and the variant IgG Fc polypeptide i) has at least one amino acid substitution at a position corresponding to position 24 and / or 30 of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, or SEQ ID NO: 145, or ii) has at least one amino acid substitution at a position corresponding to position 24 and / or 29 of SEQ ID NO: 152 or SEQ ID NO: 153 and comprises a polypeptide. Embodiment 2. i) a first therapeutic polypeptide and / or antibody (TPA1); ii) a first linker (L1); iii) a variant Fc polypeptide (Fc) of a companion animal species; iv) optionally, a second linker (L2); and v) optionally, a second therapeutic polypeptide and / or antibody (TPA2) which is an adjacent polypeptide comprising, and the variant IgG Fc polypeptide a) It contains at least one amino acid modification relative to the wild-type IgG Fc polypeptide of a companion animal species, and the variant IgG Fc polypeptide has an increased binding affinity for Protein A relative to the wild-type IgG Fc polypeptide; b) It contains at least one amino acid modification relative to the wild-type IgG Fc polypeptide of a companion animal species, and the variant IgG Fc polypeptide has a decreased binding affinity for C1q relative to the wild-type IgG Fc polypeptide; c) It contains at least one amino acid modification relative to the wild-type IgG Fc polypeptide of a companion animal species, and the variant IgG Fc polypeptide has a decreased binding affinity for CD16 relative to the wild-type IgG Fc polypeptide; d) It contains a hinge region that contains at least one amino acid modification relative to the wild-type feline or equine IgG Fc polypeptide, and the variant IgG Fc polypeptide has an increased recombinant production and / or an increased hinge disulfide formation relative to the wild-type IgG Fc polypeptide as determined by SDS-PAGE analysis under reducing and / or non-reducing conditions; e) It contains at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, and the at least one amino acid substitution is cysteine, and the variant IgG Fc polypeptide is capable of forming at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide; f) It contains at least one amino acid substitution relative to the wild-type canine IgG-A or IgG-D Fc polypeptide, and the variant IgG Fc polypeptide has an increased binding affinity for C1q and / or CD16 relative to the wild-type canine IgG-A or IgG-D Fc polypeptide; and / or g) It contains a CH1 region that contains at least one amino acid modification relative to the wild-type canine or feline IgG CH1 region, and the variant IgG Fc polypeptide i) has at least one amino acid substitution at a position corresponding to position 24 and / or 30 of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, or SEQ ID NO: 145, or ii) having at least one amino acid substitution at a position corresponding to position 24 and / or 29 of SEQ ID NO: 152 or SEQ ID NO: 153 An adjacent polypeptide comprising the same. Embodiment 3. Formula (I): TPA1-L1-Fc Formula (II): Fc-L1-TPA1; Formula (III): TPA1-L1-Fc-L2-TPA2; Formula (IV): TPA1-L1-TPA2-L2-Fc; or Formula (V): Fc-L1-TPA1-L2-TPA2 An adjacent polypeptide according to Embodiment 2, comprising the same. Embodiment 4. i) a first therapeutic polypeptide and / or antibody (TPA1) and a first variant IgG Fc polypeptide comprising at least one amino acid modification relative to a first wild-type IgG Fc polypeptide, ii) a second therapeutic polypeptide and / or antibody (TPA2) and a second variant IgG Fc polypeptide comprising at least one amino acid modification relative to a second wild-type IgG Fc polypeptide A multimeric protein comprising the same, a) the first variant IgG Fc polypeptide has i) an amino acid substitution at a position corresponding to position 138 of SEQ ID NO: 1, position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 4, or position 138 of SEQ ID NO: 6; ii) an amino acid substitution at a position corresponding to position 154 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; or iii) an amino acid substitution at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 comprising, b) the second variant IgG Fc polypeptide has i) An amino acid substitution at a position corresponding to position 138, 140, and / or 181 of SEQ ID NO: 1, position 137, 139, and / or 180 of SEQ ID NO: 2, position 137, 139, and / or 180 of SEQ ID NO: 3, or position 138, 140, and / or 181 of SEQ ID NO: 4; ii) An amino acid substitution at a position corresponding to position 154, 156, and / or 197 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; or iii) An amino acid substitution at a position corresponding to position 131, 133, and / or 174 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 A multimeric protein comprising the same. Embodiment 5. The first variant IgG Fc polypeptide and / or the second variant IgG Fc polypeptide is a) Comprising at least one amino acid modification relative to the wild-type IgG Fc polypeptide of the companion animal species, and the first and / or second variant IgG Fc polypeptides have an increased binding affinity to Protein A relative to the wild-type IgG Fc polypeptide; b) Comprising at least one amino acid modification relative to the wild-type IgG Fc polypeptide of the companion animal species, and the first and / or second variant IgG Fc polypeptides have a decreased binding affinity to C1q relative to the wild-type IgG Fc polypeptide; c) Comprising at least one amino acid modification relative to the wild-type IgG Fc polypeptide of the companion animal species, and the first and / or second variant IgG Fc polypeptides have a decreased binding affinity to CD16 relative to the wild-type IgG Fc polypeptide; d) Comprising a hinge region comprising at least one amino acid modification relative to the wild-type feline or equine IgG Fc polypeptide, and the first and / or second variant IgG Fc polypeptides have an increased recombinant production and / or an increased hinge disulfide formation relative to the wild-type IgG Fc polypeptide as determined by SDS-PAGE analysis under reducing and / or non-reducing conditions; e) comprising at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, wherein the at least one amino acid substitution is cysteine, and the first and / or second variant IgG Fc polypeptide can form at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide; f) comprising at least one amino acid substitution relative to the wild-type canine IgG-A or IgG-D Fc polypeptide, wherein the variant IgG Fc polypeptide has an increased binding affinity for C1q and / or CD16 relative to the wild-type canine IgG-A or IgG-D Fc polypeptide; and / or g) comprising a CH1 region comprising at least one amino acid modification relative to the wild-type canine or feline IgG CH1 region, wherein the variant IgG Fc polypeptide i) has at least one amino acid substitution at a position corresponding to position 24 and / or 30 of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, or SEQ ID NO: 145, or ii) has at least one amino acid substitution at a position corresponding to position 24 and / or 29 of SEQ ID NO: 152 or SEQ ID NO: 153 The multimeric protein of embodiment 4, comprising. Embodiment 6. The multimeric protein of embodiment 4 or embodiment 5, wherein the first wild-type IgG Fc polypeptide and the second wild-type IgG Fc polypeptide are derived from the same IgG subtype. Embodiment 7. The multimeric protein of embodiment 4 or embodiment 5, wherein the first wild-type IgG Fc polypeptide and the second wild-type IgG Fc polypeptide are derived from different IgG subtypes. Embodiment 8. The polypeptide, adjacent polypeptide, or multimeric protein of any one of embodiments 2 to 7, wherein TPA2, if present, comprises a different amino acid sequence compared to TPA1. Embodiment 9. The polypeptide, adjacent polypeptide, or multimeric protein of any one of embodiments 2 to 8, wherein TPA1 and TPA2 are different therapeutic polypeptides or antibodies that bind to different targets. Embodiment 10. The variant IgG Fc polypeptide binds to C1q and / or CD16 with a dissociation constant (K -6 ) greater than 5×10 -5 M, greater than 1×10 -5 M, greater than 5×10 -4 M, greater than 1×10 -4 M, greater than 5×10 -3 M, or greater than 1×10 d M, as measured by biolayer interferometry, of any one of the polypeptides, adjacent polypeptides, or multimeric proteins of the previous embodiments. Embodiment 11. The variant IgG Fc polypeptide binds to Protein A with a dissociation constant (K -6 ) less than 5×10 -6 M, less than 1×10 -7 M, less than 5×10 -7 M, less than 1×10 -8 M, less than 5×10 -8 M, less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, less than 5×10 -11 M, less than 1×10 -12 M, less than 5×10 -12 M, or less than 1×10 d M, as measured by biolayer interferometry, of any one of the polypeptides, adjacent polypeptides, or multimeric proteins of the previous embodiments. Embodiment 12. The variant canine IgG-A or variant canine IgG-D Fc polypeptide binds to Protein A with a dissociation constant (K -6 ) less than 5×10 -6 M, less than 1×10 -7 M, less than 5×10 -7 M, less than 1×10 -8 M, less than 5×10 -8 M, less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, less than 1×10-11 less than M, 5×10 -12 less than M, or 1×10 -12 dissociation constant (K d ) that binds to C1q and / or CD16, any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments. Embodiment 13. Any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments, wherein the companion animal species is a dog, a cat, or a horse. Embodiment 14. The wild-type IgG Fc polypeptide is a) canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc; b) equine IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc, or IgG7 Fc; or c) feline IgG1a Fc, IgG1b Fc, or IgG2 Fc Any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments. Embodiment 15. The variant IgG Fc polypeptide comprises a CH1 region that contains at least one amino acid modification relative to the wild-type canine or feline IgG CH1 region, and the variant IgG Fc polypeptide a) at least one amino acid substitution at position 24 and / or position 30 of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, or SEQ ID NO: 145; or b) at least one amino acid substitution at position 24 and / or position 29 of SEQ ID NO: 152 or SEQ ID NO: 153 Any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments. Embodiment 16. The variant IgG Fc polypeptide comprises a CH1 region that contains at least one amino acid modification relative to the wild-type canine or feline IgG CH1 region, and the variant IgG Fc polypeptide a) leucine at the position corresponding to position 24 of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, or SEQ ID NO: 145, and / or asparagine at the position corresponding to position 30; or b) Leucine at the position corresponding to the 24th position of SEQ ID NO: 152 or SEQ ID NO: 153, and / or asparagine at the position corresponding to the 29th position A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 17. A variant IgG Fc polypeptide comprising a CH1 region comprising at least one amino acid modification relative to the wild-type canine or feline IgG CH1 region, wherein the variant IgG Fc polypeptide a) Leucine at the 24th position and / or asparagine at the 30th position of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, or SEQ ID NO: 145; or b) Leucine at the 24th position and / or asparagine at the 29th position of SEQ ID NO: 152 or SEQ ID NO: 153 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 18. A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising a wild-type or variant canine or feline light chain constant region. Embodiment 19. A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising a wild-type or variant canine or feline kappa light chain constant region. Embodiment 20. a) At least one amino acid substitution at the position corresponding to the 11th and / or 22nd position of SEQ ID NO: 150; or b) At least one amino acid substitution at the position corresponding to the 11th and / or 22nd position of SEQ ID NO: 156 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising a variant light chain constant region comprising at least one amino acid modification relative to the wild-type canine or feline kappa light chain constant region. Embodiment 21. a) At least one amino acid substitution at the position corresponding to the 11th and / or 22nd position of SEQ ID NO: 150; or b) At least one amino acid substitution at the position corresponding to the 11th and / or 22nd position of SEQ ID NO: 156 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising a variant light chain constant region comprising at least one amino acid modification relative to the wild-type canine or feline kappa light chain constant region. Embodiment 22. a) Alanine at a position corresponding to position 11 of SEQ ID NO: 150, and / or arginine at a position corresponding to position 22; or b) Alanine at a position corresponding to position 11 of SEQ ID NO: 156, and / or arginine at a position corresponding to position 22 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising a variant light chain constant region comprising at least one amino acid modification relative to the wild-type canine or feline kappa light chain constant region. Embodiment 23. a) Alanine at position 11 of SEQ ID NO: 150, and / or arginine at position 22; or b) Alanine at position 11 of SEQ ID NO: 156, and / or arginine at position 22 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising a variant light chain constant region comprising at least one amino acid modification relative to the wild-type canine or feline kappa light chain constant region. Embodiment 24. A variant IgG Fc polypeptide, a) comprising at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; b) comprising at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 3 of SEQ ID NO: 51; and / or c) comprising at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 20 of SEQ ID NO: 51, of any one of the previous embodiments of a polypeptide, adjacent polypeptide, or multimeric protein. Embodiment 25. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a) at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; b) at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 3 of SEQ ID NO: 51; and / or c) at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 20 of SEQ ID NO: 51, a polypeptide. Embodiment 26. The variant IgG Fc polypeptide comprises a) at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; b) at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 3 of SEQ ID NO: 51; and / or c) at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 20 of SEQ ID NO: 51, any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments. Embodiment 27. The variant IgG Fc polypeptide a) contains at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide contains a proline at the position corresponding to position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69 or at position 16; b) contains at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide contains a serine at the position corresponding to position 3 of SEQ ID NO: 51 or at position 3, and / or c) a polypeptide, contiguous polypeptide, or multimeric protein that contains at least one amino acid substitution relative to the wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide contains a proline at the position corresponding to position 20 of SEQ ID NO: 51 or at position 20. Embodiment 28. A polypeptide, contiguous polypeptide, or multimeric protein according to any one of the preceding embodiments, wherein the variant IgG Fc polypeptide contains a hinge region or a portion of a hinge region from an IgG Fc polypeptide of a different isotype. Embodiment 29. A polypeptide, contiguous polypeptide, or multimeric protein according to any one of the preceding embodiments, wherein the variant IgG Fc polypeptide contains a hinge region or a portion of a hinge region from a wild-type feline IgG-1 Fc polypeptide or from a wild-type equine IgG1 Fc polypeptide. Embodiment 30. A polypeptide, contiguous polypeptide, or multimeric protein according to any one of the preceding embodiments, wherein the variant IgG Fc polypeptide contains a cysteine at a position corresponding to position 8, 9, 10, 11, 12, 13, 14, 15, or 16 of SEQ ID NO: 69. Embodiment 31. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide contains a cysteine at a position corresponding to position 8, 9, 10, 11, 12, 13, 14, 15, or 16 of SEQ ID NO: 69. Embodiment 32. A variant IgG Fc polypeptide, polypeptide adjacent thereto, or multimeric protein according to any one of the preceding embodiments, wherein the variant IgG Fc polypeptide comprises a cysteine at a position corresponding to position 14 of SEQ ID NO: 69. Embodiment 33. A polypeptide, polypeptide adjacent thereto, or multimeric protein according to any one of the preceding embodiments, wherein the variant IgG Fc polypeptide comprises a cysteine at position 14 of SEQ ID NO: 69. Embodiment 34. The variant IgG Fc polypeptide a) has an amino acid substitution at a position corresponding to position 21 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 23 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 25 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 80 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 205 of SEQ ID NO: 1, and / or an amino acid substitution at a position corresponding to position 207 of SEQ ID NO: 1; b) has an amino acid substitution at a position corresponding to position 21 of SEQ ID NO: 4, an amino acid substitution at a position corresponding to position 23 of SEQ ID NO: 4, and / or an amino acid substitution at a position corresponding to position 24 of SEQ ID NO: 4; c) has an amino acid substitution at a position corresponding to position 21 of SEQ ID NO: 6, an amino acid substitution at a position corresponding to position 23 of SEQ ID NO: 6, an amino acid substitution at a position corresponding to position 25 of SEQ ID NO: 6, an amino acid substitution at a position corresponding to position 80 of SEQ ID NO: 6, and / or an amino acid substitution at a position corresponding to position 207 of SEQ ID NO: 6; d) has an amino acid substitution at a position corresponding to position 15 of SEQ ID NO: 50 and / or an amino acid substitution at a position corresponding to position 203 of SEQ ID NO: 50; e) has an amino acid substitution at a position corresponding to position 199 of SEQ ID NO: 54 and / or an amino acid substitution at a position corresponding to position 200 of SEQ ID NO: 54; and / or f) has an amino acid substitution at a position corresponding to position 199 of SEQ ID NO: 55, an amino acid substitution at a position corresponding to position 200 of SEQ ID NO: 55, an amino acid substitution at a position corresponding to position 201 of SEQ ID NO: 55, and / or an amino acid substitution at a position corresponding to position 202 of SEQ ID NO: 55 and is a polypeptide, polypeptide adjacent thereto, or multimeric protein according to any one of the preceding embodiments. Embodiment 35. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide has a) an amino acid substitution at a position corresponding to position 21 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 23 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 25 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 80 of SEQ ID NO: 1, an amino acid substitution at a position corresponding to position 205 of SEQ ID NO: 1, and / or an amino acid substitution at a position corresponding to position 207 of SEQ ID NO: 1; b) an amino acid substitution at a position corresponding to position 21 of SEQ ID NO: 4, an amino acid substitution at a position corresponding to position 23 of SEQ ID NO: 4, and / or an amino acid substitution at a position corresponding to position 24 of SEQ ID NO: 4; c) an amino acid substitution at a position corresponding to position 21 of SEQ ID NO: 6, an amino acid substitution at a position corresponding to position 23 of SEQ ID NO: 6, an amino acid substitution at a position corresponding to position 25 of SEQ ID NO: 6, an amino acid substitution at a position corresponding to position 80 of SEQ ID NO: 6, and / or an amino acid substitution at a position corresponding to position 207 of SEQ ID NO: 6; d) an amino acid substitution at a position corresponding to position 15 of SEQ ID NO: 50, and / or an amino acid substitution at a position corresponding to position 203 of SEQ ID NO: 50; e) an amino acid substitution at a position corresponding to position 199 of SEQ ID NO: 54, and / or an amino acid substitution at a position corresponding to position 200 of SEQ ID NO: 54; and / or f) an amino acid substitution at a position corresponding to position 199 of SEQ ID NO: 55, an amino acid substitution at a position corresponding to position 200 of SEQ ID NO: 55, an amino acid substitution at a position corresponding to position 201 of SEQ ID NO: 55, and / or an amino acid substitution at a position corresponding to position 202 of SEQ ID NO: 55 comprising the polypeptide. Embodiment 36. The variant IgG Fc polypeptide has a) an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acid substitution at position 23 of SEQ ID NO: 1, an amino acid substitution at position 25 of SEQ ID NO: 1, an amino acid substitution at position 80 of SEQ ID NO: 1, an amino acid substitution at position 205 of SEQ ID NO: 1, and / or an amino acid substitution at position 207 of SEQ ID NO: 1; b) An amino acid substitution at position 21 of SEQ ID NO: 4, an amino acid substitution at position 23 of SEQ ID NO: 4, and / or an amino acid substitution at position 24 of SEQ ID NO: 4; c) An amino acid substitution at position 21 of SEQ ID NO: 4, an amino acid substitution at position 23 of SEQ ID NO: 6, an amino acid substitution at position 25 of SEQ ID NO: 6, an amino acid substitution at position 80 of SEQ ID NO: 6, and / or an amino acid substitution at position 207 of SEQ ID NO: 6; d) An amino acid substitution at position 15 of SEQ ID NO: 50, and / or an amino acid substitution at position 203 of SEQ ID NO: 50; e) An amino acid substitution at position 199 of SEQ ID NO: 54, and / or an amino acid substitution at position 200 of SEQ ID NO: 54; and / or f) An amino acid substitution at position 199 of SEQ ID NO: 55, an amino acid substitution at position 200 of SEQ ID NO: 55, an amino acid substitution at position 201 of SEQ ID NO: 55, and / or an amino acid substitution at position 202 of SEQ ID NO: 55 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising: Embodiment 37. The variant IgG Fc polypeptide is a) Threonine at the position corresponding to position 21 of SEQ ID NO: 1, leucine at the position corresponding to position 23 of SEQ ID NO: 1, alanine at the position corresponding to position 25 of SEQ ID NO: 1, glycine at the position corresponding to position 80 of SEQ ID NO: 1, alanine at the position corresponding to position 205 of SEQ ID NO: 1, and / or histidine at the position corresponding to position 207 of SEQ ID NO: 1; b) Threonine at the position corresponding to position 21 of SEQ ID NO: 4, leucine at the position corresponding to position 23 of SEQ ID NO: 4, and / or isoleucine at the position corresponding to position 24 of SEQ ID NO: 4; c) Threonine at the position corresponding to position 21 of SEQ ID NO: 6, leucine at the position corresponding to position 23 of SEQ ID NO: 6, alanine at the position corresponding to position 25 of SEQ ID NO: 6, glycine at the position corresponding to position 80 of SEQ ID NO: 6, and / or histidine at the position corresponding to position 207 of SEQ ID NO: 6; d) Threonine or valine at the position corresponding to position 15 of SEQ ID NO: 50, and / or tyrosine or valine at the position corresponding to position 203 of SEQ ID NO: 50; e) Leucine at the position corresponding to the 199th position of SEQ ID NO: 54, and / or histidine at the position corresponding to the 200th position of SEQ ID NO: 54; and / or f) Leucine at the position corresponding to the 199th position of SEQ ID NO: 55, histidine at the position corresponding to the 200th position of SEQ ID NO: 55, asparagine at the position corresponding to the 201st position of SEQ ID NO: 55, and / or histidine at the position corresponding to the 202nd position of SEQ ID NO: 55 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 38. The variant IgG Fc polypeptide is a) Threonine at the 21st position of SEQ ID NO: 1, leucine at the 23rd position of SEQ ID NO: 1, alanine at the 25th position of SEQ ID NO: 1, glycine at the 80th position of SEQ ID NO: 1, alanine at the 205th position of SEQ ID NO: 1, and / or histidine at the 207th position of SEQ ID NO: 1; b) Threonine at the 21st position of SEQ ID NO: 3, leucine at the 23rd position of SEQ ID NO: 4, and / or isoleucine at the 24th position of SEQ ID NO: 4; c) Threonine at the 21st position of SEQ ID NO: 6, leucine at the 23rd position of SEQ ID NO: 6, alanine at the 25th position of SEQ ID NO: 6, glycine at the 80th position of SEQ ID NO: 6, and / or histidine at the 207th position of SEQ ID NO: 6; d) Threonine or valine at the 15th position of SEQ ID NO: 50, and / or tyrosine or valine at the 203rd position of SEQ ID NO: 50; e) Leucine at the 199th position of SEQ ID NO: 54, and / or histidine at the 200th position of SEQ ID NO: 54; and / or f) Leucine at the 199th position of SEQ ID NO: 55, histidine at the 200th position of SEQ ID NO: 55, asparagine at the 201st position of SEQ ID NO: 55, and / or histidine at the 202nd position of SEQ ID NO: 55 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 39. The variant IgG Fc polypeptide is a) An amino acid substitution at the position corresponding to the 93rd position of SEQ ID NO: 2, or an amino acid substitution at the position corresponding to the 93rd position of SEQ ID NO: 4; b) an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 49, an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 52, an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 53, or an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 56; or c) an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 65, an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 66, an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 67, or an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 68 A polypeptide, contiguous polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 40. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide a) has an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 4; b) has an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 49, an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 52, an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 53, or an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 56; or c) has an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 65, an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 66, an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 67, or an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 68 A polypeptide comprising Embodiment 41. The variant IgG Fc polypeptide a) has an amino acid substitution at position 93 of SEQ ID NO: 2, or an amino acid substitution at position 93 of SEQ ID NO: 4; b) has an amino acid substitution at position 87 of SEQ ID NO: 49, an amino acid substitution at position 87 of SEQ ID NO: 52, an amino acid substitution at position 87 of SEQ ID NO: 53, or an amino acid substitution at position 87 of SEQ ID NO: 56; or c) has an amino acid substitution at position 198 of SEQ ID NO: 65, an amino acid substitution at position 198 of SEQ ID NO: 66, an amino acid substitution at position 198 of SEQ ID NO: 67, or an amino acid substitution at position 198 of SEQ ID NO: 68 Any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments, including Embodiment 42. The variant IgG Fc polypeptide is a) arginine at the position corresponding to position 93 of SEQ ID NO: 2, or arginine at the position corresponding to position 93 of SEQ ID NO: 4; b) serine at the position corresponding to position 87 of SEQ ID NO: 49, serine substitution at the position corresponding to position 87 of SEQ ID NO: 52, serine at the position corresponding to position 87 of SEQ ID NO: 53, or serine at the position corresponding to position 87 of SEQ ID NO: 56; or c) alanine at the position corresponding to position 198 of SEQ ID NO: 65, alanine at the position corresponding to position 198 of SEQ ID NO: 66, alanine at the position corresponding to position 198 of SEQ ID NO: 67, or alanine at the position corresponding to position 198 of SEQ ID NO: 68 Any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments, including Embodiment 43. The variant IgG Fc polypeptide is a) arginine at position 93 of SEQ ID NO: 2, or arginine at position 93 of SEQ ID NO: 4; b) serine at position 87 of SEQ ID NO: 49, serine at position 87 of SEQ ID NO: 52, serine at position 87 of SEQ ID NO: 53, or serine at position 87 of SEQ ID NO: 56; or c) alanine at position 198 of SEQ ID NO: 65, alanine at position 198 of SEQ ID NO: 66, alanine at position 198 of SEQ ID NO: 67, or alanine at position 198 of SEQ ID NO: 68 Any one polypeptide, adjacent polypeptide, or multimeric protein of the previous embodiments, including Embodiment 44. The variant IgG Fc polypeptide is a) amino acid substitution at the position corresponding to position 5 of SEQ ID NO: 2, amino acid substitution at the position corresponding to position 38 of SEQ ID NO: 2, amino acid substitution at the position corresponding to position 39 of SEQ ID NO: 2, amino acid substitution at the position corresponding to position 97 of SEQ ID NO: 2, and / or amino acid substitution at the position corresponding to position 98 of SEQ ID NO: 2; or b) An amino acid substitution at the position corresponding to the 5th position of SEQ ID NO: 4, an amino acid substitution at the position corresponding to the 38th position of SEQ ID NO: 4, an amino acid substitution at the position corresponding to the 39th position of SEQ ID NO: 4, an amino acid substitution at the position corresponding to the 97th position of SEQ ID NO: 4, and / or an amino acid substitution at the position corresponding to the 98th position of SEQ ID NO: 4 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 45. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide is a) An amino acid substitution at the position corresponding to the 5th position of SEQ ID NO: 2, an amino acid substitution at the position corresponding to the 38th position of SEQ ID NO: 2, an amino acid substitution at the position corresponding to the 39th position of SEQ ID NO: 2, an amino acid substitution at the position corresponding to the 97th position of SEQ ID NO: 2, and / or an amino acid substitution at the position corresponding to the 98th position of SEQ ID NO: 2; or b) An amino acid substitution at the position corresponding to the 5th position of SEQ ID NO: 4, an amino acid substitution at the position corresponding to the 38th position of SEQ ID NO: 4, an amino acid substitution at the position corresponding to the 39th position of SEQ ID NO: 4, an amino acid substitution at the position corresponding to the 97th position of SEQ ID NO: 4, and / or an amino acid substitution at the position corresponding to the 98th position of SEQ ID NO: 4 A polypeptide comprising Embodiment 46. The variant IgG Fc polypeptide is a) An amino acid substitution at the 5th position of SEQ ID NO: 2, an amino acid substitution at the 38th position of SEQ ID NO: 2, an amino acid substitution at the 39th position of SEQ ID NO: 2, an amino acid substitution at the 97th position of SEQ ID NO: 2, and / or an amino acid substitution at the 98th position of SEQ ID NO: 2; or b) An amino acid substitution at the 5th position of SEQ ID NO: 4, an amino acid substitution at the 38th position of SEQ ID NO: 4, an amino acid substitution at the 39th position of SEQ ID NO: 4, an amino acid substitution at the 97th position of SEQ ID NO: 4, and / or an amino acid substitution at the 98th position of SEQ ID NO: 4 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 47. The variant IgG Fc polypeptide is a) Proline at the position corresponding to the 5th position of SEQ ID NO: 2, glycine at the position corresponding to the 38th position of SEQ ID NO: 2, arginine at the position corresponding to the 39th position of SEQ ID NO: 2, isoleucine at the position corresponding to the 97th position of SEQ ID NO: 2, and / or glycine at the position corresponding to the 98th position of SEQ ID NO: 2; or b) Proline at the position corresponding to the 5th position of SEQ ID NO: 4, glycine at the position corresponding to the 38th position of SEQ ID NO: 4, arginine at the position corresponding to the 39th position of SEQ ID NO: 4, isoleucine at the position corresponding to the 97th position of SEQ ID NO: 4, and / or glycine at the position corresponding to the 98th position of SEQ ID NO: 4 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising: Embodiment 48. The variant IgG Fc polypeptide is a) Proline at the 5th position of SEQ ID NO: 2, glycine at the 38th position of SEQ ID NO: 2, arginine at the 39th position of SEQ ID NO: 2, isoleucine at the 97th position of SEQ ID NO: 2, and / or glycine at the 98th position of SEQ ID NO: 2; or b) Proline at the 5th position of SEQ ID NO: 4, glycine at the 38th position of SEQ ID NO: 4, arginine at the 39th position of SEQ ID NO: 4, isoleucine at the 97th position of SEQ ID NO: 4, and / or glycine at the 98th position of SEQ ID NO: 4 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising: Embodiment 49. a) A variant canine IgG-A Fc polypeptide comprising alanine at the position corresponding to the 2nd position of SEQ ID NO: 1, methionine or lysine at the position corresponding to the 5th position of SEQ ID NO: 1, threonine at the position corresponding to the 21st position of SEQ ID NO: 1, leucine at the position corresponding to the 23rd position of SEQ ID NO: 1, alanine at the position corresponding to the 25th position of SEQ ID NO: 1, valine at the position corresponding to the 35th position of SEQ ID NO: 1, asparagine at the position corresponding to the 38th position of SEQ ID NO: 1, proline at the position corresponding to the 39th position of SEQ ID NO: 1, glutamate at the position corresponding to the 65th position of SEQ ID NO: 1, glycine at the position corresponding to the 80th position of SEQ ID NO: 1, lysine at the position corresponding to the 93rd position of SEQ ID NO: 1, asparagine at the position corresponding to the 96th position of SEQ ID NO: 1, lysine at the position corresponding to the 97th position of SEQ ID NO: 1, alanine at the position corresponding to the 98th position of SEQ ID NO: 1, and / or histidine at the position corresponding to the 207th position of SEQ ID NO: 1; or b) A variant canine IgG-D Fc polypeptide comprising alanine at the position corresponding to the 2nd position of SEQ ID NO: 6, methionine or lysine at the position corresponding to the 5th position of SEQ ID NO: 6, threonine at the position corresponding to the 21st position of SEQ ID NO: 6, leucine at the position corresponding to the 23rd position of SEQ ID NO: 6, alanine at the position corresponding to the 25th position of SEQ ID NO: 6, valine at the position corresponding to the 35th position of SEQ ID NO: 6, asparagine at the position corresponding to the 38th position of SEQ ID NO: 6, proline at the position corresponding to the 39th position of SEQ ID NO: 6, glutamate at the position corresponding to the 65th position of SEQ ID NO: 6, glycine at the position corresponding to the 80th position of SEQ ID NO: 6, lysine at the position corresponding to the 93rd position of SEQ ID NO: 6, asparagine at the position corresponding to the 96th position of SEQ ID NO: 6, lysine at the position corresponding to the 97th position of SEQ ID NO: 6, alanine at the position corresponding to the 98th position of SEQ ID NO: 6, and / or histidine at the position corresponding to the 207th position of SEQ ID NO: 6 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 50. a) A variant canine IgG-A Fc polypeptide comprising alanine at the position corresponding to the 2nd position of SEQ ID NO: 1, methionine or lysine at the position corresponding to the 5th position of SEQ ID NO: 1, threonine at the position corresponding to the 21st position of SEQ ID NO: 1, leucine at the position corresponding to the 23rd position of SEQ ID NO: 1, alanine at the position corresponding to the 25th position of SEQ ID NO: 1, valine at the position corresponding to the 35th position of SEQ ID NO: 1, asparagine at the position corresponding to the 38th position of SEQ ID NO: 1, proline at the position corresponding to the 39th position of SEQ ID NO: 1, glutamate at the position corresponding to the 65th position of SEQ ID NO: 1, glycine at the position corresponding to the 80th position of SEQ ID NO: 1, lysine at the position corresponding to the 93rd position of SEQ ID NO: 1, asparagine at the position corresponding to the 96th position of SEQ ID NO: 1, lysine at the position corresponding to the 97th position of SEQ ID NO: 1, alanine at the position corresponding to the 98th position of SEQ ID NO: 1, and / or histidine at the position corresponding to the 207th position of SEQ ID NO: 1; or b) A variant canine IgG-D Fc polypeptide comprising alanine at the position corresponding to the 2nd position of SEQ ID NO: 6, methionine or lysine at the position corresponding to the 5th position of SEQ ID NO: 6, threonine at the position corresponding to the 21st position of SEQ ID NO: 6, leucine at the position corresponding to the 23rd position of SEQ ID NO: 6, alanine at the position corresponding to the 25th position of SEQ ID NO: 6, valine at the position corresponding to the 35th position of SEQ ID NO: 6, asparagine at the position corresponding to the 38th position of SEQ ID NO: 6, proline at the position corresponding to the 39th position of SEQ ID NO: 6, glutamate at the position corresponding to the 65th position of SEQ ID NO: 6, glycine at the position corresponding to the 80th position of SEQ ID NO: 6, lysine at the position corresponding to the 93rd position of SEQ ID NO: 6, asparagine at the position corresponding to the 96th position of SEQ ID NO: 6, lysine at the position corresponding to the 97th position of SEQ ID NO: 6, alanine at the position corresponding to the 98th position of SEQ ID NO: 6, and / or histidine at the position corresponding to the 207th position of SEQ ID NO: 6 A polypeptide comprising the same. Embodiment 51. a) A variant canine IgG-A Fc polypeptide comprising alanine at position 2 of SEQ ID NO: 1, methionine or lysine at position 5 of SEQ ID NO: 1, threonine at position 21 of SEQ ID NO: 1, leucine at position 23 of SEQ ID NO: 1, alanine at position 25 of SEQ ID NO: 1, valine at position 35 of SEQ ID NO: 1, asparagine at position 38 of SEQ ID NO: 1, proline at position 39 of SEQ ID NO: 1, glutamate at position 65 of SEQ ID NO: 1, glycine at position 80 of SEQ ID NO: 1, lysine at position 93 of SEQ ID NO: 1, asparagine at position 96 of SEQ ID NO: 1, lysine at position 97 of SEQ ID NO: 1, alanine at position 98 of SEQ ID NO: 1, alanine at position 205 of SEQ ID NO: 1, and / or histidine at position 207 of SEQ ID NO: 1; or b) A variant canine IgG-D Fc polypeptide comprising alanine at position 2 of SEQ ID NO: 6, methionine or lysine at position 5 of SEQ ID NO: 6, threonine at position 21 of SEQ ID NO: 6, leucine at position 23 of SEQ ID NO: 6, alanine at position 25 of SEQ ID NO: 6, valine at position 35 of SEQ ID NO: 6, asparagine at position 38 of SEQ ID NO: 6, proline at position 39 of SEQ ID NO: 6, glutamate at position 65 of SEQ ID NO: 6, glycine at position 80 of SEQ ID NO: 6, lysine at position 93 of SEQ ID NO: 6, asparagine at position 96 of SEQ ID NO: 6, lysine at position 97 of SEQ ID NO: 6, alanine at position 98 of SEQ ID NO: 6, and / or histidine at position 207 of SEQ ID NO: 6 A polypeptide, contiguous polypeptide, or multimeric protein of any one of the previous embodiments, comprising Embodiment 52. a) Tyrosine or tryptophan at a position corresponding to position 138 of SEQ ID NO: 1, tyrosine or tryptophan at a position corresponding to position 137 of SEQ ID NO: 2, tyrosine or tryptophan at a position corresponding to position 137 of SEQ ID NO: 4, or tyrosine or tryptophan at a position corresponding to position 138 of SEQ ID NO: 6; b) Tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 69, tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 65 or SEQ ID NO: 66, or tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 67 or SEQ ID NO: 68; or c) Tyrosine or tryptophan at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 A polypeptide, contiguous polypeptide, or multimeric protein of any one of the previous embodiments, comprising a variant IgG Fc polypeptide comprising Embodiment 53. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide a) Tyrosine or tryptophan at a position corresponding to position 138 of SEQ ID NO: 1, tyrosine or tryptophan at a position corresponding to position 137 of SEQ ID NO: 2, tyrosine or tryptophan at a position corresponding to position 137 of SEQ ID NO: 4, or tyrosine or tryptophan at a position corresponding to position 138 of SEQ ID NO: 6; b) Tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 69, tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 65 or SEQ ID NO: 66, or tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 67 or SEQ ID NO: 68; or c) Tyrosine or tryptophan at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 comprising. Embodiment 54. The variant IgG Fc polypeptide is a) Tyrosine or tryptophan at position 138 of SEQ ID NO: 1, tyrosine or tryptophan at position 137 of SEQ ID NO: 2, tyrosine or tryptophan at position 137 of SEQ ID NO: 4, or tyrosine or tryptophan at position 138 of SEQ ID NO: 6; or b) Tyrosine or tryptophan at position 154 of SEQ ID NO: 69, tyrosine or tryptophan at position 154 of SEQ ID NO: 65 or SEQ ID NO: 66, or tyrosine or tryptophan at position 154 of SEQ ID NO: 67 or SEQ ID NO: 68; and / or c) Tyrosine or tryptophan at position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising the same. Embodiment 55. a) Serine at a position corresponding to position 138 of SEQ ID NO: 1, serine at a position corresponding to position 137 of SEQ ID NO: 2, serine at a position corresponding to position 137 of SEQ ID NO: 4, serine at a position corresponding to position 138 of SEQ ID NO: 6, serine at a position corresponding to position 154 of SEQ ID NO: 69, serine at a position corresponding to position 154 of SEQ ID NO: 65 or SEQ ID NO: 66, serine at a position corresponding to position 154 of SEQ ID NO: 67 or SEQ ID NO: 68, or serine at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; b) Alanine at a position corresponding to position 140 of SEQ ID NO: 1, alanine at a position corresponding to position 139 of SEQ ID NO: 2, alanine at a position corresponding to position 139 of SEQ ID NO: 4, alanine at a position corresponding to position 140 of SEQ ID NO: 6, alanine at a position corresponding to position 156 of SEQ ID NO: 69, alanine at a position corresponding to position 156 of SEQ ID NO: 65 or SEQ ID NO: 66, alanine at a position corresponding to position 156 of SEQ ID NO: 67 or SEQ ID NO: 68, or alanine at a position corresponding to position 133 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; and / or c) Threonine at a position corresponding to position 181 of SEQ ID NO: 1, threonine at a position corresponding to position 180 of SEQ ID NO: 2, threonine at a position corresponding to position 180 of SEQ ID NO: 4, threonine at a position corresponding to position 181 of SEQ ID NO: 6, threonine at a position corresponding to position 197 of SEQ ID NO: 69, threonine at a position corresponding to position 197 of SEQ ID NO: 65 or SEQ ID NO: 66, threonine at a position corresponding to position 197 of SEQ ID NO: 67 or SEQ ID NO: 68, or threonine at a position corresponding to position 174 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 A polypeptide, an adjacent polypeptide, or a multimeric protein of any one of the previous embodiments, comprising a variant IgG Fc polypeptide comprising Embodiment 56. A polypeptide comprising a variant IgG Fc polypeptide, wherein the variant IgG Fc polypeptide a) serine at a position corresponding to position 138 of SEQ ID NO: 1, serine at a position corresponding to position 137 of SEQ ID NO: 2, serine at a position corresponding to position 137 of SEQ ID NO: 4, serine at a position corresponding to position 138 of SEQ ID NO: 6, serine at a position corresponding to position 154 of SEQ ID NO: 69, serine at a position corresponding to position 154 of SEQ ID NO: 65 or SEQ ID NO: 66, serine at a position corresponding to position 154 of SEQ ID NO: 67 or SEQ ID NO: 68, or serine at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; b) alanine at a position corresponding to position 140 of SEQ ID NO: 1, alanine at a position corresponding to position 139 of SEQ ID NO: 2, alanine at a position corresponding to position 139 of SEQ ID NO: 4, alanine at a position corresponding to position 140 of SEQ ID NO: 6, alanine at a position corresponding to position 156 of SEQ ID NO: 69, alanine at a position corresponding to position 156 of SEQ ID NO: 65 or SEQ ID NO: 66, alanine at a position corresponding to position 156 of SEQ ID NO: 67 or SEQ ID NO: 68, or alanine at a position corresponding to position 133 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; and / or c) threonine at a position corresponding to position 181 of SEQ ID NO: 1, threonine at a position corresponding to position 180 of SEQ ID NO: 2, threonine at a position corresponding to position 180 of SEQ ID NO: 4, threonine at a position corresponding to position 181 of SEQ ID NO: 6, threonine at a position corresponding to position 197 of SEQ ID NO: 69, threonine at a position corresponding to position 197 of SEQ ID NO: 65 or SEQ ID NO: 66, threonine at a position corresponding to position 197 of SEQ ID NO: 67 or SEQ ID NO: 68, or threonine at a position corresponding to position 174 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 A polypeptide comprising Embodiment 57. A variant IgG Fc polypeptide, a) serine at position 138 of SEQ ID NO: 1, serine at position 137 of SEQ ID NO: 2, serine at position 137 of SEQ ID NO: 4, serine at position 138 of SEQ ID NO: 6, serine at position 154 of SEQ ID NO: 69, serine at position 154 of SEQ ID NO: 65 or SEQ ID NO: 66, serine at position 154 of SEQ ID NO: 67 or SEQ ID NO: 68, or serine at position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; b) alanine at position 140 of SEQ ID NO: 1, alanine at position 139 of SEQ ID NO: 2, alanine at position 139 of SEQ ID NO: 4, alanine at position 140 of SEQ ID NO: 6, alanine at position 156 of SEQ ID NO: 69, alanine at position 156 of SEQ ID NO: 65 or SEQ ID NO: 66, alanine at position 156 of SEQ ID NO: 67 or SEQ ID NO: 68, or alanine at position 133 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; and / or; c) threonine at position 181 of SEQ ID NO: 1, threonine at position 181 of SEQ ID NO: 2, threonine at position 181 of SEQ ID NO: 4, threonine at position 181 of SEQ ID NO: 6, threonine at position 197 of SEQ ID NO: 69, threonine at position 197 of SEQ ID NO: 65 or SEQ ID NO: 66, threonine at position 197 of SEQ ID NO: 67 or SEQ ID NO: 68, or threonine at position 174 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 A polypeptide, contiguous polypeptide, or multimeric protein according to any one of the preceding embodiments, comprising Embodiment 58. A polypeptide, contiguous polypeptide, or multimeric protein according to any one of the preceding embodiments, wherein the polypeptide or variant Fc polypeptide is glycosylated. Embodiment 59. A polypeptide, contiguous polypeptide, or multimeric protein according to any one of the preceding embodiments, wherein the polypeptide or variant Fc polypeptide is of a non-glycosylated form. Embodiment 60. The polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, wherein L1 and L2, if present, are each independently a flexible linker. Embodiment 61. The polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, wherein the amino acid sequences of L1 and L2, if present, each independently contain 100%, at least 95%, at least 90%, or at least 85% serine and / or glycine amino acid residues. Embodiment 62. The polypeptide, adjacent polypeptide, or multimeric protein according to any one of the previous embodiments, wherein the polypeptide, adjacent polypeptide, or multimeric polypeptide contains an extension at the C-terminus. Embodiment 63. The polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, wherein the polypeptide, adjacent polypeptide, or multimeric polypeptide contains one glycine residue, two glycine residues, three glycine residues, four glycine residues, five glycine residues, six glycine residues, seven glycine residues, eight glycine residues, or more than eight glycine residues at the C-terminus. Embodiment 64. The polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, wherein the adjacent polypeptide contains the amino acid sequence of SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, or SEQ ID NO: 165 at its C-terminus. Embodiment 65. The therapeutic polypeptide, TPA1, and / or TPA2 is an NGF polypeptide, a receptor for the NGF polypeptide (e.g., the ECD of the receptor for the NGF polypeptide), a TrkA polypeptide (e.g., the ECD of the TrkA polypeptide), an LNGFR polypeptide (e.g., the ECD of the LNGFR polypeptide), a TNFα polypeptide, a receptor for the TNFα polypeptide, a TNFR polypeptide (e.g., the ECD of the TNFR polypeptide), a TNFR1 polypeptide (e.g., the ECD of the TNFR1 polypeptide), a TNFR2 polypeptide (e.g., the ECD of the TNFR2 polypeptide), an IL5 polypeptide, a receptor for the IL5 polypeptide, an IL5R polypeptide (e.g., the ECD of the IL5R polypeptide), an IL5Rα polypeptide (e.g., the ECD of the IL5Rα polypeptide), an IL6 polypeptide, a receptor for the IL6 polypeptide, an IL6R polypeptide (e.g., the ECD of the IL6R polypeptide), an IL17 polypeptide, a receptor for the IL17 polypeptide, an IL17R polypeptide (e.g., the ECD of the IL17R polypeptide), an IL17RA polypeptide (e.g., the ECD of the IL17RA polypeptide), an IL17RB polypeptide (e.g., the ECD of the IL17RB polypeptide), an IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), an IL23 polypeptide, a receptor for the IL23 polypeptide, an IL23R polypeptide (e.g., the ECD of the IL23R polypeptide), an IL12Rβ1 polypeptide (e.g., the ECD of the IL12Rβ1 polypeptide), a PDL1 polypeptide, a receptor for the PDL1 polypeptide, a PDL2 polypeptide, a receptor for the PDL2 polypeptide, a PD1 polypeptide (e.g., the ECD of the PD1 polypeptide), an integrin polypeptide (e.g., an ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, or ITGB8 polypeptide), a receptor for the integrin polypeptide, a fibronectin polypeptide (e.g., the ECD of the fibronectin polypeptide),Vitronectin polypeptide (e.g., ECD of vitronectin polypeptide), collagen polypeptide (e.g., ECD of collagen polypeptide), laminin polypeptide (e.g., ECD of laminin polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD86 polypeptide, receptor of CD86 polypeptide, CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), B7-H3 polypeptide, receptor of B7-H3 polypeptide (e.g., ECD of receptor of B7-H3 polypeptide), LAG-3 polypeptide (e.g., ECD of LAG-3 polypeptide), IL31 polypeptide, receptor of IL31 polypeptide, IL31RA polypeptide (e.g., ECD of IL31RA polypeptide), OSMR polypeptide (e.g., ECD of OSMR polypeptide), IL4 polypeptide, receptor of IL4R polypeptide, IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13 polypeptide, receptor of IL13 polypeptide, IL13RA1 polypeptide (e.g., ECD of IL13RA1 polypeptide), IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13Rα2 polypeptide (e.g., ECD of IL13Rα2 polypeptide), IL22 polypeptide, receptor of IL22 polypeptide (e.g., ECD of IL22 polypeptide), IL22Rα1 polypeptide (e.g., ECD of IL22Rα1 polypeptide), IL10Rβ2 polypeptide (e.g., ECD of IL10Rβ2 polypeptide), IL33 polypeptide, receptor of IL33 polypeptide, IL1RL1 polypeptide (e.g., ECD of IL1RL1 polypeptide), EGF polypeptide, receptor of EGF polypeptide, TGFα polypeptide, receptor of TGFα polypeptide, EGFR polypeptide (e.g., ECD of EGFR polypeptide), MMP9 polypeptide, FGF polypeptide (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23 polypeptide), receptor of FGF polypeptide,FGFR polypeptides (e.g., FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), ECDs of FGFR polypeptides (e.g., ECDs of FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), EGF polypeptides, receptors of EGF polypeptides, neuregulin polypeptides (e.g., neuregulin isoforms I, II, III, IV, V, or VI polypeptides), receptors of neuregulin polypeptides, HER polypeptides (e.g., HER1, HER2, HER3, or HER4 polypeptides), ECDs of HER polypeptides (e.g., ECDs of HER1, HER2, HER3, or HER4 polypeptides), EpCAM polypeptides (e.g., ECDs of EpCAM polypeptides), CD20 polypeptides (e.g., ECDs of CD20 polypeptides), ligands of CD20 polypeptides, CD19 polypeptides (e.g., ECDs of CD19 polypeptides), ligands of CD19 polypeptides, CGRP polypeptides (e.g., α-CGRP polypeptide or β-CGRP polypeptide), receptors of CGRP polypeptides, receptors of α-CGRP polypeptide, receptors of β-CGRP polypeptide, CALCRL polypeptides (e.g., ECDs of CALCRL polypeptides), RAMP polypeptides (e.g., RAMP1, RAMP2, or RAMP3 polypeptides), ECDs of RAMP polypeptides (e.g., ECDs of RAMP1, RAMP2, or RAMP3 polypeptides), IGF polypeptides (e.g., IGF-1 or IGF-2 polypeptides), receptors of IGF polypeptides (e.g., receptors of IGF1 or IGF-2 polypeptides), IGFR polypeptides (e.g., IGFR1 or IGFR2 polypeptides), ECDs of IGFR polypeptides (e.g., ECDs of IGFR1 or IGFR2 polypeptides), IGFBP polypeptides (e.g., IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6 polypeptides), VEGF polypeptides (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides), receptors of VEGF polypeptides (e.g., receptors of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides),VEGFR polypeptides (e.g., VEGFR1, VEGFR2, or VEGFR3 polypeptides), ECDs of VEGFR polypeptides (e.g., ECDs of VEGFR1, VEGFR2, or VEGFR3 polypeptides), FLT1 receptor polypeptides (e.g., ECDs of FLT1 receptor polypeptides), IL36 polypeptides (e.g., IL36A, IL36B, or IL36G polypeptides), receptors for IL36 polypeptides (e.g., receptors for IL36A, IL36B, or IL36G polypeptides), IL36R polypeptides (e.g., ECDs of IL36R polypeptides), IL1R1 polypeptides (e.g., ECDs of IL1R1 polypeptides), IL1R2 polypeptides (e.g., ECDs of IL1R2 polypeptides), IL1RL1 polypeptides (ECDs of IL1RL1 polypeptides), IL18R1 polypeptides (ECDs of IL18R1 polypeptides), bacterial toxin polypeptides, exotoxin polypeptides, endotoxin polypeptides, botulinum neurotoxin polypeptides, tetanus toxin polypeptides, staphylococcal toxin polypeptides, CD52 polypeptides (e.g., ECDs of CD52 polypeptides), ligands for CD52 polypeptides, SIGLEC10 polypeptides, PCSK9 polypeptides, receptors for PCSK9 polypeptides, LDLR polypeptides (e.g., ECDs of LDLR polypeptides), CEA polypeptides (e.g., CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptides), ECDs of CEA polypeptides (e.g., ECDs of CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptides), BAFF polypeptides, receptors for BAFF polypeptides, TRAF polypeptides (e.g., TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRAF7 polypeptides), receptors for TRAF polypeptides (e.g., receptors for TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 polypeptides), BCMA polypeptides, ECDs of BCMA polypeptides, SOST polypeptides, receptors for SOST polypeptides, LRP polypeptides (e.g., LRP5 or LRP6 polypeptides), ECDs of LRP polypeptides (e.g., ECDs of LRP5 or LRP6 polypeptides), DLL polypeptides (e.g.,DLL4 polypeptide), a receptor for the DLL polypeptide, a Jagged polypeptide (e.g., JAG1 or JAG polypeptide), a receptor for the Jagged polypeptide (e.g., a receptor for JAG1 or JAG polypeptide), a NOTCH polypeptide (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), a ligand for the NOTCH polypeptide (e.g., a ligand for NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), VWF polypeptide, a receptor for the VWF polypeptide, factor VIII polypeptide, a receptor for the factor VIII polypeptide, platelet GP1b receptor polypeptide (e.g., ECD of platelet GP1b receptor polypeptide), integrin α IIb β 3 polypeptide (e.g., integrin α IIb β 3ECD of polypeptide), IL2 polypeptide, receptor of IL2 polypeptide, IL2R polypeptide (e.g., IL2Rα, IL2Rβ, or IL2Rγ polypeptide), ECD of IL2R polypeptide (e.g., ECD of IL2Rα, IL2Rβ, or IL2Rγ polypeptide), TGFβ polypeptide, receptor of TGFβ polypeptide, decorin polypeptide, EIF3I polypeptide, LTBP1 polypeptide, TGFβR1 polypeptide (e.g., ECD of TGFβR1 polypeptide), YWHAE polypeptide, IgE polypeptide, receptor or IgE polypeptide, Fc receptor polypeptide (e.g., FcεRI or FcεRII polypeptide), ECD of Fc receptor polypeptide (e.g., ECD of FcεRI or FcεRII polypeptide), KLK polypeptide (e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, or KLK15 polypeptide), Rank1 polypeptide, receptor of Rank1 polypeptide, RANK polypeptide (e.g., ECD of RANK polypeptide), TSLP polypeptide, receptor of TSLP polypeptide, CRLF2 polypeptide (e.g., ECD of CRLF2 polypeptide), IL7Rα polypeptide (e.g., ECD of IL7Rα polypeptide), S1P polypeptide, CD3 polypeptide (e.g., CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), ECD of CD3 polypeptide (e.g., ECD of CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD28 polypeptide (e.g., ECD of CD28 polypeptide), CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), GnRH polypeptide, receptor of GNRH polypeptide, GnRHR polypeptide (e.g., ECD of GnRHR polypeptide), ICAM polypeptide (e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), receptor of ICAM polypeptide (e.g., receptor of ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide),JAM-A polypeptide, a receptor for the JAM-A polypeptide, an LFA-1 polypeptide (e.g., the ECD of the LFA-1 polypeptide), Na, v 1.7 polypeptide, a C5 polypeptide (e.g., a C5a or C5b polypeptide), a receptor for the C5 polypeptide (e.g., a receptor for the C5a or C5b polypeptide), a C5aR polypeptide (e.g., the ECD of the C5aR polypeptide), a C5L2 polypeptide (e.g., the ECD of the C5L2 polypeptide), an IL17 polypeptide, a receptor for the IL17 polypeptide, an IL17Ra polypeptide (e.g., the ECD of the IL17Ra polypeptide), an IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), an EPO polypeptide, a somatostatin polypeptide, a GLP1 polypeptide, and a glucagon polypeptide, a polypeptide, an adjacent polypeptide, or a multimeric protein of any one of the previous embodiments selected from Embodiment 66. A polypeptide, TPA1, and / or TPA2 of any one of the previous embodiments, an adjacent polypeptide, or a multimeric protein, wherein the therapeutic polypeptide, TPA1, and / or TPA2 is a canine polypeptide, a feline polypeptide, or a equine polypeptide. Embodiment 67. The antibody, TPA1, and / or TPA2 is / are an NGF polypeptide, a receptor for the NGF polypeptide (e.g., the ECD of the receptor for the NGF polypeptide), a TrkA polypeptide (e.g., the ECD of the TrkA polypeptide), an LNGFR polypeptide (e.g., the ECD of the LNGFR polypeptide), a TNFα polypeptide, a receptor for the TNFα polypeptide, a TNFR polypeptide (e.g., the ECD of the TNFR polypeptide), a TNFR1 polypeptide (e.g., the ECD of the TNFR1 polypeptide), a TNFR2 polypeptide (e.g., the ECD of the TNFR2 polypeptide), an IL5 polypeptide, a receptor for the IL5 polypeptide, an IL5R polypeptide (e.g., the ECD of the IL5R polypeptide), an IL5Rα polypeptide (e.g., the ECD of the IL5Rα polypeptide), an IL6 polypeptide, a receptor for the IL6 polypeptide, an IL6R polypeptide (e.g., the ECD of the IL6R polypeptide), an IL17 polypeptide, a receptor for the IL17 polypeptide, an IL17R polypeptide (e.g., the ECD of the IL17R polypeptide), an IL17RA polypeptide (e.g., the ECD of the IL17RA polypeptide), an IL17RB polypeptide (e.g., the ECD of the IL17RB polypeptide), an IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), an IL23 polypeptide, a receptor for the IL23 polypeptide, an IL23R polypeptide (e.g., the ECD of the IL23R polypeptide), an IL12Rβ1 polypeptide (e.g., the ECD of the IL12Rβ1 polypeptide), a PDL1 polypeptide, a receptor for the PDL1 polypeptide, a PDL2 polypeptide, a receptor for the PDL2 polypeptide, a PD1 polypeptide (e.g., the ECD of the PD1 polypeptide), an integrin polypeptide (e.g., an ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, or ITGB8 polypeptide), a receptor for the integrin polypeptide, a fibronectin polypeptide (e.g., the ECD of the fibronectin polypeptide),Vitronectin polypeptide (e.g., ECD of vitronectin polypeptide), collagen polypeptide (e.g., ECD of collagen polypeptide), laminin polypeptide (e.g., ECD of laminin polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD86 polypeptide, receptor of CD86 polypeptide, CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), B7-H3 polypeptide, receptor of B7-H3 polypeptide (e.g., ECD of receptor of B7-H3 polypeptide), LAG-3 polypeptide (e.g., ECD of LAG-3 polypeptide), IL31 polypeptide, receptor of IL31 polypeptide, IL31RA polypeptide (e.g., ECD of IL31RA polypeptide), OSMR polypeptide (e.g., ECD of OSMR polypeptide), IL4 polypeptide, receptor of IL4R polypeptide, IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13 polypeptide, receptor of IL13 polypeptide, IL13RA1 polypeptide (e.g., ECD of IL13RA1 polypeptide), IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13Rα2 polypeptide (e.g., ECD of IL13Rα2 polypeptide), IL22 polypeptide, receptor of IL22 polypeptide (e.g., ECD of IL22 polypeptide), IL22Rα1 polypeptide (e.g., ECD of IL22Rα1 polypeptide), IL10Rβ2 polypeptide (e.g., ECD of IL10Rβ2 polypeptide), IL33 polypeptide, receptor of IL33 polypeptide, IL1RL1 polypeptide (e.g., ECD of IL1RL1 polypeptide), EGF polypeptide, receptor of EGF polypeptide, TGFα polypeptide, receptor of TGFα polypeptide, EGFR polypeptide (e.g., ECD of EGFR polypeptide), MMP9 polypeptide, FGF polypeptide (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23 polypeptide), receptor of FGF polypeptide,FGFR polypeptides (e.g., FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), ECDs of FGFR polypeptides (e.g., ECDs of FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), EGF polypeptides, receptors of EGF polypeptides, neuregulin polypeptides (e.g., neuregulin isoform I, II, III, IV, V, or VI polypeptides), receptors of neuregulin polypeptides, HER polypeptides (e.g., HER1, HER2, HER3, or HER4 polypeptides), ECDs of HER polypeptides (e.g., ECDs of HER1, HER2, HER3, or HER4 polypeptides), EpCAM polypeptides (e.g., ECDs of EpCAM polypeptides), CD20 polypeptides (e.g., ECDs of CD20 polypeptides), ligands of CD20 polypeptides, CD19 polypeptides (e.g., ECDs of CD19 polypeptides), ligands of CD19 polypeptides, CGRP polypeptides (e.g., α-CGRP polypeptide or β-CGRP polypeptide), receptors of CGRP polypeptides, receptors of α-CGRP polypeptide, receptors of β-CGRP polypeptide, CALCRL polypeptides (e.g., ECDs of CALCRL polypeptides), RAMP polypeptides (e.g., RAMP1, RAMP2, or RAMP3 polypeptides), ECDs of RAMP polypeptides (e.g., ECDs of RAMP1, RAMP2, or RAMP3 polypeptides), IGF polypeptides (e.g., IGF-1 or IGF-2 polypeptides), receptors of IGF polypeptides (e.g., receptors of IGF1 or IGF-2 polypeptides), IGFR polypeptides (e.g., IGFR1 or IGFR2 polypeptides), ECDs of IGFR polypeptides (e.g., ECDs of IGFR1 or IGFR2 polypeptides), IGFBP polypeptides (e.g., IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6 polypeptides), VEGF polypeptides (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides), receptors of VEGF polypeptides (e.g., receptors of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides),VEGFR polypeptides (e.g., VEGFR1, VEGFR2, or VEGFR3 polypeptides), ECDs of VEGFR polypeptides (e.g., ECDs of VEGFR1, VEGFR2, or VEGFR3 polypeptides), FLT1 receptor polypeptides (e.g., ECDs of FLT1 receptor polypeptides), IL36 polypeptides (e.g., IL36A, IL36B, or IL36G polypeptides), receptors for IL36 polypeptides (e.g., receptors for IL36A, IL36B, or IL36G polypeptides), IL36R polypeptides (e.g., ECDs of IL36R polypeptides), IL1R1 polypeptides (e.g., ECDs of IL1R1 polypeptides), IL1R2 polypeptides (e.g., ECDs of IL1R2 polypeptides), IL1RL1 polypeptides (ECDs of IL1RL1 polypeptides), IL18R1 polypeptides (ECDs of IL18R1 polypeptides), bacterial toxin polypeptides, exotoxin polypeptides, endotoxin polypeptides, botulinum neurotoxin polypeptides, tetanus toxin polypeptides, staphylococcal toxin polypeptides, CD52 polypeptide (e.g., ECD of CD52 polypeptide), ligand of CD52 polypeptide, SIGLEC10 polypeptide, PCSK9 polypeptide, receptor of PCSK9 polypeptide, LDLR polypeptide (e.g., ECD of LDLR polypeptide), CEA polypeptide (e.g., CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), ECD of CEA polypeptide (e.g., ECD of CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), BAFF polypeptide, receptor of BAFF polypeptide, TRAF polypeptide (e.g., TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRAF7 polypeptide), receptor of TRAF polypeptide (e.g., receptor of TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 polypeptide), BCMA polypeptide, ECD of BCMA polypeptide, SOST polypeptide, receptor of SOST polypeptide, LRP polypeptide (e.g., LRP5 or LRP6 polypeptide), ECD of LRP polypeptide (e.g., ECD of LRP5 or LRP6 polypeptide), DLL polypeptide (e.g., DLL4 polypeptide), receptor of DLL polypeptide, Jagged polypeptide (e.g., JAG1 or JAG polypeptide), receptor of Jagged polypeptide (e.g., receptor of JAG1 or JAG polypeptide), NOTCH polypeptide (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), ligand of NOTCH polypeptide (e.g., ligand of NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), VWF polypeptide, receptor of VWF polypeptide, Factor VIII polypeptide, receptor of Factor VIII polypeptide, platelet GP1b receptor polypeptide (e.g., ECD of platelet GP1b receptor polypeptide), integrin α IIb β 3 polypeptide (e.g., integrin α IIb β 3ECD of a polypeptide), an IL2 polypeptide, a receptor of an IL2 polypeptide, an IL2R polypeptide (e.g., an IL2Rα, IL2Rβ, or IL2Rγ polypeptide), an ECD of an IL2R polypeptide (e.g., an ECD of an IL2Rα, IL2Rβ, or IL2Rγ polypeptide), a TGFβ polypeptide, a receptor of a TGFβ polypeptide, a decorin polypeptide, an EIF3I polypeptide, an LTBP1 polypeptide, a TGFβR1 polypeptide (e.g., an ECD of a TGFβR1 polypeptide), a YWHAE polypeptide, an IgE polypeptide, a receptor or an IgE polypeptide, an Fc receptor polypeptide (e.g., an FcεRI or FcεRII polypeptide), an ECD of an Fc receptor polypeptide (e.g., an ECD of an FcεRI or FcεRII polypeptide), a KLK polypeptide (e.g., a KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, or KLK15 polypeptide), a Rank1 polypeptide, a receptor of a Rank1 polypeptide, a RANK polypeptide (e.g., an ECD of a RANK polypeptide), a TSLP polypeptide, a receptor of a TSLP polypeptide, a CRLF2 polypeptide (e.g., an ECD of a CRLF2 polypeptide), an IL7Rα polypeptide (e.g., an ECD of an IL7Rα polypeptide), an S1P polypeptide, a CD3 polypeptide (e.g., a CD3γ polypeptide, a CD3δ polypeptide, or a CD3ε polypeptide), an ECD of a CD3 polypeptide (e.g., an ECD of a CD3γ polypeptide, a CD3δ polypeptide, or a CD3ε polypeptide), a CD80 polypeptide, a receptor of a CD80 polypeptide, a CD28 polypeptide (e.g., an ECD of a CD28 polypeptide), a CTLA-4 polypeptide (e.g., an ECD of a CTLA-4 polypeptide), a GnRH polypeptide, a receptor of a GNRH polypeptide, a GnRHR polypeptide (e.g., an ECD of a GnRHR polypeptide), an ICAM polypeptide (e.g., an ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), a receptor of an ICAM polypeptide (e.g., a receptor of an ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide),JAM-A polypeptide, a receptor for the JAM-A polypeptide, an LFA-1 polypeptide (e.g., the ECD of the LFA-1 polypeptide), Na, v 1.7 polypeptide, a C5 polypeptide (e.g., a C5a or C5b polypeptide), a receptor for the C5 polypeptide (e.g., a receptor for the C5a or C5b polypeptide), a C5aR polypeptide (e.g., the ECD of the C5aR polypeptide), a C5L2 polypeptide (e.g., the ECD of the C5L2 polypeptide), an IL17 polypeptide, a receptor for the IL17 polypeptide, an IL17Ra polypeptide (e.g., the ECD of the IL17Ra polypeptide), an IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), an EPO polypeptide, a somatostatin polypeptide, a GLP1 polypeptide, and a glucagon polypeptide, an antibody that binds to a target polypeptide selected from any one of the polypeptides, adjacent polypeptides, or multimeric proteins of the previous embodiments. Embodiment 68. An antibody that binds to a canine target polypeptide, a feline target polypeptide, or a equine target polypeptide, any one of the polypeptides, adjacent polypeptides, or multimeric proteins of the previous embodiments. Embodiment 69. The variant IgG Fc polypeptide is at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, and / or 250, and includes any polypeptide, adjacent polypeptide, or multimeric protein of any of the previous embodiments having an amino acid sequence. Embodiment 70. A polypeptide, adjacent polypeptide, or multimeric protein of any one of the previous embodiments, comprising the amino acid sequences of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 146, 147, 148, 149, 150, 151, 154, 155, 157, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 271, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, and / or 250. Embodiment 71. A polypeptide comprising the amino acid sequences of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 146, 147, 148, 149, 150, 151, 154, 155, 157, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 271, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, and / or 250. Embodiment 72. Any polypeptide, multimeric protein, or contiguous polypeptide of the previous embodiments, wherein at least one amino acid modification or substitution comprises an amino acid substitution with an amino acid derivative. Embodiment 73. An isolated nucleic acid encoding any polypeptide, multimeric protein, or contiguous polypeptide of the previous embodiments. Embodiment 74. A host cell comprising the nucleic acid of Embodiment 74. Embodiment 75. A method for producing a polypeptide, comprising culturing the host cell of Embodiment 74 and isolating the polypeptide. Embodiment 76. A pharmaceutical composition comprising any one of the polypeptides, multimeric proteins, or adjacent polypeptides of Embodiments 1 to 72 and a pharmaceutically acceptable carrier. Embodiment 77. A method of exposing a cell to any one of the polypeptides, multimeric proteins, adjacent polypeptides, or pharmaceutical compositions of Embodiments 1 to 72 or 76. Embodiment 78. The method of Embodiment 77, wherein the cell is exposed to the polypeptide, heterodimeric protein, adjacent polypeptide, or pharmaceutical composition ex vivo. Embodiment 79. The method of Embodiment 77, wherein the cell is exposed to the polypeptide, heterodimeric protein, adjacent polypeptide, or pharmaceutical composition in vivo. Embodiment 80. The method according to any one of Embodiments 77 to 79, wherein the cell is a human cell, a canine cell, a feline cell, or a equine cell. Embodiment 81. A method of delivering a polypeptide to a subject, comprising parenterally administering any one of the polypeptides, multimeric proteins, adjacent polypeptides, or pharmaceutical compositions of Embodiments 1 to 72 or 76. Embodiment 82. A method of delivering a polypeptide to a subject, comprising administering any one of the polypeptides, multimeric proteins, adjacent polypeptides, or pharmaceutical compositions of Embodiments 1 to 72 or 76 by intramuscular route, intraperitoneal route, intrathecal route, subcutaneous route, intraarterial route, intra-articular route, intrathecal route, or inhalation route. Embodiment 83. A method of treating a subject having diabetes or obesity, comprising administering to the subject a therapeutically effective amount of any one of the polypeptides, multimeric proteins, adjacent polypeptides, or pharmaceutical compositions of Embodiments 1 to 72 or 76. Embodiment 84. The method according to any one of Embodiments 81 to 83, wherein the subject is a human subject. Embodiment 85. The method according to any one of Embodiments 81 to 83, wherein the subject is a companion animal species. Embodiment 86. The method according to Embodiment 85, wherein the companion animal species is a dog, a horse, or a cat.

Brief Description of the Drawings

[0006]

Figure 1

[0007]

Figure 2

[0008] Table 1 provides a list of exemplary sequences referred to herein. JPEG0007691365000001.jpg 250158 JPEG0007691365000002.jpg 255160 JPEG0007691365000003.jpg 255161 JPEG0007691365000004.jpg 255161 JPEG0007691365000005.jpg 255161 JPEG0007691365000006.jpg 255163 JPEG0007691365000007.jpg 255161 JPEG0007691365000008.jpg 255159 JPEG0007691365000009.jpg 255160 JPEG0007691365000010.jpg 255161 JPEG0007691365000011.jpg 255161 JPEG0007691365000012.jpg 255160 JPEG0007691365000013.jpg 255160 JPEG0007691365000014.jpg 255163 JPEG0007691365000015.jpg 255159 JPEG0007691365000016.jpg 255160 JPEG0007691365000017.jpg 255160 JPEG0007691365000018.jpg 255161 JPEG0007691365000019.jpg 255160 JPEG0007691365000020.jpg 255158 JPEG0007691365000021.jpg 255163 JPEG0007691365000022.jpg 255160 JPEG0007691365000023.jpg 255163 JPEG0007691365000024.jpg 255160 JPEG0007691365000025.jpg 255162 JPEG0007691365000026.jpg 255163 JPEG0007691365000027.jpg 255164 JPEG0007691365000028.jpg 255160 JPEG0007691365000029.jpg 255160 JPEG0007691365000030.jpg 255162 JPEG0007691365000031.jpg 255159 JPEG0007691365000032.jpg 255162 JPEG0007691365000033.jpg255164JPEG0007691365000034.jpg255160JPEG0007691365000035.jpg255165JPEG0007691365000036.jpg255162JPEG0007691365000037.jpg255161JPEG0007691365000038.jpg255161JPEG0007691365000039.jpg255164JPEG0007691365000040.jpg255161JPEG0007691365000041.jpg255163JPEG0007691365000042.jpg255160JPEG0007691365000043.jpg255161JPEG0007691365000044.jpg255160JPEG0007691365000045.jpg115170.

[0009] Variant IgG Fc polypeptides from companion animals such as dogs, horses, and cats are described. In some embodiments, the variant IgG Fc polypeptide has increased binding to Protein A, decreased binding to C1q, decreased binding to CD16, increased stability, increased recombinant productivity, increased hinge disulfide formation, and / or forms a heterodimeric polypeptide. In some embodiments, an antibody, antibody fragment, or fusion protein comprises the variant IgG Fc polypeptide. Also provided are assays for methods of producing or purifying the variant IgG Fc polypeptide and methods of administering the variant IgG Fc polypeptide to a companion animal.

[0010] For the convenience of the reader, the following definitions are provided for the terms used herein.

[0011] As used herein, K D Numerical terms such as etc. are calculated based on scientific measurements and are thus subject to the effects of appropriate measurement errors. In some cases, numerical terms may include values rounded to the nearest significant figure. ​

[0012] As used herein, "a" or "an" means "at least one" or "one or more" unless otherwise specified. As used herein, the term "or" means "and / or" unless otherwise stated. In the context of claims that depend on a plurality of items, the use of "or" in citing other claims refers only to the alternative claims.

[0013] [Exemplary variant IgG Fc polypeptides] Provided are novel variant IgG Fc polypeptides, e.g., having increased binding to Protein A, decreased binding to C1q, decreased binding to CD16, increased stability, increased recombinant productivity, increased hinge disulfide formation, and / or variant IgG Fc polypeptides for heterodimeric protein formation assays.

[0014] "Amino acid sequence" means 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 include natural or unnatural amino acid residues and include, without limitation, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) with respect to the native sequence, as long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host producing the protein or errors in PCR amplification.

[0015] "IgX Fc" or "IgX Fc polypeptide" refers to an Fc polypeptide derived from a specific 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 γ chain, "IgA Fc" indicates that the Fc polypeptide is derived from the α chain, "IgD Fc" indicates that the Fc polypeptide is derived from the δ chain, "IgE Fc" indicates that the Fc polypeptide is derived from the ε chain, "IgM Fc" indicates that the Fc polypeptide is derived from the μ chain, and so on. In some embodiments, the IgG Fc polypeptide includes the hinge, CH2, and CH3, but does not include CH1 or CL. In some embodiments, the IgG Fc polypeptide includes CH2 and CH3, but does not include CH1, the hinge, or CL. In some embodiments, the IgG Fc polypeptide includes CH1, the hinge, CH2, and CH3, with or without CL1. In some embodiments, an Fc polypeptide such as the IgG Fc polypeptide lacks one or more C-terminal amino acids such as from 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, the ability to bind to C1q, the ability to bind to CD16, and / or the ability to bind to protein A. The "effector function" of an Fc polypeptide is an action or activity that is wholly or partially carried out by any antibody in response to stimulation and may include complement binding and / or ADCC (antibody-dependent cell cytotoxicity) induction. "IgX-N Fc" or "IgGXN Fc" indicates that the Fc polypeptide is derived from a specific subclass of an 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.

[0016] "Hinge" refers to any portion of an Fc polypeptide or variant Fc polypeptide that is rich in proline and contains at least one cysteine residue located between CH1 and CH2 of the full-length heavy chain constant region.

[0017] In some embodiments, the hinge can form disulfide bonds within the same hinge region, within the same Fc polypeptide, with the hinge region of a separate Fc polypeptide, or with a separate Fc polypeptide. In some embodiments, the hinge contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten proline residues.

[0018] The term "companion animal species" refers to animals that are suitable as companions for humans. In some embodiments, the companion animal species is a dog (or canine), a cat (or feline), or a horse (or equine). In some embodiments, the companion animal species is a small mammal such as a dog, a cat, a canine, a feline, a rabbit, a ferret, a guinea pig, or a rodent. In some embodiments, the companion animal species is a domestic animal such as a horse, a cow, or a pig.

[0019] In some embodiments, the IgX Fc polypeptide or IgX-N Fc polypeptide is derived from a companion animal such as a dog, a cat, or a horse. In some embodiments, the IgG Fc polypeptide is isolated from a canine γ heavy chain such as IgG-A, IgG-B, IgG-C, or IgG-D. In some cases, the IgG Fc polypeptide is isolated from a feline γ heavy chain such as IgG1a, IgG1b, or IgG2. In other examples, the IgG Fc polypeptide is isolated from an equine γ heavy chain such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7.

[0020] The terms "IgX Fc" and "IgX Fc polypeptide" include wild-type IgX Fc polypeptides and variant IgX Fc polypeptides unless otherwise indicated.

[0021] "Wild type" refers to the non-mutated form of a naturally occurring polypeptide, or a fragment thereof. A wild type polypeptide can be produced recombinantly.

[0022] In some embodiments, the wild type IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 152, or SEQ ID NO: 153.

[0023] "Variant" is a polypeptide that differs from a reference polypeptide by substitution, deletion, and / or addition of one or more non-natural amino acids. In some embodiments, the variant retains at least one biological activity of the reference polypeptide. In some embodiments, a variant (e.g., variant canine IgG-A Fc, variant canine IgG-C Fc, variant canine IgG-D Fc, variant equine IgG2 Fc, variant equine IgG5 Fc, or variant equine IgG6 Fc) has an activity that the reference polypeptide substantially lacks. For example, in some embodiments, variant canine IgG-A Fc, variant canine IgG-C Fc, variant canine IgG-D Fc, variant equine IgG2 Fc, variant equine IgG5 Fc, or variant equine IgG6 Fc binds to protein A.

[0024] As used herein, "percent amino acid sequence identity (%)" and "homology" with respect to a nucleic acid molecule or polypeptide sequence are defined as the percentage of nucleotide or amino acid residues in a reference sequence that are identical to the nucleotide 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, and without considering conservative substitutions as part of sequence identity. Alignments for the purpose of determining percent sequence identity can be achieved in various ways within the skill in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEGALINE TM (DNASTAR) software and can be achieved in a variety of ways within the skill in the art. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0025] 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, and without considering conservative substitutions as part of sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus 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 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity with the sequence of a reference nucleic acid or polypeptide.

[0026] As used herein, the "position corresponding to position n" (where n is any number) refers to the amino acid position of the subject polypeptide that aligns with position n of the 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 in the subject polypeptide corresponds to position n of the reference polypeptide can be achieved in a variety of ways within the skill of the art using publicly available computer software such as, for example, BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN TM (DNASTAR) software and the like, and can be achieved in a variety of ways within the skill of the art. One of ordinary skill in the art can determine appropriate parameters for alignment, including any parameters necessary to achieve a maximum alignment over the full length of the two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.

[0027] A "point mutation" is a mutation that involves a single amino acid residue. The mutation can be a loss of an amino acid, a substitution of one amino acid residue for another, or an insertion of an additional amino acid residue.

[0028] An "amino acid substitution" refers to the replacement of one amino acid in a polypeptide with another amino acid. In some embodiments, the amino acid substitution is a conservative substitution. Non-limiting exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions can be introduced into the molecule of interest, and the product can be screened for retention / improvement of the desired activity, e.g., antigen-binding, reduction of immunogenicity, or improvement of ADCC or CDC or enhancement of pharmacokinetics.

[0029] JPEG0007691365000046.jpg193169

[0030] Amino acids can be grouped according to common side-chain characteristics: (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 affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0031] Non-conservative substitutions involve exchanging one member of these classes for another.

[0032] As used herein, "variant IgG Fc" is an IgG Fc polypeptide that differs from a reference IgG Fc polypeptide by one or more amino acid substitutions, deletions, and / or additions and that substantially retains at least one biological activity of the reference IgG Fc polypeptide.

[0033] As used herein, "amino acid derivative" refers to any amino acid, modified amino acid, and / or amino acid analog that is not one of the 20 common natural amino acids found in humans. Exemplary amino acid derivatives include natural amino acids not found in humans (e.g., selenocysteine and pyrrolysine, which may be found in some microorganisms) and non-natural amino acids. Exemplary amino acid derivatives include, but are not limited to, amino acid derivatives commercially available through chemical manufacturers (e.g., sigmaaldrich.com / chemistry / chemistry-products.html?TablePage=16274965, accessed May 6, 2017, which is hereby incorporated by reference). One or more amino acid derivatives can be introduced at specific positions in a polypeptide using a translation system that utilizes a host cell, an orthogonal aminoacyl-tRNA synthetase derived from an orthologous bacterium synthetase, an orthogonal tRNA, and an amino acid derivative. For a detailed description, see, for example, U.S. Patent No. 9,624,485.

[0034] In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions with amino acid derivatives. In some embodiments, the amino acid derivative is an alanine derivative, a cysteine derivative, an aspartic acid derivative, a glutamic acid derivative, a phenylalanine derivative, a glycine derivative, a histidine derivative, an isoleucine derivative, a lysine derivative, a leucine derivative, a methionine derivative, an asparagine derivative, a proline derivative, a glutamine derivative, an arginine derivative, a serine derivative, a threonine derivative, a valine derivative, a tryptophan derivative, or a tyrosine derivative.

[0035] In some embodiments, the variant IgG Fc polypeptide comprises a variant IgG Fc polypeptide of a companion animal species. In some embodiments, the variant IgG Fc polypeptide comprises a variant canine IgG Fc polypeptide, a variant equine IgG Fc polypeptide, or a feline IgG Fc polypeptide.

[0036] [Exemplary variant IgG Fc polypeptides with modified protein A binding] In some embodiments, the variant IgG Fc polypeptide has a modified protein A binding affinity. In some embodiments, the variant IgG Fc polypeptide has an increased binding affinity to protein A. In some embodiments, the variant IgG Fc polypeptide can be purified using protein A column chromatography.

[0037] In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions at positions corresponding to positions 21, 23, 25, 80, 205, and / or 207 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions at positions corresponding to positions 21, 23, and / or 24 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions at positions corresponding to positions 21, 23, 25, 80, and / or 207 of SEQ ID NO: 6.

[0038] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 15 and / or 203 of SEQ ID NO: 50. In some embodiments, the variant IgG Fc polypeptide comprises a substituted amino acid at a position corresponding to position 199 and / or 200 of SEQ ID NO: 54. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to positions 199, 200, 201, and / or 202 of SEQ ID NO: 55.

[0039] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at positions 21, 23, 25, 80, 205, and / or 207 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at positions 21, 23, and / or 24 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at positions 21, 23, 25, 80, and / or 207 of SEQ ID NO: 6.

[0040] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 15 and / or 203 of SEQ ID NO: 50. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 199 and / or 200 of SEQ ID NO: 54. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at positions 199, 200, 201, and / or 202 of SEQ ID NO: 55.

[0041] In some embodiments, the variant IgG Fc polypeptide comprises threonine at a position corresponding to position 21 of SEQ ID NO: 1, leucine at a position corresponding to position 23 of SEQ ID NO: 1, alanine at a position corresponding to position 25 of SEQ ID NO: 1, glycine at a position corresponding to position 80 of SEQ ID NO: 1, alanine at a position corresponding to position 205 of SEQ ID NO: 1, and / or histidine at a position corresponding to position 207 of SEQ ID NO: 1. In some embodiments, the variant IgG Fc polypeptide comprises threonine at a position corresponding to position 21 of SEQ ID NO: 4, leucine at a position corresponding to position 23 of SEQ ID NO: 4, and / or isoleucine at a position corresponding to position 24 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises threonine at a position corresponding to position 21 of SEQ ID NO: 6, leucine at a position corresponding to position 23 of SEQ ID NO: 6, alanine at a position corresponding to position 25 of SEQ ID NO: 6, glycine at a position corresponding to position 80 of SEQ ID NO: 6, and / or histidine at a position corresponding to position 207 of SEQ ID NO: 6.

[0042] In some embodiments, the variant IgG Fc polypeptide comprises threonine or valine at a position corresponding to position 15 of SEQ ID NO: 50, and / or tyrosine or valine at a position corresponding to position 203 of SEQ ID NO: 50. In some embodiments, the variant IgG Fc polypeptide comprises leucine at a position corresponding to position 199 of SEQ ID NO: 54, and / or histidine at a position corresponding to position 200 of SEQ ID NO: 54. In some embodiments, the variant IgG Fc polypeptide comprises isoleucine at a position corresponding to position 199 of SEQ ID NO: 55, histidine at a position corresponding to position 200 of SEQ ID NO: 55, asparagine at a position corresponding to position 201 of SEQ ID NO: 55, and / or histidine at a position corresponding to position 202 of SEQ ID NO: 55.

[0043] In some embodiments, the variant IgG Fc polypeptide comprises threonine at position 21 of SEQ ID NO: 1, leucine at position 23 of SEQ ID NO: 1, alanine at position 25 of SEQ ID NO: 1, glycine at position 80 of SEQ ID NO: 1, alanine at position 205 of SEQ ID NO: 1, and / or histidine at position 207 of SEQ ID NO: 1. In some embodiments, the variant IgG Fc polypeptide comprises threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, and / or isoleucine at position 24 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises threonine at position 21 of SEQ ID NO: 6, leucine at position 23 of SEQ ID NO: 6, alanine at position 25 of SEQ ID NO: 6, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 6.

[0044] In some embodiments, the variant IgG Fc polypeptide comprises threonine or valine at position 15 of SEQ ID NO: 50, and / or tyrosine or valine at position 203 of SEQ ID NO: 50. In some embodiments, the variant IgG Fc polypeptide comprises leucine at position 199 of SEQ ID NO: 54, and / or histidine at position 200 of SEQ ID NO: 54. In some embodiments, the variant IgG Fc polypeptide comprises isoleucine at position 199 of SEQ ID NO: 55, histidine at position 200 of SEQ ID NO: 55, asparagine at position 201 of SEQ ID NO: 55, and / or histidine at position 202 of SEQ ID NO: 55.

[0045] [Exemplary variant IgG Fc polypeptides with modified CD16 binding] In some embodiments, the variant IgG Fc polypeptide has a modified CD16 binding affinity. In some embodiments, the variant IgG Fc polypeptide has a decreased binding affinity for CD16. In some embodiments, the variant IgG Fc may have a reduced ADCC immunoreactivity.

[0046] In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions at positions corresponding to positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 2. The variant IgG Fc polypeptide comprises amino acid substitutions at positions corresponding to positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 3.

[0047] In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions at positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises amino acid substitutions at positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 4.

[0048] In some embodiments, the variant IgG Fc polypeptide comprises proline at the position corresponding to position 5 of SEQ ID NO: 2, glycine at the position corresponding to position 38, arginine at the position corresponding to position 39, isoleucine at the position corresponding to position 97, and / or glycine at the position corresponding to position 98. In some embodiments, the variant IgG Fc polypeptide comprises proline at the position corresponding to position 5 of SEQ ID NO: 4, glycine at the position corresponding to position 38, arginine at the position corresponding to position 39, isoleucine at the position corresponding to position 97, and / or glycine at the position corresponding to position 98.

[0049] In some embodiments, the variant IgG Fc polypeptide comprises proline at position 5, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises proline at position 5, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98 of SEQ ID NO: 4.

[0050] [Exemplary variant IgG Fc polypeptides with modified C1q binding] In some embodiments, the variant IgG Fc polypeptide has an altered C1q binding affinity. In some embodiments, the variant IgG Fc polypeptide has a reduced binding affinity for C1q. In some embodiments, the variant IgG Fc polypeptide may have reduced complement binding. In some embodiments, the variant IgG Fc may have reduced complement-mediated immune responsiveness.

[0051] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 49. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 52. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 53. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 87 of SEQ ID NO: 56. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0052] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 93 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 93 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 87 of SEQ ID NO: 49. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 87 of SEQ ID NO: 52. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 87 of SEQ ID NO: 53. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 87 of SEQ ID NO: 56. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 198 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0053] In some embodiments, the variant IgG Fc polypeptide comprises arginine at the position corresponding to position 93 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises arginine at the position corresponding to position 93 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises serine at the position corresponding to position 87 of SEQ ID NO: 49. In some embodiments, the variant IgG Fc polypeptide comprises a serine substitution at the position corresponding to position 87 of SEQ ID NO: 52. In some embodiments, the variant IgG Fc polypeptide comprises serine at the position corresponding to position 87 of SEQ ID NO: 53. In some embodiments, the variant IgG Fc polypeptide comprises serine at the position corresponding to position 87 of SEQ ID NO: 56. In some embodiments, the variant IgG Fc polypeptide comprises alanine at the position corresponding to position 198 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0054] In some embodiments, the variant IgG Fc polypeptide comprises arginine at position 93 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 93 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 87 of SEQ ID NO: 49. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 87 of SEQ ID NO: 52. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 87 of SEQ ID NO: 53. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 87 of SEQ ID NO: 56. In some embodiments, the variant IgG Fc polypeptide comprises alanine at position 198 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0055] [Exemplary variant IgG Fc polypeptides having modified interchain disulfide bond properties] In some embodiments, the variant feline IgG Fc polypeptide has at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide. In some embodiments, the variant feline IgG Fc polypeptide has at least one additional interchain disulfide bond in the hinge region. In some embodiments, the variant feline IgG2 Fc polypeptide having at least one additional interchain disulfide bond has increased interchain stability relative to the wild-type feline IgG Fc polypeptide. In some embodiments, the variant IgG polypeptide has at least one amino acid modification in the hinge region relative to the wild-type IgG Fc polypeptide. In some embodiments, the wild-type IgG Fc polypeptide is a wild-type feline or equine IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide comprises a hinge region or a portion of the hinge region from an IgG Fc polypeptide of a different isotype. In some embodiments, the variant IgG Fc polypeptide comprises a hinge region from a wild-type feline IgG-1a Fc polypeptide, a wild-type feline IgG-1b Fc polypeptide, or a wild-type equine IgG1 Fc polypeptide. In some embodiments, the variant IgG2 Fc polypeptide has increased recombinant productivity and / or increased hinge disulfide-forming ability relative to the wild-type IgG Fc polypeptide. In some embodiments, the increased recombinant production and / or increased hinge disulfide formation can be determined by SDS-PAGE analysis under reducing and / or non-reducing conditions.

[0056] In some embodiments, the variant IgG Fc polypeptide comprises a cysteine at a position corresponding to positions 8, 9, 10, 11, 12, 13, 14, 15, or 16 of SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises a cysteine at positions 8, 9, 10, 11, 12, 13, 14, 15, or 16 of SEQ ID NO: 69.

[0057] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 3 and / or position 20 of SEQ ID NO: 51.

[0058] In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 3 and / or position 20 of SEQ ID NO: 51.

[0059] In some embodiments, the variant IgG Fc polypeptide comprises a proline at a position corresponding to position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises a serine at a position corresponding to position 3 of SEQ ID NO: 51 and / or a proline at a position corresponding to position 20 of SEQ ID NO: 51.

[0060] In some embodiments, the variant IgG Fc polypeptide comprises a proline at position 16 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises a serine at position 3 of SEQ ID NO: 51 and / or a proline at position 20 of SEQ ID NO: 51.

[0061] [Exemplary Variant IgG Fc Polypeptides for Multimeric Polypeptides] In certain embodiments, the multimeric polypeptide provided herein is a bispecific antibody. A bispecific antibody has binding specificity for two different epitopes or target molecules. In some embodiments, the bispecific antibody binds to two different epitopes of the same target molecule. The bispecific antibody can be a full-length antibody or an antibody fragment.

[0062] In some embodiments, the multimeric polypeptide comprises 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, A. M. et al., An efficient route to human bispecific IgG. Nat Biotechnol, 16(7):677-81 (1998).

[0063] In some embodiments, the variant canine or variant feline IgG Fc polypeptide comprises a knob mutation. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at a position corresponding to position 138 of SEQ ID NO: 1. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at a position corresponding to position 137 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at a position corresponding to position 137 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at a position corresponding to position 138 of SEQ ID NO: 6. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at a position corresponding to position 154 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

[0064] In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at position 138 of SEQ ID NO: 1. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at position 137 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at position 137 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at position 138 of SEQ ID NO: 6. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at position 154 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises tyrosine or tryptophan at position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

[0065] In some embodiments, the variant canine or variant feline IgG Fc polypeptide comprises a whole mutation. In some embodiments, the variant IgG Fc polypeptide comprises serine at a position corresponding to position 138 of SEQ ID NO: 1, alanine at a position corresponding to position 140, and / or threonine at a position corresponding to position 181. In some embodiments, the variant IgG Fc polypeptide comprises serine at a position corresponding to position 137 of SEQ ID NO: 2, alanine at a position corresponding to position 139, and / or threonine at a position corresponding to position 180. In some embodiments, the variant IgG Fc polypeptide comprises serine at a position corresponding to position 137 of SEQ ID NO: 4, alanine at a position corresponding to position 139, and / or threonine at a position corresponding to position 180. In some embodiments, the variant IgG Fc polypeptide comprises serine at a position corresponding to position 138 of SEQ ID NO: 6, alanine at a position corresponding to position 140, and / or threonine at a position corresponding to position 181. In some embodiments, the variant IgG Fc polypeptide comprises serine at a position corresponding to position 154 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69, alanine at a position corresponding to position 156, and / or threonine at a position corresponding to position 197. In some embodiments, the variant IgG Fc polypeptide comprises serine at a position corresponding to position 131 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, alanine at a position corresponding to position 133, and / or threonine at a position corresponding to position 174.

[0066] In some embodiments, the variant IgG Fc polypeptide comprises serine at position 138, alanine at position 140, and / or threonine at position 181 of SEQ ID NO: 1. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 137, alanine at position 139, and / or threonine at position 181 of SEQ ID NO: 2. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 137, alanine at position 139, and / or threonine at position 181 of SEQ ID NO: 4. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 138, alanine at position 140, and / or threonine at position 181 of SEQ ID NO: 6. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 154, alanine at position 156, and / or threonine at position 197 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the variant IgG Fc polypeptide comprises serine at position 131, alanine at position 133, and / or threonine at position 174 of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

[0067] In some embodiments, the adjacent polypeptide comprises a first therapeutic polypeptide or a first antibody and a variant canine, feline, or equine IgG Fc polypeptide comprising a knob mutation. In some embodiments, the adjacent polypeptide comprises a second therapeutic polypeptide or a second antibody and a variant canine, feline, or equine IgG Fc polypeptide comprising a hole mutation.

[0068] [Exemplary Therapeutic Polypeptides and Antibodies] The "extracellular domain" ("ECD") is the portion of a polypeptide that extends into the extracellular space beyond the transmembrane domain. As used herein, the term "extracellular domain" can include the complete extracellular domain or a truncated extracellular domain lacking one or more amino acids that binds to its ligand. The composition of the extracellular domain can depend on the algorithm used to determine which amino acids are in the membrane. Different algorithms can predict different extracellular domains of a given protein, and different systems can express them.

[0069] As used herein, "therapeutic polypeptide" includes, unless otherwise specified, a polypeptide comprising all or a portion of an identified polypeptide from any vertebrate, 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).

[0070] As used herein, the term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (e.g., bispecific T cell engagers) and trispecific antibodies), and antibody fragments (such as Fab, F(ab’)2, ScFv, minibodies, diabodies, triabodies, and tetrabody) as long as they exhibit the desired antigen-binding activity. Dog, cat, and horse species have different classes of antibodies that are shared by many mammals.

[0071] The term antibody includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, di-scFv, sdAb (single domain antibody), and (Fab’)2 (including chemically conjugated F(ab’)2). Papain digestion of an antibody produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and the remaining “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab’)2 fragment that has two antigen-binding sites and can still cross-link antigens. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, cynomolgus monkey, dog, cat, horse, etc. Furthermore, for all antibody constructs provided herein, variants having sequences from other organisms are also contemplated. Thus, when a mouse form of an antibody is disclosed, one of ordinary skill in the art will know how to convert an antibody based on a mouse sequence to a sequence from a cat, dog, horse, etc. Antibody fragments also include single-chain scFv, tandem di-scFv, diabody, tandem tri-sdcFv, minibody, etc. in any orientation. Antibody fragments also include nanobodies (antibodies having a single monomeric domain such as sdAb, variable domain pair of a heavy chain without a light chain). Antibody fragments can be said to be of a particular species in some embodiments (e.g., mouse scFv or dog scFv). This indicates the sequence of at least a portion of the non-CDR regions, rather than the construct source. In some embodiments, the antibody includes a label or is conjugated to a second moiety.

[0072] In some embodiments, the therapeutic polypeptide is an NGF (or nerve growth factor) polypeptide, a receptor for the NGF polypeptide (e.g., the ECD of the receptor for the NGF polypeptide), a TrkA polypeptide (e.g., the ECD of the TrkA polypeptide), an LNGFR polypeptide (e.g., the ECD of the LNGFR polypeptide), a TNFα (or tumor necrosis factor alpha) polypeptide, a receptor for the TNFα polypeptide, a TNFR (or tumor necrosis factor receptor) polypeptide (e.g., the ECD of the TNFR polypeptide), a TNFR1 polypeptide (e.g., the ECD of the TNFR1 polypeptide), a TNFR2 polypeptide (e.g., the ECD of the TNFR2 polypeptide), an IL5 (or interleukin 5) polypeptide, a receptor for the IL5 polypeptide, an IL5R (or interleukin 5 receptor) polypeptide (e.g., the ECD of the IL5R polypeptide), an IL5Rα polypeptide (e.g., the ECD of the IL5Rα polypeptide), an IL6 (or interleukin 6) polypeptide, a receptor for the IL6 polypeptide, an IL6R (or interleukin 6 receptor) polypeptide (e.g., the ECD of the IL6R polypeptide), an IL17 (or interleukin 17) polypeptide, a receptor for the IL17 polypeptide, an IL17R (or interleukin 17 receptor) polypeptide (e.g., the ECD of the IL17R polypeptide), an IL17RA polypeptide (e.g., the ECD of the IL17RA polypeptide), an IL17RB polypeptide (e.g., the ECD of the IL17RB polypeptide), an IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), an IL23 (or interleukin 23) polypeptide, a receptor for the IL23 polypeptide, an IL23R (or interleukin 23 receptor) polypeptide (e.g., the ECD of the IL23R polypeptide), an IL12Rβ1 polypeptide (e.g., the ECD of the IL12Rβ1 polypeptide), a PDL (or programmed cell death ligand) polypeptide, a PDL1 polypeptide, a receptor for the PDL1 polypeptide, a PDL2 polypeptide, a receptor for the PDL2 polypeptide, a PD1 polypeptide (e.g., the ECD of the PD1 polypeptide), an integrin polypeptide (e.g., ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10,ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, or ITGB8 polypeptide), a receptor for an integrin polypeptide, a fibronectin polypeptide (e.g., the ECD of a fibronectin polypeptide), a vitronectin polypeptide (e.g., the ECD of a vitronectin polypeptide), a collagen polypeptide (e.g., the ECD of a collagen polypeptide), a laminin polypeptide (e.g., the ECD of a laminin polypeptide), a CD80 polypeptide, a receptor for a CD80 polypeptide, a CD86 polypeptide, a receptor for a CD86 polypeptide, CTLA-4 (or cytotoxic T lymphocyte-associated protein 4) polypeptide (e.g., the ECD of a CTLA-4 polypeptide), a B7-H3 polypeptide, a receptor for a B7-H3 polypeptide (e.g., the ECD of a receptor for a B7-H3 polypeptide), LAG-3 (or lymphocyte activation gene 3) polypeptide (e.g., the ECD of a LAG-3 polypeptide), IL31 (or interleukin 31) polypeptide, a receptor for an IL31 polypeptide, an IL31RA polypeptide (e.g., the ECD of an IL31RA polypeptide), OSMR (or oncostatin M receptor) polypeptide (e.g., the ECD of an OSMR polypeptide), IL4 (or interleukin 4) polypeptide, a receptor for an IL4R polypeptide, an IL4R (or interleukin 4 receptor) polypeptide (e.g., the ECD of an IL4R polypeptide), an IL13 (or interleukin 13 receptor) polypeptide, a receptor for an IL13 polypeptide, an IL13RA1 (or interleukin 13 receptor A1) polypeptide (e.g., the ECD of an IL13RA1 polypeptide), an IL4R (or interleukin 4 receptor) polypeptide (e.g., the ECD of an IL4R polypeptide), an IL13Rα2 (or interleukin 13 receptor α2) polypeptide (e.g., the ECD of an IL13Rα2 polypeptide), an IL22 (or interleukin 22) polypeptide, a receptor for an IL22 polypeptide (e.g., the ECD of an IL22 polypeptide), an IL22Rα1 (or interleukin 22 receptor α1) polypeptide (e.g., the ECD of an IL22Rα1 polypeptide),IL10Rβ2 (or interleukin 10 receptor β2) polypeptide (e.g., ECD of IL10Rβ2 polypeptide), IL33 (or interleukin 33) polypeptide, receptor of IL33 polypeptide, IL1RL1 polypeptide (e.g., ED of IL1RL1 polypeptide), EGF (or epidermal growth factor) polypeptide, receptor of EGF polypeptide, TGFα (or transforming growth factor α) polypeptide, receptor of TGFα polypeptide, EGFR (or epidermal growth factor receptor) polypeptide (e.g., ECD of EGFR polypeptide), MMP9 (or matrix metallopeptidase 9) polypeptide, FGF (or fibroblast growth factor) polypeptide (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23 polypeptide), receptor of FGF polypeptide, FGFR (or fibroblast growth factor receptor) polypeptide (e.g., FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptide), ECD of FGFR polypeptide (e.g., ECD of FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptide), EGF (or epidermal growth factor) polypeptide, receptor of EGF polypeptide, neuregulin polypeptide (e.g., neuregulin isoform I, II, III, IV, V, or VI polypeptide), receptor of neuregulin polypeptide, HER (human epidermal growth factor receptor) polypeptide (e.g., HER1, HER2, HER3, or HER4 polypeptide), ECD of HER polypeptide (e.g., ECD of HER1, HER2, HER3, or HER4 polypeptide), EpCAM (or epithelial cell adhesion molecule) polypeptide (e.g., ECD of EpCAM polypeptide), CD20 polypeptide (e.g., ECD of CD20 polypeptide), ligand of CD20 polypeptide, CD19 polypeptide (e.g., ECD of CD19 polypeptide), ligand of CD19 polypeptide, CGRP (or calcitonin gene-related peptide) polypeptide (e.g.,α-CGRP polypeptide or β-CGRP polypeptide), the receptor of CGRP polypeptide, the receptor of α-CGRP polypeptide, the receptor of β-CGRP polypeptide, CALCRL (or calcitonin receptor-like) polypeptide (e.g., the ECD of CALCRL polypeptide), RAMP (or receptor activity-modifying protein) polypeptide (e.g., RAMP1, RAMP2, or RAMP3 polypeptide), the ECD of RAMP polypeptide (e.g., the ECD of RAMP1, RAMP2, or RAMP3 polypeptide), IGF (or insulin-like growth factor) polypeptide (e.g., IGF-1 or IGF-2 polypeptide), the receptor of IGF polypeptide (e.g., the receptor of IGF-1 or IGF-2 polypeptide), IGFR (or insulin-like growth factor receptor) polypeptide (e.g., IGFR1 or IGFR2 polypeptide), the ECD of IGFR polypeptide (e.g., the ECD of IGFR1 or IGFR2 polypeptide), IGFBP (or insulin-like growth factor binding protein) polypeptide (e.g., IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6 polypeptide), VEGF (or vascular endothelial growth factor) polypeptide (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptide), the receptor of VEGF polypeptide (e.g., the receptor of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF (or placental growth factor) polypeptide), VEGFR (or vascular endothelial growth factor receptor) polypeptide (e.g., VEGFR1, VEGFR2, or VEGFR3 polypeptide), the ECD of VEGFR polypeptide (e.g., the ECD of VEGFR1, VEGFR2, or VEGFR3 polypeptide), FLT1 (or FMS-like tyrosine kinase 1) receptor polypeptide (e.g., the ECD of FLT1 receptor polypeptide), IL36 (or interleukin 36) polypeptide (e.g., IL36A, IL36B, or IL36G polypeptide), the receptor of IL36 polypeptide (e.g., the receptor of IL36A, IL36B, or IL36G polypeptide), IL36R (or interleukin 36 receptor) polypeptide (e.g., the ECD of IL36R polypeptide), IL1R1 polypeptide (e.g.,The ECD of the IL1R1 polypeptide), the IL1R2 polypeptide (e.g., the ECD of the IL1R2 polypeptide), the IL1RL1 polypeptide (the ECD of the IL1RL1 polypeptide), the IL18R1 polypeptide (the ECD of the IL18R1 polypeptide), a bacterial toxin polypeptide, an exotoxin polypeptide, an endotoxin polypeptide, a botulinum neurotoxin polypeptide, a tetanus toxin polypeptide, a staphylococcal toxin polypeptide, the CD52 polypeptide (e.g., the ECD of the CD52 polypeptide), a ligand of the CD52 polypeptide, the SIGLEC10 (or sialic acid-binding Ig-like lectin 10) polypeptide, the PCSK9 (or proprotein convertase subtilisin / kexin type 9) polypeptide, a receptor of the PCSK9 polypeptide, the LDLR (or low density lipoprotein receptor) polypeptide (e.g., the ECD of the LDLR polypeptide), the CEA (or carcinoembryonic antigen) polypeptide (e.g., the CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), the ECD of the CEA polypeptide (e.g., the ECD of the CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), the BAFF (or B cell activating factor) polypeptide, a receptor of the BAFF polypeptide, the TRAF (or TNF receptor-associated factor) polypeptide (e.g., the TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRAF7 polypeptide), Receptors for TRAF polypeptides (e.g., receptors for TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 polypeptides), BCMA polypeptides, ECD of BCMA (or B cell maturation antigen) polypeptides, SOST polypeptides, receptors for SOST (or sclerostin) polypeptides, LRP (or low density lipoprotein receptor-related protein) polypeptides (e.g., LRP5 or LRP6 polypeptides), ECD of LRP polypeptides (e.g., ECD of LRP5 or LRP6 polypeptides), DLL (or delta-like) polypeptides (e.g., DLL4 polypeptides), receptors for DLL polypeptides, Jagged polypeptides (e.g., JAG1 or JAG polypeptides), receptors for Jagged polypeptides (e.g., receptors for JAG1 or JAG polypeptides), NOTCH polypeptides (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptides), ligands for NOTCH polypeptides (e.g., ligands for NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptides), VWF (or von Willebrand factor) polypeptides, receptors for VWF polypeptides, factor VIII polypeptides, receptors for factor VIII polypeptides, platelet GP1b receptor polypeptides (e.g., ECD of platelet GP1b receptor polypeptides), integrin α IIb β 3 polypeptides (e.g., integrin α IIb β 3ECD of a polypeptide), IL2 (or interleukin 2) polypeptide, receptor of IL2 polypeptide, IL2R (or interleukin 2 receptor) polypeptide (e.g., IL2Rα, IL2Rβ, or IL2Rγ polypeptide), ECD of IL2R polypeptide (e.g., ECD of IL2Rα, IL2Rβ, or IL2Rγ polypeptide), TGFβ (or transforming growth factor β) polypeptide, receptor of TGFβ polypeptide, decorin polypeptide, EIF3I (or eukaryotic translation initiation factor 3 subunit 1) polypeptide, LTBP1 (or latent transforming growth factor beta-binding protein 1) polypeptide, TGFβR1 polypeptide (e.g., ECD of TGFβR1 polypeptide), YWHAE polypeptide, IgE polypeptide, receptor or IgE polypeptide, Fc receptor polypeptide (e.g., FcεRI or FcεRII polypeptide), ECD of Fc receptor polypeptide (e.g., ECD of FcεRI or FcεRII polypeptide), KLK (or kallikrein) polypeptide (e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, or KLK15 polypeptide), Rank1 (or receptor activator of nuclear factor kappa-B ligand) polypeptide, receptor of Rank1 polypeptide, RANK (or receptor activator of nuclear factor kappa-B ligand) polypeptide (e.g., ECD of RANK polypeptide), TSLP (or thymic stromal lymphopoietin) polypeptide, receptor of TSLP polypeptide, CRLF2 (or cytokine receptor-like factor 2) polypeptide (e.g., ECD of CRLF2 polypeptide), IL7Rα polypeptide (e.g., ECD of IL7Rα polypeptide), S1P (or specificity protein 1) polypeptide, CD3 polypeptide (e.g., CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), ECD of CD3 polypeptide (e.g., ECD of CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD28 polypeptide (e.g., ECD of CD28 polypeptide),CTLA-4 (or cytotoxic T lymphocyte-associated protein 4) polypeptide (e.g., the ECD of CTLA-4 polypeptide), GnRH (or gonadotropin-releasing hormone) polypeptide, the receptor of GnRH polypeptide, GnRHR (or gonadotropin-releasing hormone receptor) polypeptide (e.g., the ECD of GnRHR polypeptide), ICAM (or intercellular adhesion molecule) polypeptide (e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), the receptor of ICAM polypeptide (e.g., the receptor of ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), JAM-A polypeptide, the receptor of JAM-A polypeptide, LFA-1 polypeptide (e.g., the ECD of LFA-1 polypeptide), Na, v 1.7 polypeptide, C5 (or complement component 5) polypeptide (e.g., C5a or C5b polypeptide), the receptor of C5 polypeptide (e.g., the receptor of C5a or C5b polypeptide), C5aR polypeptide (e.g., the ECD of C5aR polypeptide), C5L2 polypeptide (e.g., the ECD of C5L2 polypeptide), IL17 polypeptide, the receptor of IL17 polypeptide, IL17Ra polypeptide (e.g., the ECD of IL17Ra polypeptide), IL17RC polypeptide (e.g., the ECD of IL17RC polypeptide), EPO polypeptide, somatostatin polypeptide, GLP1 polypeptide, glucagon polypeptide, and so on.

[0073] In some embodiments, the antibody recognizes one or more of the following polypeptides: NGF polypeptide, a receptor for the NGF polypeptide (e.g., the ECD of a receptor for the NGF polypeptide), TrkA polypeptide (e.g., the ECD of the TrkA polypeptide), LNGFR polypeptide (e.g., the ECD of the LNGFR polypeptide), TNFα polypeptide, a receptor for the TNFα polypeptide, TNFR polypeptide (e.g., the ECD of the TNFR polypeptide), TNFR1 polypeptide (e.g., the ECD of the TNFR1 polypeptide), TNFR2 polypeptide (e.g., the ECD of the TNFR2 polypeptide), IL5 polypeptide, a receptor for the IL5 polypeptide, IL5R polypeptide (e.g., the ECD of the IL5R polypeptide), IL5Rα polypeptide (e.g., the ECD of the IL5Rα polypeptide), IL6 polypeptide, a receptor for the IL6 polypeptide, IL6R polypeptide (e.g., the ECD of the IL6R polypeptide), IL17 polypeptide, a receptor for the IL17 polypeptide, IL17R polypeptide (e.g., the ECD of the IL17R polypeptide), IL17RA polypeptide (e.g., the ECD of the IL17RA polypeptide), IL17RB polypeptide (e.g., the ECD of the IL17RB polypeptide), IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), IL23 polypeptide, a receptor for the IL23 polypeptide, IL23R polypeptide (e.g., the ECD of the IL23R polypeptide), IL12Rβ1 polypeptide (e.g., the ECD of the IL12Rβ1 polypeptide), PDL1 polypeptide, a receptor for the PDL1 polypeptide, PDL2 polypeptide, a receptor for the PDL2 polypeptide, PD1 polypeptide (e.g., the ECD of the PD1 polypeptide), integrin polypeptide (e.g., ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, or ITGB8 polypeptide), a receptor for the integrin polypeptide, fibronectin polypeptide (e.g.,Fibronectin polypeptide (e.g., ECD of fibronectin polypeptide), vitronectin polypeptide (e.g., ECD of vitronectin polypeptide), collagen polypeptide (e.g., ECD of collagen polypeptide), laminin polypeptide (e.g., ECD of laminin polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD86 polypeptide, receptor of CD86 polypeptide, CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), B7-H3 polypeptide, receptor of B7-H3 polypeptide (e.g., ECD of receptor of B7-H3 polypeptide), LAG-3 polypeptide (e.g., ECD of LAG-3 polypeptide), IL31 polypeptide, receptor of IL31 polypeptide, IL31RA polypeptide (e.g., ECD of IL31RA polypeptide), OSMR polypeptide (e.g., ECD of OSMR polypeptide), IL4 polypeptide, receptor of IL4R polypeptide, IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13 polypeptide, receptor of IL13 polypeptide, IL13RA1 polypeptide (e.g., ECD of IL13RA1 polypeptide), IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13Rα2 polypeptide (e.g., ECD of IL13Rα2 polypeptide), IL22 polypeptide, receptor of IL22 polypeptide (e.g., ECD of IL22 polypeptide), IL22Rα1 polypeptide (e.g., ECD of IL22Rα1 polypeptide), IL10Rβ2 polypeptide (e.g., ECD of IL10Rβ2 polypeptide), IL33 polypeptide, receptor of IL33 polypeptide, IL1RL1 polypeptide (e.g., ECD of IL1RL1 polypeptide), EGF polypeptide, receptor of EGF polypeptide, TGFα polypeptide, receptor of TGFα polypeptide, EGFR polypeptide (e.g., ECD of EGFR polypeptide), MMP9 polypeptide, FGF polypeptide (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23 polypeptide),Receptors for FGF polypeptides, FGFR polypeptides (e.g., FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), ECDs of FGFR polypeptides (e.g., ECDs of FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), EGF polypeptides, receptors for EGF polypeptides, neuregulin polypeptides (e.g., neuregulin isoform I, II, III, IV, V, or VI polypeptides), receptors for neuregulin polypeptides, HER polypeptides (e.g., HER1, HER2, HER3, or HER4 polypeptides), ECDs of HER polypeptides (e.g., ECDs of HER1, HER2, HER3, or HER4 polypeptides), EpCAM polypeptides (e.g., ECDs of EpCAM polypeptides), CD20 polypeptides (e.g., ECDs of CD20 polypeptides), ligands for CD20 polypeptides, CD19 polypeptides (e.g., ECDs of CD19 polypeptides), ligands for CD19 polypeptides, CGRP polypeptides (e.g., α-CGRP polypeptide or β-CGRP polypeptide), receptors for CGRP polypeptides, receptors for α-CGRP polypeptide, receptors for β-CGRP polypeptide, CALCRL polypeptides (e.g., ECDs of CALCRL polypeptides), RAMP polypeptides (e.g., RAMP1, RAMP2, or RAMP3 polypeptides), ECDs of RAMP polypeptides (e.g., ECDs of RAMP1, RAMP2, or RAMP3 polypeptides), IGF polypeptides (e.g., IGF-1 or IGF-2 polypeptides), receptors for IGF polypeptides (e.g., receptors for IGF1 or IGF-2 polypeptides), IGFR polypeptides (e.g., IGFR1 or IGFR2 polypeptides), ECDs of IGFR polypeptides (e.g., ECDs of IGFR1 or IGFR2 polypeptides), IGFBP polypeptides (e.g., IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6 polypeptides), VEGF polypeptides (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides), receptors for VEGF polypeptides (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D,or a receptor for a PGF polypeptide), a VEGFR polypeptide (e.g., a VEGFR1, VEGFR2, or VEGFR3 polypeptide), an ECD of a VEGFR polypeptide (e.g., an ECD of a VEGFR1, VEGFR2, or VEGFR3 polypeptide), an FLT1 receptor polypeptide (e.g., an ECD of an FLT1 receptor polypeptide), an IL36 polypeptide (e.g., an IL36A, IL36B, or IL36G polypeptide), a receptor for an IL36 polypeptide (e.g., a receptor for an IL36A, IL36B, or IL36G polypeptide), an IL36R polypeptide (e.g., an ECD of an IL36R polypeptide), an IL1R1 polypeptide (e.g., an ECD of an IL1R1 polypeptide), an IL1R2 polypeptide (e.g., an ECD of an IL1R2 polypeptide), , IL1RL1 polypeptide (ECD of IL1RL1 polypeptide), IL18R1 polypeptide (ECD of IL18R1 polypeptide), bacterial toxin polypeptide, exotoxin polypeptide, endotoxin polypeptide, botulinum neurotoxin polypeptide, tetanus toxin polypeptide, staphylococcal toxin polypeptide, CD52 polypeptide (e.g., ECD of CD52 polypeptide), ligand of CD52 polypeptide, SIGLEC10 polypeptide, PCSK9 polypeptide, receptor of PCSK9 polypeptide, LDLR polypeptide (e.g., ECD of LDLR polypeptide), CEA polypeptide (e.g., CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), ECD of CEA polypeptide (e.g., ECD of CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), BAFF polypeptide, receptor of BAFF polypeptide, TRAF polypeptide (e.g., TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRAF7 polypeptide), receptor of TRAF polypeptide (e.g., receptor of TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 polypeptide), BCMA polypeptide, ECD of BCMA polypeptide, SOST polypeptide, receptor of SOST polypeptide, LRP polypeptide (e.g., LRP5 or LRP6 polypeptide), ECD of LRP polypeptide (e.g., ECD of LRP5 or LRP6 polypeptide), DLL polypeptide (e.g., DLL4 polypeptide), receptor of DLL polypeptide, Jagged polypeptide (e.g., JAG1 or JAG polypeptide), receptor of Jagged polypeptide (e.g., receptor of JAG1 or JAG polypeptide), NOTCH polypeptide (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), ligand of NOTCH polypeptide (e.g., ligand of NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), VWF polypeptide, receptor of VWF polypeptide, Factor VIII polypeptide, receptor of Factor VIII polypeptide, platelet GP1b receptor polypeptide (e.g., ECD of platelet GP1b receptor polypeptide), integrin αIIb β 3 polypeptide (for example, integrin α IIb β 3ECD of polypeptide), IL2 polypeptide, receptor of IL2 polypeptide, IL2R polypeptide (e.g., IL2Rα, IL2Rβ, or IL2Rγ polypeptide), ECD of IL2R polypeptide (e.g., ECD of IL2Rα, IL2Rβ, or IL2Rγ polypeptide), TGFβ polypeptide, receptor of TGFβ polypeptide, decorin polypeptide, EIF3I polypeptide, LTBP1 polypeptide, TGFβR1 polypeptide (e.g., ECD of TGFβR1 polypeptide), YWHAE polypeptide, IgE polypeptide, receptor or IgE polypeptide, Fc receptor polypeptide (e.g., FcεRI or FcεRII polypeptide), ECD of Fc receptor polypeptide (e.g., ECD of FcεRI or FcεRII polypeptide), KLK polypeptide (e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, or KLK15 polypeptide), Rank1 polypeptide, receptor of Rank1 polypeptide, RANK polypeptide (e.g., ECD of RANK polypeptide), TSLP polypeptide, receptor of TSLP polypeptide, CRLF2 polypeptide (e.g., ECD of CRLF2 polypeptide), IL7Rα polypeptide (e.g., ECD of IL7Rα polypeptide), S1P polypeptide, CD3 polypeptide (e.g., CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), ECD of CD3 polypeptide (e.g., ECD of CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD28 polypeptide (e.g., ECD of CD28 polypeptide), CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), GnRH polypeptide, receptor of GNRH polypeptide, GnRHR polypeptide (e.g., ECD of GnRHR polypeptide), ICAM polypeptide (e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), receptor of ICAM polypeptide (e.g., receptor of ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide),JAM-A polypeptide, a receptor for the JAM-A polypeptide, an LFA-1 polypeptide (e.g., the ECD of the LFA-1 polypeptide), Na, v 1.7 polypeptide, C5 polypeptide (e.g., C5a or C5b polypeptide), a receptor for the C5 polypeptide (e.g., a receptor for the C5a or C5b polypeptide), C5aR polypeptide (e.g., the ECD of the C5aR polypeptide), C5L2 polypeptide (e.g., the ECD of the C5L2 polypeptide), IL17 polypeptide, a receptor for the IL17 polypeptide, IL17Ra polypeptide (e.g., the ECD of the IL17Ra polypeptide), IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), EPO polypeptide, somatostatin polypeptide, GLP1 polypeptide, glucagon polypeptide, and the like.

[0074] [Exemplary variant IgG Fc polypeptides and fusion molecules] Polypeptides and other molecules may include variant IgG Fc polypeptides. In some embodiments, the fusion molecule includes a variant IgG Fc polypeptide such as the variant IgG Fc polypeptides described herein. In some embodiments, the antibody or antibody fragment includes a variant IgG Fc polypeptide such as the variant IgG Fc polypeptides described herein.

[0075] As used herein, a "fusion molecule" refers to a molecule that includes one or more "fusion partners". In some embodiments, the fusion partners are covalently bound ( "fused"). If both of the two fusion partners are polypeptides, the fusion partner polypeptides can be part of an adjacent amino acid sequence (i.e., an adjacent polypeptide). The first fusion partner polypeptide can be bound to either the N-terminus or the C-terminus of the second fusion partner. In some embodiments, the fusion partners are translated as a single polypeptide from a coding sequence encoding both fusion partners. The fusion partners can be covalently bound via other means, such as a chemical bond other than a peptide bond, for example. Many known methods for covalently attaching a polypeptide to another molecule (e.g., a fusion partner) can be used. In other embodiments, the fusion partners are fused via a "linker" composed of at least one amino acid or chemical moiety. In some embodiments, the fusion partners are non-covalently bound. In some such embodiments, they can be bound, for example, using a binding pair. Exemplary binding pairs include, but are not limited to, biotin and avidin or streptavidin, an antibody and its antigen, and the like.

[0076] In some embodiments, the fusion partner includes an IgG Fc polypeptide and at least one therapeutic polypeptide and / or antibody. In some embodiments, the fusion partner includes an IgG Fc polypeptide, a first therapeutic polypeptide or antibody, and a second therapeutic polypeptide or antibody. In some embodiments, the therapeutic polypeptide can be bound to either the N-terminus or the C-terminus of the IgG Fc polypeptide. In some embodiments, the antibody can be bound to either the N-terminus or the C-terminus of the IgG Fc polypeptide.

[0077] As used herein, the term "adjacent polypeptide" is used to mean an uninterrupted amino acid sequence. An adjacent 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 in-frame the DNA sequence of a second protein, such that the DNA sequence is expressed as a single protein. Typically, this is accomplished by cloning the cDNA into an expression vector in-frame with an existing gene.

[0078] A "linker" refers to one or more amino acid residues that link a first polypeptide to a second polypeptide.

[0079] In some embodiments, the linker is a flexible unstructured linker. In some embodiments, the linker is a glycine-rich, serine-rich, or glycine-serine-rich linker. In some embodiments, the linker comprises 100%, at least 95%, at least 90%, or at least 85% serine and / or glycine amino acid residues.

[0080] As used herein, "extension" refers to one or more amino acid residues linked to the C-terminus or N-terminus of a polypeptide.

[0081] In some embodiments, the extension is flexible. In some embodiments, the extension adds flexibility to the polypeptide without interfering with the biological activity of the polypeptide. In some embodiments, the extension increases the solubility of the polypeptide. In some embodiments, the extension comprises one or more glycine residues. In some embodiments, the extension comprises one glycine residue (SEQ ID NO: 88), two glycine residues (SEQ ID NO: 89), three glycine residues (SEQ ID NO: 90), four glycine residues (SEQ ID NO: 91), five glycine residues (SEQ ID NO: 92), six glycine residues (SEQ ID NO: 93), seven glycine residues (SEQ ID NO: 94), eight glycine residues (SEQ ID NO: 95), or more glycine residues.

[0082] In some embodiments, the adjacent polypeptide comprises an IgG Fc polypeptide comprising any one amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, and a GLP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 85.In some embodiments, the adjacent polypeptide comprises an IgG Fc polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, and a GLP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 86.In some embodiments, the adjacent polypeptide comprises an IgG Fc polypeptide comprising any one amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, and a GLP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 87.In some embodiments, the adjacent polypeptide comprises an IgG Fc polypeptide comprising any one amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, and a GLP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 98.In some embodiments, the adjacent polypeptide comprises an IgG Fc polypeptide comprising any one amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, and a GLP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 99.

[0083] In some embodiments, the adjacent polypeptide comprises an IgG Fc polypeptide comprising any one amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, and a glucagon polypeptide comprising the amino acid sequence of SEQ ID NO: 21.

[0084] In some embodiments, the adjacent polypeptide is Formula (I): TPA1-L1-Fc; Formula (II): Fc-L1-TPA1; Formula (III): TPA1-L1-Fc-L2-TPA2; Formula (IV): TPA1-L1-TPA2-L2-Fc; or Formula (V): Fc-L1-TPA1-L2-TPA2 comprising, wherein TPA1 is a first therapeutic polypeptide and / or antibody, TPA2 is a second therapeutic polypeptide and / or antibody (e.g., the same therapeutic polypeptide, a different therapeutic polypeptide, the same antibody, or a different antibody), L1 and L2 are any linkers; Fc is a variant IgG Fc polypeptide of a companion animal species. Optionally, adjacent polypeptides may include a signal sequence. The constructs of Formulas I-V may include TPA3, TPA4, TPA5, etc. after or before any TPA1 or TPA2. TPA3, TPA4, TPA5, etc. are third, fourth, fifth, etc. therapeutic polypeptides and / or antibodies (e.g., the same therapeutic polypeptide, a different therapeutic polypeptide, the same antibody, or a different antibody).

[0085] In some embodiments, the Fc polypeptide is human IgG Fc. In some embodiments, the Fc polypeptide is human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In some embodiments, the Fc polypeptide is a variant human IgG Fc.

[0086] In some embodiments, the Fc polypeptide is IgG Fc derived from a companion animal. In some embodiments, the Fc polypeptide is canine IgG-A Fc, canine IgG-B Fc, canine IgG-C Fc, canine IgG-D Fc. In some embodiments, Fc is equine IgG1 Fc, equine IgG2 Fc, equine IgG3 Fc, equine IgG4 Fc, equine IgG5 Fc, equine IgG6 Fc, or equine IgG7 Fc. In some embodiments, Fc is feline IgG1a Fc, feline IgG1b Fc, or feline IgG2 Fc.

[0087] In some embodiments, the Fc polypeptide is a variant IgG Fc. In some embodiments, the FC polypeptide is a variant canine IgG-A Fc, variant canine IgG-B Fc, variant canine IgG-C Fc, variant canine IgG-D Fc. In some embodiments, the Fc is a variant equine IgG1 Fc, variant equine IgG2 Fc, variant equine IgG3 Fc, variant equine IgG4 Fc, variant equine IgG5 Fc, variant equine IgG6 Fc, or variant equine IgG7 Fc. In some embodiments, the Fc is a variant feline IgG1a Fc, variant feline IgG1b Fc, or variant feline IgG2 Fc.

[0088] In some embodiments, L1 and L2, when present, are each independently a flexible linker. In some embodiments, the amino acid sequences of L1 and L2, when present, each independently contain 100%, at least 95%, at least 90%, or at least 85% serine and / or glycine amino acid residues.

[0089] In some embodiments, the adjacent polypeptide contains an extension at its C-terminus. In some embodiments, the adjacent polypeptide contains one glycine residue, two glycine residues, three glycine residues, four glycine residues, five glycine residues, six glycine residues, seven glycine residues, eight glycine residues, or more than eight glycine residues at its C-terminus. In some embodiments, the adjacent polypeptide contains the amino acid sequence of SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, or SEQ ID NO: 165 at its C-terminus.

[0090] In some embodiments, the adjacent polypeptide comprises the amino acid sequences of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 146, 147, 148, 149, 150, 151, 154, 155, 157, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 271, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, and / or 250.

[0091] The nucleotide sequence encoding a polypeptide of interest, such as the variant IgG Fc polypeptide or other polypeptides described herein, can be inserted into an expression vector suitable for expression in a selected host cell. The variant IgG Fc polypeptide or other polypeptides described herein can be expressed by culturing a host cell transfected with an expression vector containing the nucleotide sequence.

[0092] A "vector" is a plasmid that can be used to transfer a DNA sequence from one organism to another or to express a gene of interest. Vectors typically contain an origin of replication and regulatory sequences that regulate the expression of the gene of interest, and may or may not carry a selectable marker gene such as an antibiotic resistance gene. The vector is suitable for the host cell in which it is to be expressed. If the gene of interest is present in the vector, the vector can be referred to as a "recombinant vector".

[0093] A "host cell" can be a recipient of a vector or an isolated polynucleotide, or refers to a cell that was a recipient. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting and exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, and derivatives thereof, such as 293-6E, DG44, CHO-S, and CHO-K cells. Host cells include the progeny of a single host cell, and the progeny are not necessarily identical (either morphologically or in the genomic DNA complementary strand) to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with a polynucleotide encoding the amino acid sequences provided herein.

[0094] 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 called "isolated" if it has been separated from at least some of the components of the cell in which it was produced. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered "isolating" the polypeptide. Similarly, a polynucleotide is called "isolated" if it is not part of a larger polynucleotide typically found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) or, in the case of 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 called "isolated".

[0095] "Signal sequence" refers to an amino acid residue or the sequence of a polynucleotide encoding it that promotes the secretion of a polypeptide of interest and is typically cleaved during the transport of the polypeptide to the outside of the cell surface membrane.

[0096] In some embodiments, an adjacent polypeptide comprising a variant IgG Fc polypeptide or a variant Fc polypeptide is isolated using chromatography such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0097] A label can be attached to an adjacent polypeptide comprising a variant IgG Fc polypeptide or a variant Fc polypeptide. The term "label" refers to a moiety attached to a molecule to make the molecule detectable. In some embodiments, the adjacent polypeptide comprising a variant IgG Fc polypeptide or a variant Fc polypeptide is labeled with a detectable moiety including, but not limited to, radioisotopes, fluorescent labels, and various enzyme substrate labels known in the art. In some embodiments, the label is a detectable marker that can generate a signal detectable by visual or instrumental means, such as incorporation of radiolabeled amino acids or attachment of a biotinyl moiety to a polypeptide that can be detected by attachment of labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to: radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 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., p-galactosidase, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pairing 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 a light-generating moiety, such as an acridinium compound, and a fluorescence-generating moiety, such as fluorescein. In this regard, the moiety itself is not detectably labeled but may become detectable upon reaction with yet another moiety. General techniques used in performing the various immunoassays described above are known to those of skill in the art.

[0098] [Affinity of Exemplary Variant IgG Fc Polypeptides to Protein A and / or C1q and / or CD16] The variant IgG Fc polypeptides described herein may have modified binding affinities to Protein A and / or C1q and / or CD16. In some embodiments, the variant IgG Fc polypeptide has an increased binding affinity to Protein A relative to the wild-type IgG Fc polypeptide. Such variant IgG Fc polypeptides can be purified by Protein A column chromatography. In some embodiments, the variant IgG Fc polypeptide has a decreased binding affinity to C1q relative to the wild-type IgG Fc polypeptide. Such variant IgG Fc polypeptides may have a reduced complement-mediated immune responsiveness. In some embodiments, the variant IgG Fc polypeptide has a decreased binding affinity to CD16 relative to the wild-type IgG Fc polypeptide. Such variant IgG Fc polypeptides may have a reduced ADCC immune responsiveness. In some embodiments, the variant IgG Fc polypeptide has an increased binding affinity to Protein A relative to the wild-type IgG Fc polypeptide, and / or a decreased binding affinity to C1q relative to the wild-type IgG Fc polypeptide, and / or a decreased binding affinity to CD16 relative to the wild-type IgG Fc polypeptide.

[0099] As used herein, "Protein A" is a polypeptide comprising all or a portion of Protein A that can bind to wild-type canine IgG-B Fc, wild-type equine IgG1 Fc, wild-type equine IgG3 Fc, wild-type equine IgG4 Fc, wild-type equine IgG7 Fc, wild-type feline IgG1a Fc, wild-type feline IgG1b Fc, or wild-type feline IgG2 Fc.

[0100] "C1q" or "C1q complex" is used interchangeably to refer to a protein complex involved in the complement system or a part thereof that can bind to wild-type canine IgG-B Fc, wild-type canine IgG-C Fc, wild-type equine IgG1 Fc, wild-type equine IgG3 Fc, wild-type equine IgG4 Fc, wild-type equine IgG7 Fc, wild-type feline IgG1a Fc, or wild-type feline IgG1b Fc.

[0101] As used herein, "CD16" is a polypeptide that includes all or a part of CD16 that can bind to wild-type canine IgG-A Fc or wild-type canine IgG-D Fc. The term "bind 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 an 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 immunoblotting, ELISA, KinEx A, biolayer interferometry (BLI), surface plasmon resonance apparatus, etc.

[0102] As used herein, "Protein A+" means that the Fc polypeptide has a Protein A binding affinity. In some embodiments, the Protein A+ Fc polypeptide includes at least one amino acid modification that increases the Protein A binding affinity.

[0103] As used herein, "Protein A-" means that the Protein A binding affinity of the Fc polypeptide is low or absent.

[0104] As used herein, "C1q+" means that the Fc polypeptide has a C1q binding affinity.

[0105] As used herein, "C1q-" means that the C1q binding affinity of the Fc polypeptide is low or absent. In some embodiments, the C1q-Fc polypeptide has at least one amino acid modification that reduces the C1q binding affinity.

[0106] As used herein, "CD16+" means that the Fc polypeptide has CD16 binding affinity.

[0107] As used herein, "CD16-" means that the CD16 binding affinity of the Fc polypeptide is low or absent. In some embodiments, the CD16-Fc polypeptide has at least one amino acid modification that reduces the CD16 binding affinity.

[0108] The term "affinity" means the overall strength of the 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 molecule X for partner Y can generally be expressed as the dissociation constant (K D ). Affinity can be measured by common methods known in the art, such as immunoblotting, ELISA, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance apparatus.

[0109] "Surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecule-specific interactions, for example, by detecting changes in protein concentration within a biosensor matrix using, for example, a BIAcore TM system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further explanation, see Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26.

[0110] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from a layer of immobilized protein on the tip of a biosensor and an internal reference layer. A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern that can be measured in real time. A non-limiting, exemplary apparatus for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377: 209-277.

[0111] "K D ", "K d ", "Kd" or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of a receptor-ligand interaction or an antibody-antigen interaction.

[0112] In some embodiments, the variant IgG Fc polypeptide binds to Protein A with a dissociation constant (K -6 less than 5×10 -6 M, less than 1×10 -7 M, less than 5×10 -7 M, less than 1×10 -8 M, less than 5×10 -8 M, less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, less than 5×10 -11 M, less than 1×10 -12 M, or less than 1×10 -12 M as measured by biolayer interferometry. D )

[0113] In some embodiments, the variant IgG Fc polypeptide binds to Protein A with a dissociation constant (K -6 greater than 5×10 -5 M, greater than 1×10 -5 M, greater than 5×10 -4 M, greater than 1×10 -4 M, greater than 5×10-3 binds to C1q and / or CD16 with a dissociation constant (K D ) greater than M.

[0114] In some embodiments, the variant canine IgG-A or IgG-D Fc polypeptide binds to C1q and / or CD16 with a dissociation constant (K -6 ) less than 5×10 -6 M, less than 1×10 -7 M, less than 5×10 -7 M, less than 1×10 -8 M, less than 5×10 -8 M, less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, less than 5×10 -11 M, less than 1×10 -12 M, or less than 1×10 -12 M, and binds to C1q and / or CD16 with a dissociation constant (K D ).

[0115] In some embodiments, the K D of an IgG Fc polypeptide, such as a variant IgG Fc polypeptide to Protein A or C1q or CD16, is measured by using a biolayer interferometry assay using a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont, CA) according to the supplier's instructions. Briefly, biotinylated Protein A or C1q or CD16 is bound to the sensor tip and the binding of the IgG Fc polypeptide is monitored for a specified time or until a steady state is reached. Dissociation can be monitored for a specified time or until a steady state is reached. To correct for drift, a blank curve of buffer only is subtracted. The data is fit to a 2:1 binding model using ForteBio data analysis software to determine the association rate constant (k on ), the dissociation rate constant (k off ), and K d . The equilibrium dissociation constant (K D) is calculated as the ratio of k off / k on . The term "k on " refers to the rate constant when molecule X binds to its partner Y, and the term "k off " refers to the rate constant when molecule X or partner Y dissociates from the molecule X / partner Y complex.

[0116] "Increase" or "stimulate" means to increase, improve, or enhance activity, function, or amount as compared to a reference. In some embodiments, "increase" or "stimulate" means 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 200% or more relative to a reference value. In some embodiments, "increase" or "stimulate" means the ability to cause an overall increase of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, about 25% to about 70% relative to a reference value. In some embodiments, "increase" or "stimulate" means the ability to cause an overall increase of 50% or more. In some embodiments, "increase" or "stimulate" means the ability to cause an overall increase of 75%, 85%, 90%, 95%, or more. In some embodiments, the above amount is stimulated or increased over a period of time relative to a control dosage (such as a placebo) over the same period of time.

[0117] In some embodiments, the variant IgG Fc polypeptide can bind to Protein A with an increased affinity 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 relative to the reference IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide can bind to Protein A with an increased affinity of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, about 25% to about 70% relative to the reference IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a wild-type IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a different variant IgG Fc polypeptide.

[0118] "Reduce" or "inhibit" means to cause a decrease, reduction, or suppression of activity, function, or amount as compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease 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, or about 90% or more relative to the reference IgG Fc polypeptide. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, about 25% to about 70% relative to a reference value. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the above amounts are inhibited or decreased over a period of time relative to a control dose (such as a placebo) over the same period of time.

[0119] In some embodiments, the variant IgG Fc polypeptide can bind C1q or CD16 with a reduced affinity 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 relative to the reference IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide can bind C1q or CD16 with a reduced affinity of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, about 25% to about 70% relative to the reference IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a wild-type IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a different variant IgG Fc polypeptide.

[0120] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. The reference can be a wild-type reference or a variant reference. The reference can be obtained from a healthy or non-afflicted sample. In some examples, the reference is obtained from a non-afflicted or untreated sample of a companion animal. In some examples, the reference is obtained from one or more healthy animals of a particular species that are not the animals being tested or treated.

[0121] [Exemplary Pharmaceutical Compositions] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that enables the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are toxic to an unacceptable degree to the subject to which the formulation is administered.

[0122] "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 a therapeutic agent that together constitutes a "pharmaceutical composition" to be administered to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffer solutions such as phosphate, citrate, tromethamine, or HEPES buffer; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone, cellulose-based substances; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.

[0123] The pharmaceutical composition can be stored in lyophilized form. Thus, in some embodiments, the preparation method includes a lyophilization step. Next, the lyophilized composition can typically be reformulated as an aqueous composition suitable for parenteral administration before administration to a dog, cat, or horse. In other embodiments, particularly when the variant IgG Fc polypeptides or other polypeptides described herein are very stable to heat and oxidative denaturation, the pharmaceutical composition can be administered directly to a dog, cat, or horse, or after appropriate dilution, as a liquid, i.e., as an aqueous composition, and can be stored as such. The lyophilized composition can be reconstituted with water for injection (WFI). A bacteriostatic reagent such as benzyl alcohol may be included. Thus, the present invention provides a pharmaceutical composition in solid or liquid form.

[0124] The pH of the pharmaceutical composition can be in the range of about pH 5 to about pH 8 when administered. The compositions of the present invention are sterile when they are 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 regardless of the presence or absence of antibacterial agents.

[0125] [Predetermined Use of Fc Polypeptide and Pharmaceutical Composition] Polypeptides comprising a variant Fc polypeptide, such as the variant IgG Fc polypeptides of the present invention, or pharmaceutical compositions comprising the variant Fc polypeptides of the present invention can be useful for extending the in vivo product half-life in companion animals, including but not limited to dogs, cats, or horses.

[0126] As used herein, "treatment" is an approach for obtaining a beneficial or desirable clinical outcome. As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a mammal, including companion animals. For the purposes of this disclosure, beneficial or desirable clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the degree of the disease, prevention or delay of disease spread, prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of the medical condition, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, suppression of its occurrence, and remission (partial or complete). Also included within "treatment" is a reduction in the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the foregoing, the term treatment does not require 100 percent removal of all aspects of the disorder.

[0127] 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 therapies, 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 induce the desired response in the animal. A therapeutically effective amount is also an amount where the therapeutically beneficial effects exceed the toxic or detrimental effects of the substance / molecule, agonist or antagonist. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective at the required dosage and for the period of time necessary to achieve the desired therapeutic or prophylactic result.

[0128] In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same, is administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same, is administered as a bolus injection or by continuous infusion over a predetermined period of time. In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or pharmaceutical compositions comprising them, are administered intramuscularly, intraperitoneally, intrathecally, subcutaneously, intraarterially, into the joint synovial cavity, into the subarachnoid space, or by inhalation.

[0129] In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same, is administered in an amount in the range of from 0.0001 mg / kg body weight to 100 mg / kg body weight per dose, from 0.005 mg / kg body weight to 20 mg / kg body weight per dose, from 1 mg / kg body weight to 10 mg / kg body weight per dose, from 0.5 mg / kg body weight to 100 mg / kg body weight per dose, from 1 mg / kg body weight to 100 mg / kg body weight per dose, from 5 mg / kg body weight to 100 mg / kg body weight per dose, from 10 mg / kg body weight to 100 mg / kg body weight per dose, from 20 mg / kg body weight to 100 mg / kg body weight per dose, from 50 mg / kg body weight to 100 mg / kg body weight per dose, from 1 mg / kg body weight to 10 mg / kg body weight per dose, from 5 mg / kg body weight to 10 mg / kg body weight per dose, from 0.5 mg / kg body weight to 10 mg / kg body weight per dose, or from 5 mg / kg body weight to 50 mg / kg body weight per dose.

[0130] In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same, is administered to a companion animal once or over a series of treatments. In some embodiments, the dose is administered once a week for at least 2 or 3 consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, and in some cases, one or more weeks of treatment-free periods are interspersed. In other embodiments, a therapeutically effective dose is administered once a day for 2 to 5 consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, and in some cases, one or more days or weeks of treatment-free periods are interspersed.

[0131] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (co - administration) and sequential or successive administration in any order. The term "co - administration" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administrations overlap in time, or the administration of one therapeutic agent is within a short time of the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered at intervals of approximately less than a certain number of minutes. The term "sequential" 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 has been discontinued, or 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 at intervals of approximately more than a certain number of minutes. As used herein, "in combination with" refers to performing one treatment modality in addition to another treatment modality. Thus, "in combination with" refers to performing one treatment modality before, during, or after performing another treatment modality on an animal.

[0132] In some embodiments, the dosage is administered once a week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, with periods of no treatment of one or more weeks interspersed in some cases. In other embodiments, a therapeutically effective dosage is administered once a day for 2 to 5 consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, with periods of no treatment of one or more days or weeks interspersed in some cases.

[0133] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (co - administration) and sequential or successive administration in any order. The term "co - administration" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administrations overlap in time, or the administration of one therapeutic agent occurs within a short time relative to the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered at intervals of approximately less than a certain number of minutes. The term "sequential" 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 has been discontinued, or the administration of one or more agents is initiated before the administration of one or more other agents. For example, the administrations of two or more therapeutic agents are administered at intervals of approximately more than a certain number of minutes. As used herein, "in combination with" refers to administering a certain treatment modality in addition to another treatment modality. Thus, "in combination with" refers to administering a certain treatment modality before, during, or after another treatment modality is administered to an animal.

[0134] The following examples illustrate certain aspects of the disclosure and are not intended to limit the disclosure in any way.

Examples

[0135] Example 1 Variant canine IgG Fc polypeptides with increased Protein A - binding, and / or decreased complement - binding, and / or decreased CD16 - binding The purification of antibodies using Protein A affinity is a well - developed process. However, of the four subtypes of canine IgG, only IgG - B Fc (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) has Protein A - binding affinity. The Protein A - binding affinities of canine IgG - A Fc (e.g., SEQ ID NO: 1), IgG - C Fc (e.g., SEQ ID NO: 4 or SEQ ID NO: 5), and IgG - D Fc (e.g., SEQ ID NO: 6) are weak or undetectable. To modify the Protein A - binding, variant canine IgG - A Fc, IgG - C Fc, and IgG - D Fc polypeptides were designed.

[0136] In addition, canine IgG-B Fc and IgG-C Fc have complement activity and bind to C1q, but the binding affinities of canine IgG-A Fc and IgG-D Fc for C1q are weak or cannot be measured. Variant canine IgG-B Fc and IgG-C Fc polypeptides were designed to potentially reduce C1q binding and / or potentially reduce complement-mediated immune responsiveness.

[0137] Furthermore, canine IgG-B Fc and IgG-C Fc have CD16 binding activity. Variant canine IgG-B Fc and IgG-C Fc polypeptides were designed to potentially reduce the binding of CD16 to IgG-B Fc and IgG-C Fc and / or potentially reduce ADCC.

[0138] Table 3 below summarizes the Protein A and C1q binding characteristics of canine IgG Fc subtypes. In particular, there is no wild-type canine IgG Fc subtype that lacks C1q binding and binds to Protein A.

[0139] JPEG0007691365000047.jpg61147

[0140] Three-dimensional protein modeling and protein sequence analysis were used to identify the sequences of canine IgG-B Fc that are likely to contact Protein A. Figure 1 shows the alignment of canine IgG-A, IgG-B, IgG-C, and IgG-D Fc sequences. The box indicates the region that may contact Protein A.

[0141] Two approaches were used to design variant canine IgG-A, IgG-C, and IgG-D Fc polypeptides with increased protein A binding. In the first approach, the variant canine IgG-A, IgG-C, and IgG-D Fc polypeptides were designed to have the same protein A binding motif sequences as canine IgG-B Fc (e.g., SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively). In the second approach, variant canine IgG-A Fc I(21)T / Q(207)H (SEQ ID NO:10), variant canine IgG-C Fc I(21)T (SEQ ID NO:11), and variant canine IgG-D Fc I(21)T / Q(207)H (SEQ ID NO:12) were designed to have one or two amino acid substitutions in the protein A binding region corresponding to the canine IgG-B Fc sequence.

[0142] In addition, variant canine IgG-A Fc, IgG-C Fc, and IgG-D Fc polypeptides with increased protein A binding having one or more of the amino acid substitutions listed in Table 4 can be prepared.

[0143] JPEG0007691365000048.jpg73157

[0144] To potentially reduce the binding of C1q to canine IgG-B Fc and IgG-C Fc, and / or to potentially reduce complement-mediated immunoreactivity, variant canine IgG-B Fc and IgG-C Fc polypeptides can be prepared having an amino acid substitution of Lys with any amino acid other than Lys at the amino acid position corresponding to position 93 of SEQ ID NO: 2 or SEQ ID NO: 4, respectively. These amino acid substitutions were identified after protein sequence and 3D structure modeling analysis of canine IgG-B Fc and IgG-C Fc compared to canine IgG-A Fc and IgG-D Fc, which are understood not to exhibit complement activity. For example, variant canine IgG-B Fc K(93)R (SEQ ID NO: 13) and variant canine IgG-C Fc K(93)R (SEQ ID NO: 14) can be prepared. A decrease in binding was observed between human C1q and a fusion protein containing variant canine IgG-B Fc K(93)R compared to a fusion protein containing wild-type canine IgG-B Fc.

[0145] To potentially reduce the binding of CD16 to canine IgG-B Fc and IgG-C Fc, and / or to potentially reduce ADCC, variant canine IgG-B Fc and IgG-C Fc polypeptides can be prepared having one or more of the amino acid substitutions listed in Table 5 (e.g., 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: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and / or SEQ ID NO: 29). The amino acid substitutions were identified after protein sequence and 3D structure modeling analysis of canine IgG-B and IgG-C compared to IgG-A and IgG-D, which are understood not to exhibit ADCC activity.

[0146] JPEG0007691365000049.jpg117170

[0147] Wild-type canine IgG-C Fc lacks protein A binding ability and has C1q binding ability. Therefore, the double-variant canine IgG-C Fc that binds to protein A and has reduced binding ability to C1q can be prepared by combining one or more of the amino acid substitutions listed in Table 4 with K(93)R substitution or K(93)X substitution, where X is any amino acid other than Lys (for example, SEQ ID NO: 30). The double-variant canine IgG-B Fc or double-variant canine IgG-C Fc with reduced binding ability to C1q and reduced binding ability to CD16 can be prepared by combining one or more of the amino acid substitutions listed in Table 5 with K(93)R substitution or K(93)X substitution, where X is any amino acid other than Lys (for example, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and / or SEQ ID NO: 34). The triple-variant canine-IgG-C Fc that binds to protein A and has reduced binding ability to C1q and CD16 can be prepared by combining one or more of the amino acid substitutions listed in Table 4 and one or more of the amino acid substitutions listed in Table 5 with K(93)R substitution or K(93)X substitution, where X is any amino acid except Lys.

[0148] Determine the binding ability of any variant canine IgG Fc to protein A, CD16, and / or C1q, and compare it with the binding ability of another IgG Fc (for example, the corresponding wild-type canine IgG Fc, another wild-type or variant canine IgG Fc, or the wild-type or variant IgG Fc of another companion animal, etc.) to protein A, CD16, and / or C1q.

[0149] Binding analysis can be performed using an Octet biosensor. Briefly, a target molecule (e.g., Protein A, C1q, CD16, etc.) can be biotinylated and free unreacted biotin can be removed (e.g., by dialysis). The biotinylated target molecule is captured at the tip of a streptavidin sensor. The binding of the target molecule to IgG Fc polypeptides at various concentrations (e.g., 10 μg / mL) is monitored for a specified time or until a steady state is reached. Dissociation is monitored for a specified time or until a steady state is reached. To correct for drift, a blank curve of buffer alone can be subtracted. The data is fit to a 1:1 binding model using ForteBio TM data analysis software to determine k on , k off , and K d .

[0150] Example 2 Variant canine IgG-A and IgG-D Fc polypeptides with increased Protein A binding, and / or increased complement binding, and / or increased CD16 binding Based on the amino acid positions identified as being involved in Protein A, C1q, and CD16 binding as described in Example 1, gain-of-function canine IgG-A Fc and IgG-D Fc polypeptides were designed to potentially increase the binding of Protein A, C1q, and / or CD16 to canine IgG-A Fc and IgG-D Fc. For example, variant canine IgG-A and IgG-D Fc polypeptides can be designed that have one or more amino acid substitutions in the Protein A binding region, C1q binding region, and / or CD16 binding region corresponding to the sequence of a wild-type canine IgG Fc polypeptide that binds Protein A, C1q, and / or CD16.

[0151] By combining one or more of the amino acid substitutions listed in Table 6, single, double, or triple variant canine IgG-A and / or IgG-D polypeptides can be prepared. For example, a variant canine IgG-A Fc polypeptide of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and / or SEQ ID NO: 41 and a variant canine IgG-D Fc polypeptide of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and / or SEQ ID NO: 48 can be prepared.

[0152] JPEG0007691365000050.jpg142164

[0153] Determine the binding of any variant canine IgG-A or IgG-D Fc polypeptide to Protein A, C1q, and / or CD16 and compare it to the binding of another IgG Fc (e.g., the corresponding wild-type canine IgG Fc, another wild-type or variant canine IgG Fc, or the wild-type or variant IgG Fc of another companion animal) to Protein A, C1q, and / or CD16. The binding assay described in Example 1 can be used.

[0154] Example 3 Variant equine IgG Fc polypeptides with increased Protein A binding and / or decreased complement binding Of the seven subtypes of equine IgG, IgG1 Fc (e.g., SEQ ID NO: 49), IgG3 Fc (e.g., SEQ ID NO: 52), IgG4 Fc (e.g., SEQ ID NO: 53), IgG7 Fc (e.g., SEQ ID NO: 56) have Protein A binding affinity. The Protein A binding affinity of equine IgG2 Fc (e.g., SEQ ID NO: 50, SEQ ID NO: 51), IgG5 Fc (e.g., SEQ ID NO: 54), and IgG6 Fc (e.g., SEQ ID NO: 55) is weak or not measurable. Variant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides with modified Protein A binding were designed.

[0155] In addition, the binding affinities of equine IgG2 Fc, IgG5 Fc, and IgG6 Fc for C1q are weak or not measurable, while equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc bind to C1q. Variant equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc polypeptides were designed to potentially reduce C1q binding and / or potentially reduce complement-mediated immune responsiveness.

[0156] Table 7 below summarizes the protein A and C1q binding characteristics of equine IgG Fc subtypes. In particular, there is no wild-type equine IgG Fc subtype that lacks C1q binding and binds to protein A.

[0157] JPEG0007691365000051.jpg87158

[0158] Three-dimensional protein modeling and protein sequence analysis were used to identify the sequences of equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc that are likely to be in contact with protein A. Variant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides with increased protein A binding, having one or more of the amino acid substitutions listed in Table 8, can be prepared.

[0159] JPEG0007691365000052.jpg61170

[0160] For example, variant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides having one or more amino acid substitutions in the protein A binding region corresponding to the sequence of wild-type equine IgG Fc that binds to a protein were designed. Variant equine IgG2 Fc F(203)Y (SEQ ID NO: 57); variant equine IgG2 Fc A(15)T / F(203)Y (SEQ ID NO: 58); variant equine IgG5 Fc V(199)L / E(200)Y (SEQ ID NO: 59); and variant equine IgG6 Fc I(199)L / R(200)H / H(201)N / T(202)H (SEQ ID NO: 60) with increased protein A binding properties can be prepared.

[0161] To potentially reduce the binding of C1q to equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc, and / or to potentially reduce the complement-mediated immune responsiveness, variant canine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc polypeptides having an amino acid substitution of Lys with any amino acid other than Lys at the amino acid position corresponding to position 87 of SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 56, respectively, can be prepared. These amino acid substitutions were identified after protein sequence and 3D structure modeling analysis of equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc compared to equine IgG2 Fc, IgG5 Fc, and IgG6 Fc, which are understood not to exhibit complement activity. For example, variant equine IgG1 Fc K(87)S (SEQ ID NO: 61), variant equine IgG3 Fc K(87)S (SEQ ID NO: 62), variant equine IgG4 Fc K(87)S (SEQ ID NO: 63), and variant equine IgG7 Fc K(87)S (SEQ ID NO: 64) can be prepared.

[0162] Determine the binding of any variant equine IgG Fc to Protein A and / or C1q and compare it with the binding of another IgG Fc (e.g., the corresponding wild-type equine IgG Fc, another wild-type or variant equine IgG Fc, or the wild-type or variant IgG Fc of another companion animal, etc.) to Protein A and / or C1q. The binding assay described in Example 1 can be used.

[0163] Example 4 Variant feline IgG Fc polypeptide with reduced complement binding Each of the three subtypes of feline IgG, IgG1a Fc (SEQ ID NO: 65 or SEQ ID NO: 66), IgG1b Fc (SEQ ID NO: 67 or SEQ ID NO: 68), and IgG2 Fc (SEQ ID NO: 69), has Protein A binding affinity. However, the binding affinity to C1q is weak or not measurable only for feline IgG2 Fc, and feline IgG1a Fc and IgG1b Fc bind to C1q. Variant feline IgG1a Fc and IgG1b Fc polypeptides were designed to potentially reduce C1q binding and / or potentially reduce complement-mediated immune responsiveness.

[0164] Table 9 below summarizes the Protein A and C1q binding characteristics of feline IgG Fc subtypes. In particular, there is no wild-type equine IgG Fc subtype that lacks C1q binding and binds to Protein A.

[0165] JPEG0007691365000053.jpg56162

[0166] To potentially reduce the binding of C1q to feline IgG1a Fc and IgG1b Fc, and / or to potentially reduce complement-mediated immune responsiveness, variant feline IgG1a Fc and IgG1b Fc polypeptides can be prepared having an amino acid substitution of Pro with any amino acid other than Pro at the amino acid position corresponding to position 198 of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68. These amino acid substitutions were identified after protein sequence and 3D structure modeling analysis of feline IgG1a Fc and IgG1b Fc compared to feline IgG2 Fc, which is understood not to exhibit complement activity. For example, variant feline IgG1a Fc P(198)A (e.g., SEQ ID NO: 70 or SEQ ID NO: 71) and variant feline IgG1b Fc P(198)A (e.g., SEQ ID NO: 72 or SEQ ID NO: 73) can be prepared.

[0167] Determine the binding of any variant feline IgG Fc to C1q and compare it to the binding of another IgG Fc (e.g., the corresponding wild-type feline IgG Fc, another wild-type or variant feline IgG Fc, or the wild-type or variant IgG Fc of another companion animal, etc.) to C1q. The binding assay described in Example 1 can be used.

[0168] Example 5 Variant canine, feline, and equine IgG Fc polypeptides of heterodimeric proteins Using the knob-in-hole heterodimerization approach, the pairing of variant canine IgG Fc polypeptides, variant feline IgG Fc polypeptides, and variant equine IgG Fc polypeptides was examined to enable the preparation of bispecific canine, feline, or equine antibodies or bifunctional canine, feline, or equine Fc fusion proteins. The pairing of two Fc polypeptides was 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 small amino acid at the same general position (hole).

[0169] Amino acid substitutions of threonine to tyrosine or tryptophan at positions corresponding to position 138 of canine IgG-A Fc (SEQ ID NO: 1) or canine IgG-D Fc (SEQ ID NO: 6) (T138Y or T138W), or at positions corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 2) or canine IgG-C Fc (SEQ ID NO: 4) (T137Y or T137W) can be introduced as a knob (heterodimer chain 1) into one Fc chain. Examples of the amino acid sequences of variant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 1 are 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, and SEQ ID NO: 81.

[0170] Amino acid substitutions of threonine to serine at positions corresponding to position 138 of canine IgG-A (SEQ ID NO: 1) or IgG-D (SEQ ID NO: 6), and / or leucine to alanine at positions corresponding to position 140, and / or tyrosine to threonine at positions corresponding to position 180 (T138S, L140A, and / or Y180T), or amino acid substitutions of threonine to serine at positions corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 2) or IgG-C (SEQ ID NO: 4), and / or leucine to alanine at positions corresponding to position 139, and / or tyrosine to threonine at positions corresponding to position 179 (T137S, L139A, and / or Y180T) can be introduced as a hole (heterodimer chain 2) into a second Fc chain. Examples of the amino acid sequences of variant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 2 are 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, or SEQ ID NO: 93.

[0171] An amino acid substitution from threonine to tyrosine or tryptophan (T154Y or T154W) at the position corresponding to position 154 of feline IgG1a Fc (SEQ ID NO: 65 or SEQ ID NO: 66), feline IgG1b Fc (SEQ ID NO: 67 or SEQ ID NO: 68), or feline IgG2 (SEQ ID NO: 69) can be introduced as a knob (heterodimeric chain 1) into one Fc chain. Examples of the amino acid sequences of variant feline IgG1a Fc, IgG1b Fc, and IgG2 heterodimeric chain 1 are 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, and SEQ ID NO: 103.

[0172] An amino acid substitution from threonine to serine at the position corresponding to position 154 of IgG-1a (SEQ ID NO: 65 or SEQ ID NO: 66), or IgG-1b Fc (SEQ ID NO: 67 or SEQ ID NO: 68), or IgG2 (SEQ ID NO: 69), and / or from leucine to alanine at the position corresponding to position 156, and / or from tyrosine to threonine at the position corresponding to position 197 (T154S, L156A, and / or Y197T) can be introduced as a hole (heterodimeric chain 2) into the second Fc chain. Examples of the amino acid sequences of variant feline IgG1a Fc, IgG1b Fc, and IgG2 Fc heterodimeric chain 2 are SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113.

[0173] An amino acid substitution from threonine to tyrosine or tryptophan (T131Y or T131W) at the position corresponding to position 131 of equine IgG1 Fc (SEQ ID NO: 49), equine IgG2 Fc (SEQ ID NO: 50), equine IgG3 Fc (SEQ ID NO: 52), equine IgG4 Fc (SEQ ID NO: 53), equine IgG5 Fc (SEQ ID NO: 54), equine IgG6 Fc (SEQ ID NO: 55), or equine IgG7 Fc (SEQ ID NO: 56) can be introduced as a knob (heterodimeric chain 1) into one Fc chain. Examples of the amino acid sequences of variant IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc, and IgG7 Fc heterodimeric chain 1 are SEQ ID NO: 114, SEQ ID NO: 115, 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, and SEQ ID NO: 127.

[0174] An amino acid substitution from threonine to serine at the position corresponding to position 131 of equine IgG1 Fc (SEQ ID NO: 49), equine IgG2 Fc (SEQ ID NO: 50), equine IgG3 Fc (SEQ ID NO: 52), equine IgG4 Fc (SEQ ID NO: 53), equine IgG5 Fc (SEQ ID NO: 54), equine IgG6 Fc (SEQ ID NO: 55), or equine IgG7 Fc (SEQ ID NO: 56), and / or from leucine to alanine at the position corresponding to position 133, and / or from tyrosine to threonine at the position corresponding to position 174 (T131W, L133A, and / or Y174T) can be introduced as a hole (heterodimeric chain 2) into the second Fc chain. Examples of the amino acid sequences of variant IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc, and IgG7 Fc heterodimeric chain 2 are SEQ ID NO: 128, SEQ ID NO: 129, 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, and SEQ ID NO: 141.

[0175] Pairing of variant canine IgG Fc heterodimer chains 1 and 2, pairing of variant feline IgG Fc heterodimer chains 1 and 2, and pairing of variant equine IgG Fc heterodimer chains 1 and 2 can enable Fc heterodimerization and prevent or reduce Fc homodimerization. Heterodimer chain 1 of one canine IgG subtype can be combined with heterodimer chain 2 of the same or a different canine IgG subtype. Heterodimer chain 1 of one feline IgG subtype can be combined with heterodimer chain 2 of the same or a different feline IgG subtype. Heterodimer chain 1 of one equine IgG subtype can be combined with heterodimer chain 2 of the same or a different equine IgG subtype. This design enables dimerization of bispecific canine, feline, or equine antibodies. In addition, combinations of two different peptides or proteins, or different proteins (e.g., therapeutic proteins) can be fused to the heterodimeric Fc chains.

[0176] Using variant canine IgG Fc heterodimer chains or variant feline IgG Fc heterodimer chains, for example, bispecific GLP1 and glucagon molecules can be generated, such as a GLP1 polypeptide (e.g., SEQ ID NO: 181) fused to variant canine IgG Fc heterodimer chain 1 (e.g., SEQ ID NO: 74, 75, 76, 77, 78, 79, 80, or 81) and a glucagon polypeptide (e.g., SEQ ID NO: 182) fused to variant canine IgG Fc heterodimer chain 2 (e.g., SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93).

[0177] Bispecific antibodies combine the specificities of two antibodies. To facilitate the specific pairing of the heavy chain with its intended light chain, the interfacial amino acids between CH1 and the light chain can be mutated to be complementary in shape and / or charge-charge interactions. Amino acid substitutions from alanine to leucine at the position corresponding to position 24 of canine IgG-A CH1 (SEQ ID NO: 142), canine IgG-B CH1 (SEQ ID NO: 143), canine IgG-C CH1 (SEQ ID NO: 144), or canine IgG-D CH1 (SEQ ID NO: 145), and / or from serine to asparagine at the position corresponding to position 30 (A24L and / or S30D) can be introduced. Examples of the amino acid sequences of variant canine IgG-A CH1, IgG-B CH1, IgG-C CH1, and IgG-D CH1 are SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149, respectively.

[0178] Amino acid substitutions from phenylalanine to alanine at the position corresponding to position 11 of the canine κ constant region (SEQ ID NO: 150), and / or from serine to arginine at the position corresponding to position 22 (F11A and / or S22R) can be introduced. An example of the amino acid sequence of the variant canine κ constant region is SEQ ID NO: 151.

[0179] Amino acid substitutions from alanine to leucine at the position corresponding to position 24 of feline IgG1 CH1 (SEQ ID NO: 152), and / or from serine to asparagine at the position corresponding to position 30, or amino acid substitutions from alanine to leucine at the position corresponding to position 24 of feline IgG2 CH1 (SEQ ID NO: 153), and / or from serine to asparagine at the position corresponding to position 29 can be introduced. Examples of the amino acid sequences of variant feline IgG1 CH1 and IgG2 CH1 are SEQ ID NO: 154 and SEQ ID NO: 155, respectively.

[0180] Amino acid substitutions from phenylalanine to alanine at the position corresponding to the 11th position of the feline κ constant region (SEQ ID NO: 156) and / or from serine to arginine at the position corresponding to the 22nd position (F11A and / or S22R) can be introduced. An example of the amino acid sequence of the variant feline κ constant region is SEQ ID NO: 157.

[0181] Example 6 Variant IgG Fc fusion protein An adjacent polypeptide comprising at least one therapeutic polypeptide and / or at least one antibody and a variant feline, canine, or equine IgG Fc polypeptide as described herein (e.g., an IgG Fc with modified C1q, CD16, and / or protein A binding affinity) can be prepared.

[0182] For example, the following constructs can be designed: Formula (I): TPA1-L1-Fc; Formula (II): Fc-L1-TPA1; Formula (III): TPA1-L1-Fc-L2-TPA2; Formula (IV): TPA1-L1-TPA2-L2-Fc; or Formula (V): Fc-L1-TPA1-L2-TPA2 Here, TPA1 is a first therapeutic polypeptide and / or antibody, TPA2 is a second therapeutic polypeptide and / or antibody (e.g., the same therapeutic polypeptide, a different therapeutic polypeptide, the same antibody, or a different antibody), L1 and L2 are any linkers; Fc is a variant IgG Fc polypeptide of a companion animal species. Optionally, the adjacent polypeptide contains a signal sequence. The constructs of Formulas I-V can include TPA3, TPA4, TPA5, etc. after or before any TPA1 or TPA2. TPA3, TPA4, TPA5, etc. are third, fourth, fifth, etc. therapeutic polypeptides and / or antibodies (e.g., the same therapeutic polypeptide, a different therapeutic polypeptide, the same antibody, or a different antibody).

[0183] For example, the adjacent polypeptide can include a therapeutic polypeptide and a variant feline IgG1a Fc polypeptide (e.g., SEQ ID NO: 70, 71, 94, 95, 99, 100, 104, 105, 106, 107, 154, 167, or 168), a variant feline IgG1b Fc polypeptide (e.g., SEQ ID NO: 72, 73, 96, 97, 101, 102, 108, 109, 110, 111, 154, 169, or 170), or a variant feline IgG2 Fc polypeptide (e.g., SEQ ID NO: 98, 103, 112, 113, 155, 166, 171, or 178) described herein.

[0184] The adjacent polypeptide can include a variant canine IgG-A Fc polypeptide (e.g., SEQ ID NO: 7, 10, 35, 36, 37, 38, 39, 40, 41, 74, 78, 82, 86, 90, or 146), a variant canine IgG-B Fc polypeptide (e.g., SEQ ID NO: 13, 15, 16, 17, 18, 19, 20, 21, 22, 31, 32, 75, 79, 83, 87, 91, or 147), a variant canine IgG-C Fc polypeptide (e.g., SEQ ID NO: 8, 11, 14, 23, 24, 25, 26, 27, 28, 29, 30, 33, 34, 76, 80, 84, 88, 92, or 148), or a variant canine IgG-D Fc polypeptide (e.g., SEQ ID NO: 9, 12, 42, 43, 44, 45, 46, 47, 48, 77, 81, 85, 89, 93, or 149) described herein.

[0185] The adjacent polypeptide can include a variant equine IgG1 Fc polypeptide (e.g., SEQ ID NO: 61, 114, 121, 128, or 135), a variant equine IgG2 Fc polypeptide (e.g., SEQ ID NO: 57, 58, 115, 122, 129, 136, 172, 173, 174, 175, 176, or 177), a variant equine IgG3 Fc polypeptide (e.g., SEQ ID NO: 62, 116, 123, 130, or 137), a variant equine IgG4 Fc polypeptide (e.g., SEQ ID NO: 63, 117, 124, 131, or 138), a variant equine IgG5 Fc polypeptide (e.g., SEQ ID NO: 59, 118, 125, 132, or 139), a variant equine IgG6 Fc polypeptide (e.g., SEQ ID NO: 60, 119, 126, 133, or 140), or a variant equine IgG7 Fc polypeptide (e.g., SEQ ID NO: 64, 120, 127, 134, or 141).

[0186] The linker can be a flexible unstructured linker such as a glycine and serine rich linker. A flexible extension can be added to the C-terminus of the adjacent polypeptide. The extension can include one glycine residue (SEQ ID NO: 158), two glycine residues (SEQ ID NO: 159), three glycine residues (SEQ ID NO: 160), four glycine residues (SEQ ID NO: 161), five glycine residues (SEQ ID NO: 162), six glycine residues (SEQ ID NO: 163), seven glycine residues (SEQ ID NO: 164), eight glycine residues (SEQ ID NO: 165), or more glycine residues.

[0187] Adjacent polypeptides include TPA1, TPA2, TPA3, TPA4, TPA5, etc., or NGF polypeptides, receptors for NGF polypeptides (e.g., ECD of receptors for NGF polypeptides), TrkA polypeptides (e.g., ECD of TrkA polypeptides), LNGFR polypeptides (e.g., ECD of LNGFR polypeptides), TNFα polypeptides, receptors for TNFα polypeptides, TNFR polypeptides (e.g., ECD of TNFR polypeptides), TNFR1 polypeptides (e.g., ECD of TNFR1 polypeptides), TNFR2 polypeptides (e.g., ECD of TNFR2 polypeptides), IL5 polypeptides, receptors for IL5 polypeptides, IL5R polypeptides (e.g., ECD of IL5R polypeptides), IL5Rα polypeptides (e.g., ECD of IL5Rα polypeptides), IL6 polypeptides, receptors for IL6 polypeptides, IL6R polypeptides (e.g., ECD of IL6R polypeptides), IL17 polypeptides, receptors for IL17 polypeptides, IL17R polypeptides (e.g., ECD of IL17R polypeptides), IL17RA polypeptides (e.g., ECD of IL17RA polypeptides), IL17RB polypeptides (e.g., ECD of IL17RB polypeptides), IL17RC polypeptides (e.g., ECD of IL17RC polypeptides), IL23 polypeptides, receptors for IL23 polypeptides, IL23R polypeptides (e.g., ECD of IL23R polypeptides), IL12Rβ1 polypeptides (e.g., ECD of IL12Rβ1 polypeptides), PDL1 polypeptides, receptors for PDL1 polypeptides, PDL2 polypeptides, receptors for PDL2 polypeptides, PD1 polypeptides (e.g., ECD of PD1 polypeptides), integrin polypeptides (e.g., ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, or ITGB8 polypeptides), receptors for integrin polypeptides, fibronectin polypeptides (e.g., ECD of fibronectin polypeptides),Vitronectin polypeptide (e.g., ECD of vitronectin polypeptide), collagen polypeptide (e.g., ECD of collagen polypeptide), laminin polypeptide (e.g., ECD of laminin polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD86 polypeptide, receptor of CD86 polypeptide, CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), B7-H3 polypeptide, receptor of B7-H3 polypeptide (e.g., ECD of receptor of B7-H3 polypeptide), LAG-3 polypeptide (e.g., ECD of LAG-3 polypeptide), IL31 polypeptide, receptor of IL31 polypeptide, IL31RA polypeptide (e.g., ECD of IL31RA polypeptide), OSMR polypeptide (e.g., ECD of OSMR polypeptide), IL4 polypeptide, receptor of IL4R polypeptide, IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13 polypeptide, receptor of IL13 polypeptide, IL13RA1 polypeptide (e.g., ECD of IL13RA1 polypeptide), IL4R polypeptide (e.g., ECD of IL4R polypeptide), IL13Rα2 polypeptide (e.g., ECD of IL13Rα2 polypeptide), IL22 polypeptide, receptor of IL22 polypeptide (e.g., ECD of IL22 polypeptide), IL22Rα1 polypeptide (e.g., ECD of IL22Rα1 polypeptide), IL10Rβ2 polypeptide (e.g., ECD of IL10Rβ2 polypeptide), IL33 polypeptide, receptor of IL33 polypeptide, IL1RL1 polypeptide (e.g., ECD of IL1RL1 polypeptide), EGF polypeptide, receptor of EGF polypeptide, TGFα polypeptide, receptor of TGFα polypeptide, EGFR polypeptide (e.g., ECD of EGFR polypeptide), MMP9 polypeptide, FGF polypeptide (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23 polypeptide), receptor of FGF polypeptide,FGFR polypeptides (e.g., FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), ECDs of FGFR polypeptides (e.g., ECDs of FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptides), EGF polypeptides, receptors of EGF polypeptides, neuregulin polypeptides (e.g., neuregulin isoforms I, II, III, IV, V, or VI polypeptides), receptors of neuregulin polypeptides, HER polypeptides (e.g., HER1, HER2, HER3, or HER4 polypeptides), ECDs of HER polypeptides (e.g., ECDs of HER1, HER2, HER3, or HER4 polypeptides), EpCAM polypeptides (e.g., ECDs of EpCAM polypeptides), CD20 polypeptides (e.g., ECDs of CD20 polypeptides), ligands of CD20 polypeptides, CD19 polypeptides (e.g., ECDs of CD19 polypeptides), ligands of CD19 polypeptides, CGRP polypeptides (e.g., α-CGRP polypeptide or β-CGRP polypeptide), receptors of CGRP polypeptides, receptors of α-CGRP polypeptide, receptors of β-CGRP polypeptide, CALCRL polypeptides (e.g., ECDs of CALCRL polypeptides), RAMP polypeptides (e.g., RAMP1, RAMP2, or RAMP3 polypeptides), ECDs of RAMP polypeptides (e.g., ECDs of RAMP1, RAMP2, or RAMP3 polypeptides), IGF polypeptides (e.g., IGF-1 or IGF-2 polypeptides), receptors of IGF polypeptides (e.g., receptors of IGF1 or IGF-2 polypeptides), IGFR polypeptides (e.g., IGFR1 or IGFR2 polypeptides), ECDs of IGFR polypeptides (e.g., ECDs of IGFR1 or IGFR2 polypeptides), IGFBP polypeptides (e.g., IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6 polypeptides), VEGF polypeptides (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides), receptors of VEGF polypeptides (e.g., receptors of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides),VEGFR polypeptides (e.g., VEGFR1, VEGFR2, or VEGFR3 polypeptides), ECDs of VEGFR polypeptides (e.g., ECDs of VEGFR1, VEGFR2, or VEGFR3 polypeptides), FLT1 receptor polypeptides (e.g., ECDs of FLT1 receptor polypeptides), IL36 polypeptides (e.g., IL36A, IL36B, or IL36G polypeptides), receptors for IL36 polypeptides (e.g., receptors for IL36A, IL36B, or IL36G polypeptides), IL36R polypeptides (e.g., ECDs of IL36R polypeptides), IL1R1 polypeptides (e.g., ECDs of IL1R1 polypeptides), IL1R2 polypeptides (e.g., ECDs of IL1R2 polypeptides), IL1RL1 polypeptides (ECDs of IL1RL1 polypeptides), IL18R1 polypeptides (ECDs of IL18R1 polypeptides), bacterial toxin polypeptides, exotoxin polypeptides, endotoxin polypeptides, botulinum neurotoxin polypeptides, tetanus toxin polypeptides, staphylococcal toxin polypeptides, CD52 polypeptides (e.g., ECDs of CD52 polypeptides), ligands for CD52 polypeptides, SIGLEC10 polypeptides, PCSK9 polypeptides, receptors for PCSK9 polypeptides, LDLR polypeptides (e.g., ECDs of LDLR polypeptides), CEA polypeptides (e.g., CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptides), ECDs of CEA polypeptides (e.g., CD66a, CD66b, CD66c, CD66d, CD66e, or the ECD of a CD66f polypeptide), a BAFF polypeptide, a receptor for a BAFF polypeptide, a TRAF polypeptide (e.g., a TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRAF7 polypeptide), a receptor for a TRAF polypeptide (e.g., a receptor for a TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 polypeptide), a BCMA polypeptide, the ECD of a BCMA polypeptide, a SOST polypeptide, a receptor for a SOST polypeptide, an LRP polypeptide (e.g., an LRP5 or LRP6 polypeptide), the ECD of an LRP polypeptide (e.g., the ECD of an LRP5 or LRP6 polypeptide), a DLL polypeptide (e.g., a DLL4 polypeptide), a receptor for a DLL polypeptide, a Jagged polypeptide (e.g., a JAG1 or JAG polypeptide), a receptor for a Jagged polypeptide (e.g., a receptor for a JAG1 or JAG polypeptide), a NOTCH polypeptide (e.g., a NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), a ligand for a NOTCH polypeptide (e.g., a ligand for a NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), a VWF polypeptide, a receptor for a VWF polypeptide, a Factor VIII polypeptide, a receptor for a Factor VIII polypeptide, a platelet GP1b receptor polypeptide (e.g., the ECD of a platelet GP1b receptor polypeptide), an integrin α IIb β 3 polypeptide (e.g., integrin α IIb β 3ECD of a polypeptide), IL2 polypeptide, receptor of the IL2 polypeptide, IL2R polypeptide (e.g., IL2Rα, IL2Rβ, or IL2Rγ polypeptide), ECD of the IL2R polypeptide (e.g., ECD of the IL2Rα, IL2Rβ, or IL2Rγ polypeptide), TGFβ polypeptide, receptor of the TGFβ polypeptide, decorin polypeptide, EIF3I polypeptide, LTBP1 polypeptide, TGFβR1 polypeptide (e.g., ECD of the TGFβR1 polypeptide), YWHAE polypeptide, IgE polypeptide, receptor or IgE polypeptide, Fc receptor polypeptide (e.g., FcεRI or FcεRII polypeptide), ECD of the Fc receptor polypeptide (e.g., ECD of the FcεRI or FcεRII polypeptide), KLK polypeptide (e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, or KLK15 polypeptide), Rank1 polypeptide, receptor of the Rank1 polypeptide, RANK polypeptide (e.g., ECD of the RANK polypeptide), TSLP polypeptide, receptor of the TSLP polypeptide, CRLF2 polypeptide (e.g., ECD of the CRLF2 polypeptide), IL7Rα polypeptide (e.g., ECD of the IL7Rα polypeptide), S1P polypeptide, CD3 polypeptide (e.g., CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), ECD of the CD3 polypeptide (e.g., ECD of the CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), CD80 polypeptide, receptor of the CD80 polypeptide, CD28 polypeptide (e.g., ECD of the CD28 polypeptide), CTLA-4 polypeptide (e.g., ECD of the CTLA-4 polypeptide), GnRH polypeptide, receptor of the GNRH polypeptide, GnRHR polypeptide (e.g., ECD of the GnRHR polypeptide), ICAM polypeptide (e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), receptor of the ICAM polypeptide (e.g., receptor of the ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide),JAM-A polypeptide, a receptor for the JAM-A polypeptide, an LFA-1 polypeptide (e.g., the ECD of the LFA-1 polypeptide), Na, v a 1.7 polypeptide, a C5 polypeptide (e.g., a C5a or C5b polypeptide), a receptor for the C5 polypeptide (e.g., a receptor for the C5a or C5b polypeptide), a C5aR polypeptide (e.g., the ECD of the C5aR polypeptide), a C5L2 polypeptide (e.g., the ECD of the C5L2 polypeptide), an IL17 polypeptide, a receptor for the IL17 polypeptide, an IL17Ra polypeptide (e.g., the ECD of the IL17Ra polypeptide), an IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), an EPO polypeptide, a somatostatin polypeptide, a GLP1 polypeptide, a glucagon polypeptide, etc., and may contain at least one therapeutic polypeptide selected therefrom.

[0188] The adjacent polypeptide may comprise at least one antibody selected from antibodies that recognize TPA1, TPA2, TPA3, TPA4, TPA5, etc., or one or more of the following polypeptides: NGF polypeptide, receptor for NGF polypeptide (e.g., ECD of receptor for NGF polypeptide), TrkA polypeptide (e.g., ECD of TrkA polypeptide), LNGFR polypeptide (e.g., ECD of LNGFR polypeptide), TNFα polypeptide, receptor for TNFα polypeptide, TNFR polypeptide (e.g., ECD of TNFR polypeptide), TNFR1 polypeptide (e.g., ECD of TNFR1 polypeptide), TNFR2 polypeptide (e.g., ECD of TNFR2 polypeptide), IL5 polypeptide, receptor for IL5 polypeptide, IL5R polypeptide (e.g., ECD of IL5R polypeptide), IL5Rα polypeptide (e.g., ECD of IL5Rα polypeptide), IL6 polypeptide, receptor for IL6 polypeptide, IL6R polypeptide (e.g., ECD of IL6R polypeptide), IL17 polypeptide, receptor for IL17 polypeptide, IL17R polypeptide (e.g., ECD of IL17R polypeptide), IL17RA polypeptide (e.g., ECD of IL17RA polypeptide), IL17RB polypeptide (e.g., ECD of IL17RB polypeptide), IL17RC polypeptide (e.g., ECD of IL17RC polypeptide), IL23 polypeptide, receptor for IL23 polypeptide, IL23R polypeptide (e.g., ECD of IL23R polypeptide), IL12Rβ1 polypeptide (e.g., ECD of IL12Rβ1 polypeptide), PDL1 polypeptide, receptor for PDL1 polypeptide, PDL2 polypeptide, receptor for PDL2 polypeptide, PD1 polypeptide (e.g., ECD of PD1 polypeptide), integrin polypeptide (e.g., ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, or ITGB8 polypeptide),Receptors for integrin polypeptides, fibronectin polypeptides (e.g., ECD of fibronectin polypeptides), vitronectin polypeptides (e.g., ECD of vitronectin polypeptides), collagen polypeptides (e.g., ECD of collagen polypeptides), laminin polypeptides (e.g., ECD of laminin polypeptides), CD80 polypeptides, receptors for CD80 polypeptides, CD86 polypeptides, receptors for CD86 polypeptides, CTLA-4 polypeptides (e.g., ECD of CTLA-4 polypeptides), B7-H3 polypeptides, receptors for B7-H3 polypeptides (e.g., ECD of receptors for B7-H3 polypeptides), LAG-3 polypeptides (e.g., ECD of LAG-3 polypeptides), IL31 polypeptides, receptors for IL31 polypeptides, IL31RA polypeptides (e.g., ECD of IL31RA polypeptides), OSMR polypeptides (e.g., ECD of OSMR polypeptides), IL4 polypeptides, receptors for IL4R polypeptides, IL4R polypeptides (e.g., ECD of IL4R polypeptides), IL13 polypeptides, receptors for IL13 polypeptides, IL13RA1 polypeptides (e.g., ECD of IL13RA1 polypeptides), IL4R polypeptides (e.g., ECD of IL4R polypeptides), IL13Rα2 polypeptides (e.g., ECD of IL13Rα2 polypeptides), IL22 polypeptides, receptors for IL22 polypeptides (e.g., ECD of IL22 polypeptides), IL22Rα1 polypeptides (e.g., ECD of IL22Rα1 polypeptides), IL10Rβ2 polypeptides (e.g., ECD of IL10Rβ2 polypeptides), IL33 polypeptides, receptors for IL33 polypeptides, IL1RL1 polypeptides (e.g., ECD of IL1RL1 polypeptides), EGF polypeptides, receptors for EGF polypeptides, TGFα polypeptides, receptors for TGFα polypeptides, EGFR polypeptides (e.g., ECD of EGFR polypeptides), MMP9 polypeptides, FGF polypeptides (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19,FGF20, FGF21, FGF22, or FGF23 polypeptide), a receptor for the FGF polypeptide, an FGFR polypeptide (e.g., FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptide), an ECD of the FGFR polypeptide (e.g., an ECD of FGFR1, FGFR2, FGFR3, FGFR4, or FGFRL1 polypeptide), an EGF polypeptide, a receptor for the EGF polypeptide, a neuregulin polypeptide (e.g., neuregulin isoform I, II, III, IV, V, or VI polypeptide), a receptor for the neuregulin polypeptide, a HER polypeptide (e.g., HER1, HER2, HER3, or HER4 polypeptide), an ECD of the HER polypeptide (e.g., an ECD of HER1, HER2, HER3, or HER4 polypeptide), an EpCAM polypeptide (e.g., an ECD of the EpCAM polypeptide), a CD20 polypeptide (e.g., an ECD of the CD20 polypeptide), a ligand for the CD20 polypeptide, a CD19 polypeptide (e.g., an ECD of the CD19 polypeptide), a ligand for the CD19 polypeptide, a CGRP polypeptide (e.g., an α-CGRP polypeptide or a β-CGRP polypeptide), a receptor for the CGRP polypeptide, a receptor for the α-CGRP polypeptide, a receptor for the β-CGRP polypeptide, a CALCRL polypeptide (e.g., an ECD of the CALCRL polypeptide), a RAMP polypeptide (e.g., RAMP1, RAMP2, or RAMP3 polypeptide), an ECD of the RAMP polypeptide (e.g., an ECD of RAMP1, RAMP2, or RAMP3 polypeptide), an IGF polypeptide (e.g., IGF-1 or IGF-2 polypeptide), a receptor for the IGF polypeptide (e.g., a receptor for the IGF1 or IGF-2 polypeptide), an IGFR polypeptide (e.g., IGFR1 or IGFR2 polypeptide), an ECD of the IGFR polypeptide (e.g., an ECD of IGFR1 or IGFR2 polypeptide), an IGFBP polypeptide (e.g., IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6 polypeptide), a VEGF polypeptide (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptide), a receptor for the VEGF polypeptide (e.g.,Receptors for VEGF-A, VEGF-B, VEGF-C, VEGF-D, or PGF polypeptides), VEGFR polypeptides (e.g., VEGFR1, VEGFR2, or VEGFR3 polypeptides), EC Ds of VEGFR polypeptides (e.g., EC Ds of VEGFR1, VEGFR2, or VEGFR3 polypeptides), FLT1 receptor polypeptides (e.g., EC D of the FLT1 receptor polypeptide), IL36 polypeptides (e.g., IL36A, IL36B, or IL36G polypeptides), receptors for IL36 polypeptides (e.g., receptors for IL36A, IL36B, or IL36G polypeptides), IL36R polypeptides (e.g., EC D of the IL36R polypeptide), IL1R1 polypeptides (e.g., EC D of the IL1R1 polypeptide), IL1R2 polypeptides (e.g., EC D of the IL1R2 polypeptide), IL1RL1 polypeptide (ECD of IL1RL1 polypeptide), IL18R1 polypeptide (ECD of IL18R1 polypeptide), bacterial toxin polypeptide, exotoxin polypeptide, endotoxin polypeptide, botulinum neurotoxin polypeptide, tetanus toxin polypeptide, staphylococcal toxin polypeptide, CD52 polypeptide (e.g., ECD of CD52 polypeptide), ligand of CD52 polypeptide, SIGLEC10 polypeptide, PCSK9 polypeptide, receptor of PCSK9 polypeptide, LDLR polypeptide (e.g., ECD of LDLR polypeptide), CEA polypeptide (e.g., CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), ECD of CEA polypeptide (e.g., ECD of CD66a, CD66b, CD66c, CD66d, CD66e, or CD66f polypeptide), BAFF polypeptide, receptor of BAFF polypeptide, TRAF polypeptide (e.g., TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRAF7 polypeptide), receptor of TRAF polypeptide (e.g., receptor of TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 polypeptide), BCMA polypeptide, ECD of BCMA polypeptide, SOST polypeptide, receptor of SOST polypeptide, LRP polypeptide (e.g., LRP5 or LRP6 polypeptide), ECD of LRP polypeptide (e.g., ECD of LRP5 or LRP6 polypeptide), DLL polypeptide (e.g., DLL4 polypeptide), receptor of DLL polypeptide, Jagged polypeptide (e.g., JAG1 or JAG polypeptide), receptor of Jagged polypeptide (e.g., receptor of JAG1 or JAG polypeptide), NOTCH polypeptide (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), ligand of NOTCH polypeptide (e.g., ligand of NOTCH1, NOTCH2, NOTCH3, or NOTCH4 polypeptide), VWF polypeptide, receptor of VWF polypeptide, Factor VIII polypeptide, receptor of Factor VIII polypeptide, platelet GP1b receptor polypeptide (e.g., ECD of platelet GP1b receptor polypeptide), integrin αIIb β 3 polypeptide (e.g., integrin α IIb β 3ECD of polypeptide), IL2 polypeptide, receptor of IL2 polypeptide, IL2R polypeptide (e.g., IL2Rα, IL2Rβ, or IL2Rγ polypeptide), ECD of IL2R polypeptide (e.g., ECD of IL2Rα, IL2Rβ, or IL2Rγ polypeptide), TGFβ polypeptide, receptor of TGFβ polypeptide, decorin polypeptide, EIF3I polypeptide, LTBP1 polypeptide, TGFβR1 polypeptide (e.g., ECD of TGFβR1 polypeptide), YWHAE polypeptide, IgE polypeptide, receptor or IgE polypeptide, Fc receptor polypeptide (e.g., FcεRI or FcεRII polypeptide), ECD of Fc receptor polypeptide (e.g., ECD of FcεRI or FcεRII polypeptide), KLK polypeptide (e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, or KLK15 polypeptide), Rank1 polypeptide, receptor of Rank1 polypeptide, RANK polypeptide (e.g., ECD of RANK polypeptide), TSLP polypeptide, receptor of TSLP polypeptide, CRLF2 polypeptide (e.g., ECD of CRLF2 polypeptide), IL7Rα polypeptide (e.g., ECD of IL7Rα polypeptide), S1P polypeptide, CD3 polypeptide (e.g., CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), ECD of CD3 polypeptide (e.g., ECD of CD3γ polypeptide, CD3δ polypeptide, or CD3ε polypeptide), CD80 polypeptide, receptor of CD80 polypeptide, CD28 polypeptide (e.g., ECD of CD28 polypeptide), CTLA-4 polypeptide (e.g., ECD of CTLA-4 polypeptide), GnRH polypeptide, receptor of GNRH polypeptide, GnRHR polypeptide (e.g., ECD of GnRHR polypeptide), ICAM polypeptide (e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide), receptor of ICAM polypeptide (e.g., receptor of ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5 polypeptide),JAM-A polypeptide, a receptor for the JAM-A polypeptide, an LFA-1 polypeptide (e.g., the ECD of the LFA-1 polypeptide), Na, v 1.7 polypeptide, C5 polypeptide (e.g., C5a or C5b polypeptide), a receptor for the C5 polypeptide (e.g., a receptor for the C5a or C5b polypeptide), C5aR polypeptide (e.g., the ECD of the C5aR polypeptide), C5L2 polypeptide (e.g., the ECD of the C5L2 polypeptide), IL17 polypeptide, a receptor for the IL17 polypeptide, IL17Ra polypeptide (e.g., the ECD of the IL17Ra polypeptide), IL17RC polypeptide (e.g., the ECD of the IL17RC polypeptide), EPO polypeptide, somatostatin polypeptide, GLP1 polypeptide, glucagon polypeptide, and the like.

[0189] Example 7 Isolation of Variant IgG Fc Fusion Protein A nucleotide sequence encoding an adjacent polypeptide comprising at least one therapeutic polypeptide or antibody and a variant feline, canine, or equine IgG Fc polypeptide (e.g., IgG Fc with modified C1q, CD16, and / or protein A binding affinity) described herein, such as an adjacent polypeptide of Formula I, II, III, IV, and / or V, can be synthesized and cloned into separate mammalian expression vectors.

[0190] The resulting vectors can be individually transfected into CHO cells. In the case of an adjacent polypeptide containing a signal sequence, the supernatant containing the adjacent polypeptide without the signal peptide can be collected and filtered. Adjacent polypeptides containing an Fc IgG polypeptide having protein A binding properties can be affinity purified using a protein A column (CaptivA® Protein A Affinity Resin, Repligen). The dimerization, aggregation, and / or presence of disulfide bonds of the resulting protein can be evaluated by HPLC gel filtration and / or SDS-PAGE analysis in the absence and presence of a reducing agent (DTT).

[0191] Example 8 Variant IgG Fc polypeptides with increased and / or enhanced disulfide formation Using three-dimensional protein modeling analysis of several ortholog hinge structures, approximate positions for modifying the feline IgG2 hinge to increase disulfide formation were determined. To increase disulfide formation in the feline IgG2 hinge, the hinge sequence can be modified by substituting amino acids with cysteine. For example, variant feline IgG2 Fc with a modified hinge (SEQ ID NO: 166) was prepared by substituting glycine with cysteine at the amino acid position corresponding to position 14 of SEQ ID NO: 69.

[0192] Using additional three-dimensional protein modeling analysis of several ortholog hinge structures, the feline and equine IgG hinges were modified to enhance disulfide formation. To enhance disulfide formation in the feline IgG hinge, the hinge sequence can be modified by substituting lysine with proline (e.g., K16P) at the position corresponding to position 16 of wild-type or variant feline IgG1a (SEQ ID NO: 65 or SEQ ID NO: 66), feline IgG1b (SEQ ID NO: 67 or SEQ ID NO: 68), or feline IgG2 (SEQ ID NO: 69). Examples of the amino acid sequences of variant feline IgG polypeptides with a modified hinge include SEQ ID NO: 167, SEQ ID NO: 168, and SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171.

[0193] To enhance disulfide formation in equine IgG hinge, the hinge is modified by substituting cysteine with serine at the position corresponding to position 3 of wild-type or variant equine IgG having a hinge (e.g., IgG2 Fc (SEQ ID NO: 51)), and / or substituting glutamine with proline at the position corresponding to position 20 of equine IgG having a hinge (e.g., IgG2 Fc (SEQ ID NO: 51)) (e.g., C3S and / or Q20P). Examples of the amino acid sequences of variant equine IgG polypeptide having a modified hinge include SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, and SEQ ID NO: 177.

[0194] The above amino acid substitutions can be incorporated into the hinge of the wild-type or variant Fc polypeptide described herein.

[0195] Using three-dimensional protein modeling, feline and equine variant IgG Fc polypeptides containing sequences from hinge regions from different IgG isotypes were designed for enhanced recombinant productivity and improved hinge disulfide-forming properties. Variant feline IgG2Fc polypeptides containing sequences from the hinge region of feline IgG1a or IgG1b (e.g., SEQ ID NO: 178) can be prepared. In addition, variant equine IgG2Fc polypeptides containing sequences from the hinge region of equine IgG1 (e.g., SEQ ID NO: 179 and SEQ ID NO: 180) can be prepared.

[0196] The recombinant production level and / or hinge disulfide formation level of the variant IgG Fc polypeptide can be determined and compared to the level of another IgG Fc (e.g., the corresponding wild-type IgG Fc of the same or different isotype, or the wild-type or variant IgG Fc of another companion animal) by SDS-PAGE analysis under reducing and non-reducing conditions.

[0197] Example 9 Exemplary contiguous polypeptide comprising GLP1 and a variant Fc polypeptide Exemplary adjacent polypeptides comprising a glucagon-like peptide-1 (GLP1) polypeptide and a variant feline IgG Fc with a cysteine hinge modification were designed based on Formula I (ssGLP1-G8_I_VARfeIgG2 (SEQ ID NO: 184)) and Formula III (ssGLP1-G8 / GLP1-2G_III_WTfeIgG2 (SEQ ID NO: 185)), expressed in CHO cells, and purified by protein A chromatography. The amino acid sequences of the secreted proteins after cleavage of the signal sequence are SEQ ID NO: 186 and 187, respectively. SDS-PAGE analysis of the variant feline IgG2 constructs showed a decrease in the protein amount of the low molecular weight band in the absence of a reducing agent compared to the wild-type feline IgG2 construct (compare Figure 2B with Figure 2A). These results suggest that Fc covalent pair formation was improved in both variant feline IgG2 constructs.

[0198] Furthermore, differential scanning fluorimetry was used to evaluate the stability of the adjacent polypeptides at various pHs, as reflected by the average melting temperature (n = 3) (Table 10 below). The increase in the stability of the variant feline IgG2 hinge is most evident at pH 6. For example, constructs with variant feline IgG2 (SEQ ID NO: 186 and 187) showed higher Tm (56.9 and 59.7 °C) at pH 6 than the corresponding constructs with wild-type feline IgG2 (SEQ ID NO: 188 and 189), which had Tm of 55.2 and 56.9 °C, respectively.

[0199] JPEG0007691365000054.jpg73170

[0200] Example 10 Protein Binding Kinetics The binding affinity of the adjacent polypeptides described herein to the target molecule can be evaluated using the Biolayer Interference method (Octet). Briefly, the biotinylated adjacent polypeptide or target molecule can be captured on the streptavidin sensor tip. The binding of different concentrations of the second binding partner can be monitored for 90 seconds. Dissociation can be monitored for 600 seconds. The drift is corrected by subtracting the blank curve of buffer only, and ForteBio TM data analysis software is used to fit the data to a 1:1 binding model, and k on , k off , and K d can be determined. The buffer for dilution and all binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2.

[0201] Example 11 Exemplary adjacent polypeptides comprising IL13R ECD, IL4R ECD, and variant canine IgG Fc polypeptide An adjacent polypeptide comprising the extracellular domain of the IL13 receptor (IL13R ECD; e.g., SEQ ID NO: 190, 191, 192, 193, 194, or 195), the extracellular domain of the IL4R (IL4R ECD; e.g., SEQ ID NO: 196, 197, 198, 199, 200, or 201), and the variant IgG Fc polypeptide described herein can be prepared. For example, the canine IL13R ECD of SEQ ID NO: 190, a linker, the canine IL4R ECD of SEQ ID NO: 196, and either a) a wild-type canine IgG-B Fc polypeptide comprising a hinge and the amino acid sequence of SEQ ID NO: 2, or b) a C1q-variant canine IgG-B Fc polypeptide comprising a hinge and the amino acid sequence of SEQ ID NO: 13 were tested (SEQ ID NO: 271 and 202, respectively).

[0202] To determine the C1q binding affinity of IL13R(ECD)-IL4R(ECD)-wild-type canine IgG-B Fc (SEQ ID NO: 271) compared to IL13R(ECD)-IL4R(ECD)-variant canine IgG-B Fc (SEQ ID NO: 202), a biosensor binding assay was performed. Briefly, canine IL4 was biotinylated and captured on a streptavidin sensor tip. Either IL13R(ECD)-IL4R(ECD)-wild-type canine IgG-B Fc (25 μg / mL) or IL13R(ECD)-IL4R(ECD)-variant canine IgG-B Fc (25 μg / mL) was complexed to the IL4-binding biosensor. Subsequently, the complex was used to bind to human C1q (Catalog No. 204876-1MG; Sigma Aldrich) at 250 μg / mL. The ability of human C1q to bind to either complex was measured. A decrease in binding between human C1q and IL13R(ECD)-IL4R(ECD)-variant canine IgG-B Fc was observed when compared to IL13R(ECD)-IL4R(ECD)-wild-type canine IgG-B Fc.

[0203] Example 12 Long-term stability The long-term stability of an adjacent polypeptide comprising a variant Fc IgG polypeptide described herein can be evaluated. For example, samples can be stored in PBS (pH 7.2) at 2-8°C at different concentrations (e.g., concentrations of 1 mg / mL, 1.3 mg / mL, 5 mg / mL, and / or 10 mg / mL) for a certain period of time (e.g., 1 day, 6 months, and / or 1 year). To evaluate stability, the stored samples can be analyzed by protein binding assays and / or cell-based assays.

[0204] Example 13 Serum stability The serum stability of an adjacent polypeptide comprising a variant Fc IgG polypeptide described herein can be evaluated. For example, samples can be stored with serum in PBS (pH 7.2) at physiological temperature (e.g., 37°C) for a period of time (e.g., 6 hours, 12 hours, and / or 24 hours) to test in vitro serum stability. To evaluate stability, the stored samples can be analyzed by protein binding assays and / or cell-based assays.

[0205] Example 14 In Vivo Pharmacokinetics The in vivo pharmacokinetics of an adjacent polypeptide comprising a variant Fc IgG polypeptide described herein can be evaluated after administering a single dose of the adjacent polypeptide to companion animals by injection (e.g., subcutaneous or intravenous). Serum samples can be taken before dosing (0 hour) and at certain times (e.g., 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and / or 168 hours) later, and the concentration of the adjacent polypeptide can be measured by quantitative ELISA or other means. The serum concentration of the adjacent polypeptide can be plotted against time, and the mean serum half-life (t 1 / 2 ), mean T max , mean C max , and mean area under the curve (AUC) can be determined.

[0206] Quantitative ELISA can use an antibody against the therapeutic polypeptide and an HRP-conjugated antibody against IgG-Fc for the quantification of the adjacent polypeptide in serum samples from in vivo pharmacokinetic studies. A 96-well plate can be coated with an antibody against the therapeutic target (e.g., 5 μg / mL in coating buffer, 100 μl / well). The plate can be sealed and incubated overnight at 4°C. The plate can be washed three times with 1×TBST and a blocking buffer can be added. After removing the blocking buffer, serial dilutions of the reference standard and the sample in the blocking buffer can be added (e.g., 100 μl / well) and the plate can be incubated for 2 hours at room temperature. The plate can be washed three times with 1×TBST and an HRP-conjugated antibody against IgG-Fc (e.g., 0.1 μg / mL in blocking buffer, 100 μl / well) can be added. After incubating for 1 hour at room temperature, the plate can be washed with 1×TBST. A TMB substrate (e.g., ScyTek, catalog number TM1999) can be added (100 μl / well) and can be incubated for 1 minute at room temperature. The reaction can be stopped by adding 2M H 2 SO 4 (e.g., 50 μl / well). The absorbance at 450 nm can be measured and the concentration of the adjacent polypeptide in the serum sample can be calculated.

[0207] Furthermore, the concentration of the adjacent polypeptide in the same serum sample can be evaluated using a cell-based activity assay to determine whether the adjacent polypeptide detected by ELISA is biologically active.

Claims

1. A polypeptide comprising at least one therapeutic polypeptide and / or at least one antibody fragment comprising an antigen-binding site, and a variant canine IgG-A or IgG-D Fc polypeptide, wherein the variant canine IgG-A or IgG-D Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, the variant canine IgG-A or IgG-D Fc polypeptide has an increased binding affinity for C1q and / or CD16 compared to the wild-type canine IgG-A or IgG-D Fc polypeptide, a polypeptide.

2. (a) i) A first therapeutic polypeptide and / or an antibody fragment (TPA1) comprising an antigen-binding site; ii) A first linker (L1); and iii) The variant canine IgG-A or IgG-D Fc polypeptide (Fc) according to claim 1; or (b) i) A first therapeutic polypeptide and / or an antibody fragment (TPA1) comprising an antigen-binding site; ii) A first linker (L1); iii) The variant canine IgG-A or IgG-D Fc polypeptide (Fc) according to claim 1; iv) A second linker (L2); and v) A second therapeutic polypeptide and / or an antibody fragment (TPA2) comprising an antigen-binding site The polypeptide according to claim 1, comprising.

3. Formula (I): TPA1-L1-Fc Formula (II): Fc-L1-TPA1; Formula (III): TPA1-L1-Fc-L2-TPA2; Formula (IV): TPA1-L1-TPA2-L2-Fc; or Formula (V): Fc-L1-TPA1-L2-TPA2 The polypeptide according to claim 2, comprising.

4. The polypeptide according to claim 2 or 3, wherein when TPA2 is present, it comprises a different amino acid sequence compared to TPA1.

5. The variant canine IgG-A or variant canine IgG-D Fc polypeptide binds to C1q and / or CD16 with a dissociation constant (Kd) of less than 5×10 －６ M as measured by biolayer interferometry, the polypeptide according to any one of claims 1 to 4.

6. a) A variant canine IgG-A Fc polypeptide comprising alanine at the position corresponding to position 2 of SEQ ID NO: 1, methionine or lysine at the position corresponding to position 5 of SEQ ID NO: 1, threonine at the position corresponding to position 21 of SEQ ID NO: 1, leucine at the position corresponding to position 23 of SEQ ID NO: 1, alanine at the position corresponding to position 25 of SEQ ID NO: 1, valine at the position corresponding to position 35 of SEQ ID NO: 1, asparagine at the position corresponding to position 38 of SEQ ID NO: 1, proline at the position corresponding to position 39 of SEQ ID NO: 1, glutamate at the position corresponding to position 65 of SEQ ID NO: 1, glycine at the position corresponding to position 80 of SEQ ID NO: 1, lysine at the position corresponding to position 93 of SEQ ID NO: 1, asparagine at the position corresponding to position 96 of SEQ ID NO: 1, lysine at the position corresponding to position 97 of SEQ ID NO: 1, alanine at the position corresponding to position 98 of SEQ ID NO: 1, and / or histidine at the position corresponding to position 207 of SEQ ID NO: 1; or b) A variant canine IgG-D Fc polypeptide comprising alanine at the position corresponding to position 2 of SEQ ID NO: 6, methionine or lysine at the position corresponding to position 5 of SEQ ID NO: 6, threonine at the position corresponding to position 21 of SEQ ID NO: 6, leucine at the position corresponding to position 23 of SEQ ID NO: 6, alanine at the position corresponding to position 25 of SEQ ID NO: 6, valine at the position corresponding to position 35 of SEQ ID NO: 6, asparagine at the position corresponding to position 38 of SEQ ID NO: 6, proline at the position corresponding to position 39 of SEQ ID NO: 6, glutamate at the position corresponding to position 65 of SEQ ID NO: 6, glycine at the position corresponding to position 80 of SEQ ID NO: 6, lysine at the position corresponding to position 93 of SEQ ID NO: 6, asparagine at the position corresponding to position 96 of SEQ ID NO: 6, lysine at the position corresponding to position 97 of SEQ ID NO: 6, alanine at the position corresponding to position 98 of SEQ ID NO: 6, and / or histidine at the position corresponding to position 207 of SEQ ID NO: 6 comprising a polypeptide wherein the variant canine IgG-A or IgG-D Fc polypeptide has an increased binding affinity for C1q and / or CD16 compared to the wild-type canine IgG-A or IgG-D Fc polypeptide polypeptide.

7. a) A variant canine IgG-A Fc polypeptide comprising alanine at position 2 of SEQ ID NO: 1, methionine or lysine at position 5 of SEQ ID NO: 1, threonine at position 21 of SEQ ID NO: 1, leucine at position 23 of SEQ ID NO: 1, alanine at position 25 of SEQ ID NO: 1, valine at position 35 of SEQ ID NO: 1, asparagine at position 38 of SEQ ID NO: 1, proline at position 39 of SEQ ID NO: 1, glutamate at position 65 of SEQ ID NO: 1, glycine at position 80 of SEQ ID NO: 1, lysine at position 93 of SEQ ID NO: 1, asparagine at position 96 of SEQ ID NO: 1, lysine at position 97 of SEQ ID NO: 1, alanine at position 98 of SEQ ID NO: 1, alanine at position 205 of SEQ ID NO: 1, and / or histidine at position 207 of SEQ ID NO: 1; or b) A variant canine IgG-D Fc polypeptide comprising alanine at position 2 of SEQ ID NO: 6, methionine or lysine at position 5 of SEQ ID NO: 6, threonine at position 21 of SEQ ID NO: 6, leucine at position 23 of SEQ ID NO: 6, alanine at position 25 of SEQ ID NO: 6, valine at position 35 of SEQ ID NO: 6, asparagine at position 38 of SEQ ID NO: 6, proline at position 39 of SEQ ID NO: 6, glutamate at position 65 of SEQ ID NO: 6, glycine at position 80 of SEQ ID NO: 6, lysine at position 93 of SEQ ID NO: 6, asparagine at position 96 of SEQ ID NO: 6, lysine at position 97 of SEQ ID NO: 6, alanine at position 98 of SEQ ID NO: 6, and / or histidine at position 207 of SEQ ID NO: 6 The polypeptide according to any one of claims 1 to 6, comprising the same.

8. (i) a therapeutic polypeptide, TPA1, and / or TPA2, or (ii) an antibody fragment containing an antigen-binding site, TPA1, and / or TPA2 are the NGF polypeptide, a receptor for the NGF polypeptide, the TrkA polypeptide, the LNGFR polypeptide, the TNFα polypeptide, a receptor for the TNFα polypeptide, the TNFR polypeptide, the TNFR1 polypeptide, the TNFR2 polypeptide, the IL5 polypeptide, a receptor for the IL5 polypeptide, the IL5R polypeptide, the IL5Rα polypeptide, the IL6 polypeptide, a receptor for the IL6 polypeptide, the IL6R polypeptide, the IL17 polypeptide, a receptor for the IL17 polypeptide, the IL17R polypeptide, the IL17RA polypeptide, the IL17RB polypeptide, the IL17RC polypeptide, the IL23 polypeptide, a receptor for the IL23 polypeptide, the IL23R polypeptide, the IL12Rβ1 polypeptide, the PDL1 polypeptide, a receptor for the PDL1 polypeptide, the PDL2 polypeptide, a receptor for the PDL2 polypeptide, the PD1 polypeptide, an integrin polypeptide, a receptor for the integrin polypeptide, a fibronectin polypeptide, a vitronectin polypeptide, a collagen polypeptide, a laminin polypeptide, the CD80 polypeptide, a receptor for the CD80 polypeptide, the CD86 polypeptide, a receptor for the CD86 polypeptide, the CTLA-4 polypeptide, the B7-H3 polypeptide, a receptor for the B7-H3 polypeptide, the LAG-3 polypeptide, the IL31 polypeptide, a receptor for the IL31 polypeptide, the IL31RA polypeptide, the OSMR polypeptide, the IL4 polypeptide, a receptor for the IL4R polypeptide, the IL4R polypeptide, the IL13 polypeptide, a receptor for the IL13 polypeptide, the IL13RA1 polypeptide, the IL4R polypeptide, the IL13Rα2 polypeptide, the IL22 polypeptide, a receptor for the IL22 polypeptide, the IL22Rα1 polypeptide, the IL10Rβ2 polypeptide, the IL33 polypeptide, a receptor for the IL33 polypeptide, the IL1RL1 polypeptide, the EGF polypeptide, a receptor for the EGF polypeptide, the TGFα polypeptide, a receptor for the TGFα polypeptide, the EGFR polypeptide, the MMP9 polypeptide, the FGF polypeptide, a receptor for the FGF polypeptide, the FGFR polypeptide,ECD of FGFR polypeptide, EGF polypeptide, receptor of EGF polypeptide, neuregulin polypeptide, receptor of neuregulin polypeptide, HER polypeptide, ECD of HER polypeptide, EpCAM polypeptide, CD20 polypeptide, ligand of CD20 polypeptide, CD19 polypeptide, ligand of CD19 polypeptide, CGRP polypeptide, receptor of CGRP polypeptide, receptor of α-CGRP polypeptide, receptor of β-CGRP polypeptide, CALCRL polypeptide, RAMP polypeptide, ECD of RAMP polypeptide, IGF polypeptide, receptor of IGF polypeptide, IGF receptor polypeptide, ECD of IGF receptor polypeptide, IGFBP polypeptide, VEGF polypeptide, receptor of VEGF polypeptide, VEGFR polypeptide, ECD of VEGFR polypeptide, FLT1 receptor polypeptide, IL36 polypeptide, receptor of IL36 polypeptide, IL36R polypeptide, IL1R1 polypeptide, IL1R2 polypeptide, IL1RL1 polypeptide, IL18R1 polypeptide, bacterial toxin polypeptide, exotoxin polypeptide, endotoxin polypeptide, botulinum neurotoxin polypeptide, tetanus toxin polypeptide, staphylococcal toxin polypeptide, CD52 polypeptide, ligand of CD52 polypeptide, SIGLEC10 polypeptide, PCSK9 polypeptide, receptor of PCSK9 polypeptide, LDLR polypeptide, CEA polypeptide, ECD of CEA polypeptide, BAFF polypeptide, receptor of BAFF polypeptide, TRAF polypeptide, receptor of TRAF polypeptide, BCMA polypeptide, ECD of BCMA polypeptide, sclerostin polypeptide, receptor of sclerostin polypeptide, LRP polypeptide, ECD of LRP polypeptide, DLL polypeptide, receptor of DLL polypeptide, Jagged polypeptide, receptor of Jagged polypeptide, NOTCH polypeptide, ligand of NOTCH polypeptide, VWF polypeptide, receptor of VWF polypeptide, factor VIII polypeptide, receptor of factor VIII polypeptide, platelet GP1b receptor polypeptide, integrin αIIbβ3 polypeptide, IL2 polypeptide, receptor of IL2 polypeptide, IL2R polypeptideAn antibody fragment comprising an antigen-binding site that binds to a target polypeptide selected from the ECD of an IL2R polypeptide, a TGFβ polypeptide, a receptor for a TGFβ polypeptide, a decorin polypeptide, an EIF3I polypeptide, an LTBP1 polypeptide, a TGFβR1 polypeptide, a YWHAE polypeptide, an IgE polypeptide, a receptor or an IgE polypeptide, an Fc receptor polypeptide, the ECD of an Fc receptor polypeptide, a KLK polypeptide, a Rank1 polypeptide, a receptor for a Rank1 polypeptide, a RANK polypeptide, a TSLP polypeptide, a receptor for a TSLP polypeptide, a CRLF2 polypeptide, an IL7Rα polypeptide, an S1P polypeptide, a CD3 polypeptide, the ECD of a CD3 polypeptide, a CD80 polypeptide, a receptor for a CD80 polypeptide, a CD28 polypeptide, a CTLA-4 polypeptide, a GnRH polypeptide, a receptor for a GnRH polypeptide, a GnRHR polypeptide, an ICAM polypeptide, a receptor for an ICAM polypeptide, a JAM-A polypeptide, a receptor for a JAM-A polypeptide, an LFA-1 polypeptide, a Nav1.7 polypeptide, a C5 polypeptide, a receptor for a C5 polypeptide, a C5aR polypeptide, a C5L2 polypeptide, an IL17 polypeptide, a receptor for an IL17 polypeptide, an IL17Ra polypeptide, an IL17RC polypeptide, an EPO polypeptide, a somatostatin polypeptide, a GLP1 polypeptide, and a glucagon polypeptide; and / or a therapeutic polypeptide, TPA1 and / or TPA2 is a canine polypeptide; and / or the antibody fragment comprising an antigen-binding site binds to a canine target polypeptide, the polypeptide according to any one of claims 2 to 4.

9. A polypeptide comprising the amino acid sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48.

10. An isolated nucleic acid encoding the polypeptide according to any one of claims 1 to 9.

11. A host cell comprising the nucleic acid according to claim 10.

12. A method for producing a polypeptide, comprising culturing the host cell according to claim 11 and isolating the polypeptide.

13. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

14. A method of exposing a cell to the polypeptide according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 13, wherein the cell is exposed to the polypeptide or the pharmaceutical composition ex vivo, or A method in which cells are exposed in vivo to a polypeptide or a pharmaceutical composition in a non-human animal. Method. Claim 15 The polypeptide according to claim 1, wherein the variant IgG Fc polypeptide has at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. Claim 16 The polypeptide according to claim 1, wherein the one or more amino acid substitutions are conservative substitutions.

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