Veterinary IgG FC variant

Abstract

This provides a companion animal variant IgG Fc polypeptide that has increased protein A binding for easy purification, decreased C1q binding for reduced complement-mediated immune response, decreased CD16 binding, improved stability, and / or the ability to form heterodimer proteins. [Solution] Various embodiments are provided relating to antibodies and fusion proteins containing mutant IgG Fc polypeptides, sequential polypeptides containing one or more mutant GLP1 polypeptides having an improved serum half-life, and sequential polypeptides or heterodimer polypeptides containing GLP1 polypeptide and glucagon polypeptide as dual agonists for the GLP1 receptor and glucagon receptor. Such polypeptides may be used to treat, for example, diabetes, obesity or related conditions in companion animals such as dogs, cats and horses.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 545,858, filed Aug. 15, 2017, the entire content of which is incorporated herein by reference for all purposes.

[0002] The present disclosure relates to mutant 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 reducing complement - mediated immune responses), decreased CD16 binding (e.g., for reducing induction of antibody - dependent cell cytotoxicity (ADCC)), improved stability, and / or the ability to form heterodimeric proteins. The mutant IgG Fc polypeptides of the present disclosure can have broad applicability in companion animal therapy. For example, the mutant IgG Fc polypeptides can be used in the design and manufacture of long - acting GLP1 polypeptides for treating diabetes, obesity or related symptoms in companion animals such as dogs, cats and horses. Further, the mutant IgG Fc polypeptides can 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, different IgG subtypes have differences in these functions, which are often considered when selecting specific IgG antibodies or IgG Fc fusion proteins for therapeutic or diagnostic uses. For example, the ability of an IgG subtype to have little or no measurable binding affinity for C1q or CD16 may be advantageous. Further, the ability of IgG Fc to bind Protein A can be useful for purification using Protein A affinity purification platforms.

[0004] However, most IgG Fc subtypes in dogs, cats, and horses lack protein A binding properties, have only weak or no measurable binding affinity to CD16, and have only weak or no measurable binding affinity to C1q. For example, of the four canine IgG Fc subtypes (IgG-A, IgG-B, IgG-C, and IgG-D), only canine IgG-B Fc has clear affinity to 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 mutant IgG Fc polypeptides that have reduced binding to C1q and / or CD16 and / or are capable of binding to protein A are desirable.

[0005] Glucagon-like peptide 1 (GLP1) is a potent blood glucose-lowering hormone that plays a crucial role in regulating blood glucose levels. Native GLP1 has an in vivo half-life of approximately 2 minutes. Long-acting GLP1 polypeptides can be used to treat diabetes and obesity, prevent diabetes, control hyperglycemic conditions, lower lipid levels, treat conditions where blood glucose reduction is beneficial, suppress gastric or intestinal motility, delay gastric emptying, and / or reduce food intake. There remains a need for long-acting GLP1 polypeptides to treat hyperglycemia or uncontrolled blood glucose-induced conditions in companion animals such as dogs, cats, and horses. [Overview of the Initiative]

[0006] Embodiment 1. A polypeptide comprising a mutant IgG Fc polypeptide having at least one amino acid modification to a wild-type IgG Fc polypeptide of a companion animal species, wherein the mutant IgG Fc polypeptide has increased binding affinity to protein A compared to the wild-type IgG Fc polypeptide. Embodiment 2. A polypeptide comprising a mutant IgG Fc polypeptide comprising at least one amino acid modification to the wild-type IgG Fc polypeptide of a companion animal species, wherein the mutant IgG Fc polypeptide has a reduced binding affinity for C1q and / or CD16 relative to the wild-type IgG Fc polypeptide. Embodiment 3. The polypeptide according to Embodiment 1 or Embodiment 2, wherein the mutant IgG Fc polypeptide binds to C1q and / or CD16 with a dissociation constant (K -6 d) of 5×10 -5 M or more, 1×10 -5 M or more, 5×10 -4 M or more, 1×10 -4 M or more, 5×10 -3 M or more, or 1×10 d M or more as measured by biolayer interferometry. Embodiment 4. The polypeptide according to any one of Embodiments 1 to 3, wherein the mutant IgG Fc polypeptide has an increased binding affinity for protein A relative to the wild-type IgG Fc polypeptide. Embodiment 5. The polypeptide according to any one of Embodiments 1 to 4, wherein the mutant IgG Fc polypeptide binds to protein A with a dissociation constant (K -6 d) of 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[[ID=;37]] -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. Embodiment 6. The polypeptide according to any one of Embodiments 1 to 5, wherein the companion animal species is a dog, a cat or a horse. Embodiment 7. Wild-type IgG Fc polypeptide, a) Canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc; b) Horse 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 The polypeptide according to any one of Embodiments 1 to 6. Embodiment 8. A polypeptide comprising a mutant IgG Fc polypeptide having at least one amino acid modification to the hinge region of a wild-type cat or horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide has increased recombinant production and / or increased hinge disulfide formation compared to the wild-type IgG Fc polypeptide, as determined by SDS-PAGE analysis under reducing and / or non-reducing conditions. Embodiment 9. The mutant IgG Fc polypeptide, a) Amino acid substitutions to the wild-type feline IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 3 of SEQ ID NO: 129; and / or c) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 20 of SEQ ID NO: 129. A polypeptide according to any one of embodiments 1 to 8, including the polypeptide described in any one of embodiments 1 to 8. Embodiment 10. The mutant IgG Fc polypeptide, a) Amino acid substitutions to the wild-type feline IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains an amino acid substitution at position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains an amino acid substitution at position 3 of SEQ ID NO: 129; and / or c) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains an amino acid substitution at position 20 of SEQ ID NO: 129. A polypeptide according to any one of embodiments 1 to 9, including the polypeptide described in each of embodiments 1 to 9. Embodiment 11. The mutant IgG Fc polypeptide, a) At least one amino acid substitution to the wild-type feline IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains proline at the position corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains serine at the position corresponding to position 3 of SEQ ID NO: 129 or at position 3; and / or c) At least one amino acid substitution in the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide has an amino acid substitution containing proline at the position corresponding to position 20 of SEQ ID NO: 129 or at position 20. A polypeptide according to any one of embodiments 1 to 10, including the polypeptide described in each of embodiments 1 to 10. Embodiment 12. The polypeptide according to any one of Embodiments 1 to 11, wherein the mutant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region of a different isotype of IgG Fc polypeptide. Embodiment 13. The polypeptide according to any one of Embodiments 1 to 12, wherein the mutant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region of a wild-type feline IgG-1a Fc polypeptide, a wild-type feline IgG-1b Fc polypeptide, or a wild-type horse IgG1 Fc polypeptide. Embodiment 14. The polypeptide according to any one of Embodiments 1 to 13, wherein the mutant IgG Fc polypeptide includes SEQ ID NO: 19, SEQ ID NO: 125 or SEQ ID NO: 126, SEQ ID NO: 127, 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. Embodiment 15. A polypeptide comprising the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 125, or SEQ ID NO: 126, SEQ ID NO: 127, 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. Embodiment 16. A polypeptide comprising a mutant IgG2 Fc polypeptide having at least one amino acid substitution relative to the wild-type feline IgG2 Fc polypeptide, wherein at least one amino acid substitution is cysteine, and the mutant IgG2 Fc polypeptide can form at least one additional interchain disulfide bond with respect to the wild-type feline IgG2 Fc polypeptide. Embodiment 17. The polypeptide according to any one of Embodiments 1 to 16, wherein the mutant IgG Fc polypeptide comprises at least one amino acid substitution relative to the wild-type feline IgG Fc polypeptide, the at least one amino acid substitution being cysteine, and the mutant IgG Fc polypeptide can form at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide. Embodiment 18. The polypeptide according to any one of Embodiments 1 to 17, wherein the mutant IgG Fc polypeptide contains cysteine ​​at positions corresponding to the 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or 16th positions of SEQ ID NO: 16. Embodiment 19. The polypeptide according to any one of Embodiments 1 to 18, wherein the mutant IgG Fc polypeptide contains cysteine ​​at the position corresponding to position 14 of SEQ ID NO: 16. Embodiment 20. The polypeptide according to any one of Embodiments 1 to 19, wherein the mutant IgG Fc polypeptide contains cysteine ​​at position 14 of SEQ ID NO: 16. Embodiment 21. The variant IgG Fc polypeptide is represented by SEQ ID NOs: 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, A polypeptide according to any one of Embodiments 1 to 20, which is at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to the amino acid sequence of 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, 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: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 157. Embodiment 22. The polypeptide according to any one of Embodiments 1 to 21, wherein the mutant IgG Fc polypeptide comprises SEQ ID NO: 17. Embodiment 23. A polypeptide comprising the amino acid sequence of SEQ ID NO: 17. Embodiment 24. The mutant IgG Fc polypeptide, a) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 205 of SEQ ID NO: 1, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 1; b) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 3, and / or amino acid substitutions at the position corresponding to position 24 of SEQ ID NO: 3; c) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 4, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 4; d) Amino acid substitution at the position corresponding to position 15 of SEQ ID NO: 64, and / or amino acid substitution at the position corresponding to position 203 of SEQ ID NO: 64; e) an amino acid substitution at the position corresponding to position 199 of SEQ ID NO: 67, and / or an amino acid substitution at the position corresponding to position 200 of SEQ ID NO: 67; and / or f) Amino acid substitutions at the position corresponding to position 199 of SEQ ID NO: 68, amino acid substitutions at the position corresponding to position 200 of SEQ ID NO: 68, amino acid substitutions at the position corresponding to position 201 of SEQ ID NO: 68, and / or amino acid substitutions at the position corresponding to position 202 of SEQ ID NO: 68 A polypeptide according to any one of embodiments 1 to 23, including the polypeptide described in any one of embodiments 1 to 23. Embodiment 25. The mutant IgG Fc polypeptide, a) Amino acid substitutions at position 21 of SEQ ID NO: 1, position 23 of SEQ ID NO: 1, position 25 of SEQ ID NO: 1, position 80 of SEQ ID NO: 1, position 205 of SEQ ID NO: 1, and / or position 207 of SEQ ID NO: 1; b) Amino acid substitution at position 21 of SEQ ID NO: 3, amino acid substitution at position 23 of SEQ ID NO: 3, and / or amino acid substitution at position 24 of SEQ ID NO: 3; c) Amino acid substitution at position 21 of SEQ ID NO: 4, amino acid substitution at position 23 of SEQ ID NO: 4, amino acid substitution at position 25 of SEQ ID NO: 4, amino acid substitution at position 80 of SEQ ID NO: 4, and / or amino acid substitution at position 207 of SEQ ID NO: 4; d) Amino acid substitution at position 15 of SEQ ID NO: 64, and / or amino acid substitution at position 203 of SEQ ID NO: 64; e) an amino acid substitution at position 199 of SEQ ID NO: 67, and / or an amino acid substitution at position 200 of SEQ ID NO: 67; and / or c) Amino acid substitution at position 199 of SEQ ID NO: 68, amino acid substitution at position 200 of SEQ ID NO: 68, amino acid substitution at position 201 of SEQ ID NO: 68, and / or amino acid substitution at position 202 of SEQ ID NO: 68 A polypeptide according to any one of embodiments 1 to 24, including the polypeptide described in any one of embodiments 1 to 24. Embodiment 26. The mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4; d) Threonine or valine at position 15 of sequence number 64, and / or tyrosine or valine at position 203 of sequence number 64; e) Leucine at the position corresponding to position 199 of SEQ ID NO: 67, and / or histidine at the position corresponding to position 200 of SEQ ID NO: 67; and / or f) Leucine at position 199 of SEQ ID NO: 68, histidine at position 200 of SEQ ID NO: 68, asparagine at position 201 of SEQ ID NO: 68, and / or histidine at position 202 of SEQ ID NO: 68. A polypeptide according to any one of embodiments 1 to 25, including the polypeptide described in any one of embodiments 1 to 25. Embodiment 27. The mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4; d) Threonine or valine at position 15 of sequence number 64, and / or tyrosine or valine at position 203 of sequence number 64; e) Leucine at position 199 of SEQ ID NO: 67, and / or histidine at position 200 of SEQ ID NO: 67; and / or f) Leucine at position 199 of SEQ ID NO: 68, histidine at position 200 of SEQ ID NO: 68, asparagine at position 201 of SEQ ID NO: 68, and / or histidine at position 202 of SEQ ID NO: 68 A polypeptide according to any one of embodiments 1 to 26, including the polypeptide described in any one of embodiments 1 to 26. Embodiment 28. The mutant IgG Fc polypeptide, a) Sequence ID 5, Sequence ID 6, Sequence ID 7, Sequence ID 60, Sequence ID 61, Sequence ID 62 or Sequence ID 84; or b) Sequence ID 19, Sequence ID 71, Sequence ID 72, Sequence ID 75, or Sequence ID 76 A polypeptide according to any one of embodiments 1 to 27, comprising the amino acid sequence. Embodiment 29. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 84, SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQ ID NO: 76. Embodiment 30. The mutant IgG Fc polypeptide, a) Amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 2, or amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 3; b) Amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 69; or c) Amino acid substitution at the position corresponding to position 198 of SEQ ID NO: 80, or amino acid substitution at the position corresponding to position 198 of SEQ ID NO: 81 A polypeptide according to any one of embodiments 1 to 29, including the polypeptide described in any one of embodiments 1 to 29. Embodiment 31. The mutant IgG Fc polypeptide, a) Amino acid substitution at position 93 of SEQ ID NO: 2, or amino acid substitution at position 93 of SEQ ID NO: 3; b) Amino acid substitution at position 87 of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 69; or c) Amino acid substitution at position 198 of SEQ ID NO: 80, or amino acid substitution at position 198 of SEQ ID NO: 81 A polypeptide according to any one of embodiments 1 to 30, including the polypeptide described in any one of embodiments 1 to 30. Embodiment 32. The mutant IgG Fc polypeptide, a) Arginine at position 93 of sequence number 2, or arginine at position 93 of sequence number 3; b) Serine at the position corresponding to position 87 of SEQ ID NO: 63, serine substitution at the position corresponding to position 87 of SEQ ID NO: 65, serine at the position corresponding to position 87 of SEQ ID NO: 66, or serine at the position corresponding to position 87 of SEQ ID NO: 69; or c) Alanine at position 198 of sequence number 80, or alanine at position 198 of sequence number 81. A polypeptide according to any one of embodiments 1 to 31, including the polypeptide described in any one of embodiments 1 to 31. Embodiment 33. The mutant IgG Fc polypeptide, a) Arginine at position 93 of sequence number 2, or arginine at position 93 of sequence number 3; b) Serine at position 87 of SEQ ID NO: 63, serine at position 87 of SEQ ID NO: 65, serine at position 87 of SEQ ID NO: 66, or serine at position 87 of SEQ ID NO: 69; or c) Alanine at position 198 of sequence number 80, or alanine at position 198 of sequence number 81. A polypeptide according to any one of embodiments 1 to 32, including the polypeptide described in any one of embodiments 1 to 32. Embodiment 34. The mutant IgG Fc polypeptide, a) Sequence ID 78, Sequence ID 79, or Sequence ID 84; or b) Sequence ID 70, Sequence ID 73, Sequence ID 74 or Sequence ID 77; or c) Sequence ID 82 or Sequence ID 83 A polypeptide according to any one of embodiments 1 to 33, comprising the amino acid sequence. Embodiment 35. A polypeptide comprising the amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 82, or SEQ ID NO: 83. Embodiment 36. The mutant IgG Fc polypeptide, a) an amino acid substitution at the position corresponding to position 5 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution at the position corresponding to position 98 of SEQ ID NO: 2; or b) Amino acid substitutions at the position corresponding to position 5 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 38 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 39 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 97 of SEQ ID NO: 3, and / or amino acid substitutions at the position corresponding to position 98 of SEQ ID NO: 3 A polypeptide according to any one of embodiments 1 to 35, including the polypeptide described in any one of embodiments 1 to 35. Embodiment 37. The mutant IgG Fc polypeptide, a) Amino acid substitution at position 5 of SEQ ID NO: 2, amino acid substitution at position 38 of SEQ ID NO: 2, amino acid substitution at position 39 of SEQ ID NO: 2, amino acid substitution at position 97 of SEQ ID NO: 2, and / or amino acid substitution at position 98 of SEQ ID NO: 2; or b) Amino acid substitution at position 5 of SEQ ID NO: 3, amino acid substitution at position 38 of SEQ ID NO: 3, amino acid substitution at position 39 of SEQ ID NO: 3, amino acid substitution at position 97 of SEQ ID NO: 3, and / or amino acid substitution at position 98 of SEQ ID NO: 3 A polypeptide according to any one of embodiments 1 to 36, including the polypeptide described in any one of embodiments 1 to 36. Embodiment 38. The mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO: 3. A polypeptide according to any one of embodiments 1 to 37, including the polypeptide described in any one of embodiments 1 to 37. Embodiment 39. The mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO: 3 A polypeptide according to any one of embodiments 1 to 38, including the polypeptide described in any one of embodiments 1 to 38. Embodiment 40. The mutant IgG Fc polypeptide, a) Sequence IDs 139, 140, 141, 142, 143, 144, 145, 146, or 147; or b) Sequence IDs 148, 149, 150, 151, 152, 154, 155, 156, or 157 A polypeptide according to any one of embodiments 1 to 39, comprising the amino acid sequence. Embodiment 41. A polypeptide comprising the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 157. Embodiment 42. 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: 3, or tyrosine or tryptophan at position 138 of SEQ ID NO: 4; or b) Tyrosine or tryptophan at position 154 of SEQ ID NO: 16, tyrosine or tryptophan at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or tyrosine or tryptophan at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118 Polypeptides containing mutant IgG Fc polypeptides. Embodiment 43. The mutant IgG Fc polypeptide, 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: 3, or tyrosine or tryptophan at position 138 of SEQ ID NO: 4; or b) Tyrosine or tryptophan at position 154 of SEQ ID NO: 16, tyrosine or tryptophan at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or tyrosine or tryptophan at the position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO: 118 A polypeptide according to any one of embodiments 1 to 42, including the polypeptide described in any one of embodiments 1 to 42. Embodiment 44. The polypeptide according to any one of Embodiments 1 to 43, wherein the mutant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123. Embodiment 45. A polypeptide comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123. Embodiment 46. A continuous polypeptide comprising the polypeptide described in any one of Embodiments 1 to 45 and a glucagon-like peptide 1 (GLP1) polypeptide. Embodiment 47. A continuous polypeptide comprising the polypeptide described in any one of Embodiments 1 to 46 and a glucagon polypeptide. Embodiment 48. 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: 3, serine at position 138 of SEQ ID NO: 4, serine at position 154 of SEQ ID NO: 16, serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; 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: 3, alanine at position 140 of SEQ ID NO: 4, alanine at position 156 of SEQ ID NO: 16, alanine at position 156 of SEQ ID NO: 80 or 117, or alanine at position 156 of SEQ ID NO: 81 or 118; and / or c) Threonine at position 181 of SEQ ID NO: 1, threonine at position 180 of SEQ ID NO: 2, threonine at position 180 of SEQ ID NO: 3, threonine at position 181 of SEQ ID NO: 4, threonine at position 197 of SEQ ID NO: 16, threonine at position 197 of SEQ ID NO: 80 or 117, or threonine at position 197 of SEQ ID NO: 81 or 118 Polypeptides containing mutant IgG Fc polypeptides. Embodiment 49. The mutant 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: 3, serine at position 138 of SEQ ID NO: 4, serine at position 154 of SEQ ID NO: 16, serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; 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: 3, alanine at position 140 of SEQ ID NO: 4, alanine at position 156 of SEQ ID NO: 16, alanine at position 156 of SEQ ID NO: 80 or SEQ ID NO: 117, or alanine at position 156 of SEQ ID NO: 81 or SEQ ID NO: 118; 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: 3, threonine at position 181 of SEQ ID NO: 4, threonine at position 197 of SEQ ID NO: 16, threonine at position 197 of SEQ ID NO: 80 or SEQ ID NO: 117, or threonine at position 197 of SEQ ID NO: 81 or SEQ ID NO: 118 A polypeptide according to any one of embodiments 1 to 48, including the polypeptide described in any one of embodiments 1 to 48. Embodiment 50. The polypeptide according to any one of Embodiments 1 to 49, wherein the mutant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 124. Embodiment 51. A polypeptide comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 124. Embodiment 52. The polypeptide according to any one of Embodiments 1 to 51, wherein the polypeptide is glycosylated. Embodiment 53. The polypeptide according to any one of Embodiments 1 to 51, wherein the polypeptide is nonglycosylated. Embodiment 54. A continuous polypeptide comprising the polypeptide described in any one of Embodiments 48 to 53 and a glucagon-like peptide 1 (GLP1) polypeptide. Embodiment 55. A continuous polypeptide comprising the polypeptide described in any one of Embodiments 48 to 53 and a glucagon polypeptide. Embodiment 56. A heterodimer protein comprising the serial polypeptide described in Embodiment 46 and the serial polypeptide described in Embodiment 54. Embodiment 57. A heterodimer protein comprising the continuous polypeptide described in Embodiment 47 and the continuous polypeptide described in Embodiment 55. Embodiment 58. A continuous polypeptide or heterodimer protein according to any one of Embodiments 46, 47, or 54 to 57, wherein the GLP1 polypeptide is a wild-type GLP1 polypeptide and optionally comprises the amino acid sequence of SEQ ID NO: 85. Embodiment 59. A sequential polypeptide or heterodimer protein according to any one of Embodiments 46, 47, or 54 to 58, wherein the GLP1 polypeptide is a mutant GLP1 polypeptide. Embodiment 60. A continuous polypeptide or heterodimer protein according to any one of Embodiments 46, 47, or 54 to 59, wherein the GLP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ ID NO: 99. Embodiment 61. A continuous polypeptide or heterodimer protein according to any one of Embodiments 46, 47, or 54 to 60, wherein the glucagon polypeptide is a wild-type glucagon polypeptide and optionally comprises the amino acid sequence of SEQ ID NO: 21. Embodiment 62. A sequential polypeptide or heterodimer protein according to any one of Embodiments 46, 47, or 54 to 61, wherein the glucagon polypeptide is a mutant glucagon polypeptide. Embodiment 63. i) A first mutant canine IgG Fc polypeptide comprising at least one amino acid modification to the first wild-type canine IgG Fc polypeptide, and a second mutant canine IgG Fc polypeptide comprising at least one amino acid modification to the second wild-type canine IgG Fc polypeptide; or ii) A first mutant cat IgG Fc polypeptide comprising at least one amino acid modification to the first wild-type cat IgG Fc polypeptide, and a second mutant cat IgGFc polypeptide comprising at least one amino acid modification to the second wild-type cat IgG Fc polypeptide. A heterodimeric protein containing, a) The first mutant canine IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 138 of SEQ ID NO: 1, position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position 138 of SEQ ID NO: 4; b) The second mutant canine IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 138, 140 and / or 181 of SEQ ID NO: 1, positions 137, 139 and / or 180 of SEQ ID NO: 2, positions 137, 139 and / or 180 of SEQ ID NO: 3, and positions 138, 140 and / or 181 of SEQ ID NO: 4; c) The first mutant cat IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; and / or d) The second mutant cat IgG Fc polypeptide contains amino acid substitutions at positions 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 Heterodimeric protein. Embodiment 64. A heterodimer protein according to Embodiment 63, wherein the first wild-type canine IgG Fc polypeptide and the second wild-type canine IgG Fc polypeptide are of the same IgG subtype, and / or the first wild-type feline IgG Fc polypeptide and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype. Embodiment 65. A heterodimer protein according to Embodiment 63, wherein the first wild-type canine IgG Fc polypeptide and the second wild-type canine IgG Fc polypeptide are of different IgG subtypes, and / or the first wild-type feline IgG Fc polypeptide and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype. Embodiment 66. a) The first mutant canine IgG Fc polypeptide contains tyrosine or tryptophan at the position corresponding to position 138 of SEQ ID NO: 1, position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position 138 of SEQ ID NO: 4; and / or b) The first mutant cat IgG Fc polypeptide contains tryptophan at the position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. A heterodimer protein according to any one of embodiments 63 to 65. Embodiment 67. a) The second mutant canine IgG Fc polypeptide contains serine at the position corresponding to position 138 of SEQ ID NO: 1, alanine at the position corresponding to position 140, and / or threonine at the position corresponding to position 181; serine at the position corresponding to position 137 of SEQ ID NO: 2, alanine at the position corresponding to position 139, and / or threonine at the position corresponding to position 180; serine at the position corresponding to position 137 of SEQ ID NO: 3, alanine at the position corresponding to position 139, and / or threonine at the position corresponding to position 180; and / or serine at the position corresponding to position 138 of SEQ ID NO: 4, alanine at the position corresponding to position 140, and / or threonine at the position corresponding to position 181; and / or b) The second mutant cat IgG Fc polypeptide contains serine at the position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117 or SEQ ID NO: 118, alanine at the position corresponding to position 156, and / or threonine at the position corresponding to position 197. A heterodimer protein according to any one of embodiments 63 to 66. Embodiment 68. a) The first mutant canine IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113 or SEQ ID NO: 115; and / or b) The first mutant cat IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123. A heterodimer protein according to any one of embodiments 63 to 67. Embodiment 69. a) The second mutant canine IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114 or SEQ ID NO: 116; and / or b) The second mutant cat IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 123. A heterodimer protein according to any one of embodiments 63 to 68. Embodiment 70. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 69, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises at least one additional amino acid modification compared to the wild-type IgG Fc polypeptide and has increased binding affinity to protein A compared to the wild-type IgG Fc polypeptide. Embodiment 71. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 205 of SEQ ID NO: 1, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 1; b) an amino acid substitution at the position corresponding to position 21 of SEQ ID NO: 3, an amino acid substitution at the position corresponding to position 23 of SEQ ID NO: 3, and / or an amino acid substitution at the position corresponding to position 24 of SEQ ID NO: 3; or c) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 4, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 4 A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 70, including the following: Embodiment 72. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) Amino acid substitutions at position 21 of SEQ ID NO: 1, position 23 of SEQ ID NO: 1, position 25 of SEQ ID NO: 1, position 80 of SEQ ID NO: 1, position 205 of SEQ ID NO: 1, and / or position 207 of SEQ ID NO: 1; b) an amino acid substitution at position 21 of SEQ ID NO: 3, an amino acid substitution at position 23 of SEQ ID NO: 3, and / or an amino acid substitution at position 24 of SEQ ID NO: 3; or c) Amino acid substitution at position 21 of SEQ ID NO: 4, amino acid substitution at position 23 of SEQ ID NO: 4, amino acid substitution at position 25 of SEQ ID NO: 4, amino acid substitution at position 80 of SEQ ID NO: 4, and / or amino acid substitution at position 207 of SEQ ID NO: 4 A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 71, including the following: Embodiment 73. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; or c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4. A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 72, including the following: Embodiment 74. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; or c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4 A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 73, including the following: Embodiment 75. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 74, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises at least one additional amino acid modification compared to the wild-type IgG Fc polypeptide, and has reduced binding affinity to CD16 compared to the wild-type IgG Fc polypeptide. Embodiment 76. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) an amino acid substitution at the position corresponding to position 5 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution at the position corresponding to position 98 of SEQ ID NO: 2; or b) Amino acid substitutions at the position corresponding to position 5 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 38 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 39 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 97 of SEQ ID NO: 3, and / or amino acid substitutions at the position corresponding to position 98 of SEQ ID NO: 3 A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 75, including the following: Embodiment 77. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) Amino acid substitution at position 5 of SEQ ID NO: 2, amino acid substitution at position 38 of SEQ ID NO: 2, amino acid substitution at position 39 of SEQ ID NO: 2, amino acid substitution at position 97 of SEQ ID NO: 2, and / or amino acid substitution at position 98 of SEQ ID NO: 2; or b) Amino acid substitution at position 5 of SEQ ID NO: 3, amino acid substitution at position 38 of SEQ ID NO: 3, amino acid substitution at position 39 of SEQ ID NO: 3, amino acid substitution at position 97 of SEQ ID NO: 3, and / or amino acid substitution at position 98 of SEQ ID NO: 3 A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 76, including the following: Embodiment 78. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO: 3. A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 77, including the following: Embodiment 79. A mutant IgG Fc polypeptide, a first mutant IgG Fc polypeptide and / or a second mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO: 3 A polypeptide, serial polypeptide, or heterodimer protein according to any one of embodiments 42 to 78, including the following: Embodiment 80. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 79, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises at least one additional amino acid modification compared to the wild-type canine IgG Fc polypeptide, and has reduced binding affinity to C1q compared to the wild-type canine IgG Fc polypeptide. Embodiment 81. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 80, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide include an amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 3. Embodiment 82. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 81, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide include an amino acid substitution at position 93 of SEQ ID NO: 2 or at position 93 of SEQ ID NO: 3. Embodiment 83. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 82, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide contain 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: 3. Embodiment 84. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of Embodiments 42 to 83, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide contain arginine at position 93 of SEQ ID NO: 2, or arginine at position 93 of SEQ ID NO: 3. Embodiment 85. A polypeptide, serial polypeptide, or heterodimer protein according to any one of Embodiments 1 to 84, wherein the polypeptide is an antibody, an antibody fusion, or a fusion polypeptide. Embodiment 86. a) The first glucagon-like peptide-1 (GLP1) polypeptide (GLP1A); b) First linker (L1); c) Fc polypeptide (Fc) of companion animal species; d) A second linker (L2) if applicable; and e) In some cases, a second GLP1 polypeptide (GLP1B) A continuous polypeptide containing [a specific component]. Embodiment 87. Formula (I): GLP1A-L1-Fc; or Formula (II): Fc-L1-GLP1A A continuous polypeptide according to embodiment 65, comprising: Embodiment 88. Equation (III): GLP1A-L1-Fc-L2-GLP1B A continuous polypeptide according to embodiment 65, comprising: Embodiment 89. A continuous polypeptide according to any one of Embodiments 86 to 88, wherein GLP1B, if present, has the same amino acid sequence as GLP1A. Embodiment 90. a) Glucagon-like peptide 1 (GLP1) polypeptide; b) First linker (L1); c) Fc polypeptide (Fc); d) Second linker (L2); and e) Glucagon polypeptide (Gluc) A continuous polypeptide containing [a specific component]. Embodiment 91. Formula (IV): GLP1-L1-Fc-L2-Gluc; or Formula (V): Gluc-L1-Fc-L2-GLP1 A continuous polypeptide according to embodiment 90, including the following: Embodiment 92. A continuous polypeptide according to any one of Embodiments 86 to 91, comprising GLP1A, GLP1 and / or GLP1B, if present, a wild-type GLP1 polypeptide. Embodiment 93. A sequential polypeptide according to any one of Embodiments 86 to 92, comprising, if present, a variant GLP1 polypeptide, GLP1A, GLP1, and / or GLP1B. Embodiment 94. A continuous polypeptide according to any one of Embodiments 86 to 93, wherein GLP1A, GLP1 and / or GLP1B, if present, comprises the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ ID NO: 99. Embodiment 95. A continuous polypeptide according to any one of Embodiments 86 to 94, wherein the glucagon polypeptide comprises a wild-type glucagon polypeptide and optionally comprises the amino acid sequence of SEQ ID NO: 21. Embodiment 96. A sequential polypeptide according to any one of Embodiments 86 to 95, wherein the glucagon polypeptide is a mutant glucagon polypeptide. Embodiment 97. The continuous polypeptide according to any one of Embodiments 86 to 96, wherein the Fc polypeptide is human IgG Fc. Embodiment 98. A continuous polypeptide according to any one of Embodiments 86 to 97, wherein the Fc polypeptide is human IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc. Embodiment 99. The continuous polypeptide according to any one of Embodiments 86 to 98, wherein the Fc polypeptide is the Fc of a companion animal species. Embodiment 100. The Fc polypeptide is a) Canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc; b) Horse 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 A continuous polypeptide according to any one of embodiments 86 to 97 or 99, including the following: Embodiment 101. A serial polypeptide according to any one of Embodiments 86 to 100, wherein the Fc polypeptide is a wild-type IgG Fc polypeptide. Embodiment 102. A serial polypeptide according to any one of Embodiments 86 to 100, wherein the Fc polypeptide is a mutant IgG Fc polypeptide. Embodiment 103. A continuous polypeptide according to any one of Embodiments 86 to 102, wherein the Fc polypeptide comprises a polypeptide, a continuous polypeptide, or a heterodimer protein according to any one of Embodiments 1 to 84. Embodiment 104. A serial polypeptide according to any one of Embodiments 85 to 102, having a longer serum half-life than the wild-type GLP1 polypeptide. Embodiment 105. A continuous polypeptide according to any one of Embodiments 86 to 104, wherein L1 and L2, if present, are independently flexible linkers. Embodiment 106. A continuous polypeptide according to any one of Embodiments 86 to 105, wherein the L1 and L2 amino acid sequences, if present, independently comprise 100%, at least 95%, at least 90%, and at least 85% serine and / or glycine amino acid residues, respectively. Embodiment 107. A continuous polypeptide according to any one of Embodiments 86 to 106, comprising an elongated portion at the C-terminus. Embodiment 108. A continuous polypeptide according to any one of Embodiments 86 to 107, comprising 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. Embodiment 109. A continuous polypeptide according to any one of Embodiments 86 to 108, comprising the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95 at its C-terminus. Embodiment 110. a) The amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106; or b) Amino acid sequences of SEQ ID NOs. 52, 53, 54, 55, 56, 57, 58, or 59 A continuous polypeptide according to any one of embodiments 86 to 109, including the following: Embodiment 111. A polypeptide comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. Embodiment 112. A polypeptide, heterodimer protein, or serial polypeptide according to any one of Embodiments 1 to 111, wherein at least one amino acid modification or substitution comprises an amino acid substitution with an amino acid derivative. Embodiment 113. An isolated nucleic acid encoding a polypeptide, heterodimer protein, or sequential polypeptide as described in any one of Embodiments 1 to 112. Embodiment 114. A host cell containing the nucleic acid described in Embodiment 113. Embodiment 115. A method for producing a polypeptide, comprising culturing the host cells described in Embodiment 114 and isolating the polypeptide. Embodiment 116. A pharmaceutical composition comprising a polypeptide, heterodimer protein, or serial polypeptide according to any one of Embodiments 1 to 112 and a pharmaceutically acceptable carrier. Embodiment 117. A method for increasing cAMP production in cells, comprising exposing cells to a polypeptide, heterodimer protein, serial polypeptide, or pharmaceutical composition described in any one of Embodiments 1 to 112 or 116, under conditions that allow binding of the polypeptide, heterodimer protein, or serial polypeptide to GLP1R. Embodiment 118. The method according to Embodiment 117, wherein cells are exposed ex vivo to a polypeptide, heterodimer protein, serial polypeptide, or pharmaceutical composition. Embodiment 119. The method according to Embodiment 117, wherein cells are exposed in vivo to a polypeptide, heterodimeric protein, serial polypeptide, or pharmaceutical composition. Embodiment 120. The method according to any one of Embodiments 118 to 119, wherein the cells are human cells, canine cells, feline cells, or equine cells. Embodiment 121. A method for delivering a polypeptide, comprising parenterally administering a polypeptide, heterodimer protein, continuous polypeptide, or pharmaceutical composition described in any one of Embodiments 1 to 112 or 116. Embodiment 122. A method for delivering a polypeptide, comprising administering the polypeptide, heterodimer protein, continuous polypeptide, or pharmaceutical composition described in any one of Embodiments 1 to 112 or 116, via an intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation route. Embodiment 123. A method for treating a subject having diabetes or obesity, comprising administering a therapeutically effective amount of a polypeptide, heterodimer protein, continuous polypeptide, or pharmaceutical composition described in any one of Embodiments 1 to 112 or 116 to the subject. Embodiment 124. The method according to Embodiment 123, comprising administering insulin, a DPP4 inhibitor, an SGLT2 inhibitor, biguanides, sulfonylurea, a biguanides sulfonylureas meglitinide derivative, an alpha-glucosidase inhibitor, a thiazolidinedione (TZD), an amirinomimetic, a bile acid chelating agent, or a dopamine agonist. Embodiment 125. The method according to any one of Embodiments 121 to 124, wherein the subject is a human subject. Embodiment 126. The method according to any one of Embodiments 121 to 124, wherein the subject is a companion animal species. Embodiment 127. The method according to Embodiment 126, wherein the companion animal species is a dog, horse, or cat. [Brief explanation of the drawing]

[0007] [Figure 1] Alignment of canine IgG-A, B, C, and D Fc sequences is shown. The boxes indicate regions likely to be in contact with protein A. [Figure 2A-B]Figure 2A shows SDS-PAGE analysis of GLP1-G8 / GLP-2G_III_WTfeIgG2 (SEQ ID NO: 23; "GLP1 A variant" in this figure) and GLP1-G8_I_WTfeIgG2 (SEQ ID NO: 24; "GLP1 B variant" in this figure) having a wild-type feline IgG2 hinge with one disulfide bond, in the absence and presence of the reducing agent (DTT). Figure 2B shows SDS-PAGE analysis of GLP1-G8 / GLP-2G_III_VARfeIgG2 (SEQ ID NO: 25; "GLP1 MA variant" in this figure) and GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26; "GLP1 MB variant" in this figure) having a mutant feline IgG2 hinge with two disulfide bonds, in the absence and presence of the reducing agent (DTT). [Figure 3] Figure 3 shows the cAMP CHO-K1 GLP1R bioassay for evaluating the activity of GLP1-G8 / GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) and GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) compared to controls (GLP1(7-37) and Extendin-4). [Figure 4] This figure shows a cell-based bioassay to evaluate the activity of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) (labeled "GLP1-B" in this figure) after storage of CHOK1-GLP1R cells in cAMP-glo (n=2) for one year. [Figure 5] This figure shows Western blot analysis of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) (labeled "GLP1B" in this figure) after incubation in serum at 37°C for 24 hours (lane 1), in PBS at 37°C for 24 hours (lane 2), and in PBS at 4°C for 24 hours (lane 9). A mouse anti-GLP1 antibody was used. [Figure 6] This is a plot of the time-course serum concentrations of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) in five cats after subcutaneous administration, as measured by quantitative ELISA. [Figure 7]This is a plot of serum GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) concentrations over time in five cats after subcutaneous administration, as measured by a cell-based activity assay. The mean AUC from 0 to 168 hours was approximately 840 μg(h) / mL, and the mean t1 / 2 was 36 hours.

[0008] Array description TIFF2026048634000002.tif251170TIFF2026048634000003.tif250170TIFF2026048634000004.tif250170TIFF2026048634000005.tif249170TIFF2026048634000006.tif243170TIFF2026048634000007.tif243170TIFF2026048634000008.tif249170TIFF2026048634000009.tif250170TIFF2026048634000010.tif255170TIFF2026048634000011.tif249170TIFF2026048634000012.tif250170TIFF2026048634000013.tif250170TIFF2026048634000014.tif244170TIFF2026048634000015.tif250170TIFF2026048634000016.tif250170TIFF2026048634000017.tif250170TIFF2026048634000018.tif250170TIFF2026048634000019.tif249170TIFF2026048634000020.tif250170TIFF2026048634000021.tif249170TIFF2026048634000022.tif250170TIFF2026048634000023.tif250170TIFF2026048634000024.tif250170TIFF2026048634000025.tif250170TIFF2026048634000026.tif249170TIFF2026048634000027.tif251170TIFF2026048634000028.tif251170TIFF2026048634000029.tif255170TIFF2026048634000030.tif251170TIFF2026048634000031.tif250170TIFF2026048634000032.tif249170TIFF2026048634000033.tif250170TIFF2026048634000034.tif251170TIFF2026048634000035.tif250170TIFF2026048634000036.tif250170TIFF2026 048634000037.tif250170TIFF2026048634000038.tif243170TIFF2026048634000039.tif2 50170TIFF2026048634000040.tif250170TIFF2026048634000041.tif250170TIFF20260486 34000042.tif250170TIFF2026048634000043.tif250170TIFF2026048634000044.tif71170. [Modes for carrying out the invention]

[0009] Mutant IgG Fc polypeptides from companion animals such as dogs, horses, and cats are described. In some embodiments, the mutant IgG Fc polypeptides have increased binding to protein A, decreased binding to C1q, decreased binding to CD16, increased stability, increased recombinant production, increased hinged disulfide formation, and / or form heterodimeric polypeptides. In some embodiments, antibodies, antibody fragments, or fusion proteins contain mutant IgG Fc polypeptides. Methods for producing or purifying mutant IgG Fc polypeptides and methods for administering mutant IgG Fc polypeptides to companion animals are also provided in assays.

[0010] Also provided are various embodiments relating to sequential polypeptides and heterodimer polypeptides comprising one or more mutant GLP1 polypeptides having an improved serum half-life. In some embodiments, the sequential polypeptide or heterodimer polypeptide comprises the GLP1 polypeptide and the glucagon polypeptide as dual agonists of the GLP1 receptor and the glucagon receptor. In some embodiments, such polypeptides may be used to treat, for example, diabetes, obesity, or related conditions in companion animals such as dogs, cats, and horses.

[0011] For the convenience of the reader, the following definitions of terms used herein are provided.

[0012] When used in this specification, K D Numerical terms such as these are calculated based on scientific measurements and are therefore subject to a certain degree of measurement error. In some cases, numerical terms may include numbers rounded to the nearest significant figure.

[0013] As used herein, the singular form ("a" or "an") means "at least one" or "one or more" unless otherwise specified. As used herein, the term "or" means "and / or" unless otherwise specified. In the context of multiple dependent claims, use "or" when referring back to other claims, and refer to those claims only in an alternative form.

[0014] Exemplary mutant IgG Fc polypeptide Novel mutant IgG Fc polypeptides are provided, for example, mutant IgG Fc polypeptides for increased binding to protein A, decreased binding to C1q, decreased binding to CD16, increased stability, increased recombinant production, increased hinged disulfide formation, and / or for forming heterodimer protein assays.

[0015] "Amino acid sequence" means the sequence of amino acid residues of a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to the minimum length. Such polymers of amino acid residues may include native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and their fragments are included in the definition. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, in this disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and (generally conserved in nature) substitutions of the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as due to mutations in the host producing the protein or errors resulting from PCR amplification.

[0016] A “fragmented crystalline polypeptide” or “Fc polypeptide” is a part of an antibody molecule that interacts with effector molecules and cells. It is composed of the C-terminal portion of an immunoglobulin heavy chain. As used herein, an Fc polypeptide includes a fragment of the Fc domain having one or more biological activities in the whole Fc polypeptide. In some embodiments, the biological activity of the Fc polypeptide is its ability to bind to FcRn. In some embodiments, the biological activity of the Fc polypeptide is its ability to bind to C1q. In some embodiments, the biological activity of the Fc polypeptide is its ability to bind to CD16. In some embodiments, the biological activity of the Fc polypeptide is its ability to bind to protein A. The “effector function” of the Fc polypeptide is an action or activity performed whole or partially by any antibody in response to a stimulus, including complement binding and / or ADCC (antibody-dependent cytotoxicity) induction.

[0017] "IgX Fc" 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. Therefore, "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 contains hinge, CH2, and CH3, but does not contain CH1 or CL. In some embodiments, the IgG Fc polypeptide contains CH2 and CH3, but does not contain CH1, hinge, or CL. In some embodiments, the IgG Fc polypeptide contains CH1, hinge, CH2, and CH3, and CL may or may not be present. "IgX-N Fc" or "IgGXN Fc" (where "N" indicates a subclass) indicates that the Fc polypeptide originates from a specific subclass of the 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.).

[0018] A "hinge" refers to any portion of an Fc polypeptide or mutant Fc polypeptide that is proline-rich and contains at least one cysteine ​​residue located between CH1 and CH2 in the full-length heavy chain constant region.

[0019] In some embodiments, the hinge can form a disulfide bond with a hinge region of a separate Fc polypeptide, or with a separate Fc polypeptide, within the same hinge region, within the same 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.

[0020] The term "companion animal species" refers to animals suitable to serve as human companions. In some embodiments, companion animal species are canines (or dogs), felines (or cats), or equines (or horses). In some embodiments, companion animal species are small mammals such as dogs, cats, rabbits, ferrets, guinea pigs, and rodents. In some embodiments, companion animal species are domesticated animals such as horses, cattle, and pigs.

[0021] In some embodiments, the IgX Fc polypeptide or IgX-N Fc polypeptide is derived from a companion animal such as a dog, cat, or 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 examples, the IgG Fc polypeptide is isolated from a cat γ heavy chain such as IgG1a, IgG1b, or IgG2. In some examples, the IgG Fc polypeptide is isolated from a horse γ heavy chain such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7.

[0022] The terms "IgX Fc" and "IgX Fc polypeptide" include wild-type IgX Fc polypeptide and mutant IgX Fc polypeptide unless otherwise specified.

[0023] "Wild-type" refers to the unmutated form or fragment of a polypeptide found in nature. Wild-type polypeptides may also be produced through recombinant DNA synthesis.

[0024] In some embodiments, the wild-type IgG Fc polypeptide contains the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 100, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 117, and SEQ ID NO: 118.

[0025] A “mutant” is a polypeptide that differs from a reference polypeptide by one or more non-natural amino acid substitutions, deletions, and / or additions. In some embodiments, the mutant retains at least one biological activity of the reference polypeptide. In some embodiments, the mutant (e.g., mutant canine IgG-A Fc, mutant canine IgG-C Fc, mutant canine IgG-D Fc, mutant horse IgG2 Fc, mutant horse IgG5 Fc, or mutant horse IgG6 Fc) has an activity that is qualitatively lacking in the reference polypeptide. For example, in some embodiments, mutant canine IgG-A Fc, mutant canine IgG-C Fc, mutant canine IgG-D Fc, mutant horse IgG2 Fc, mutant horse IgG5 Fc, or mutant horse IgG6 Fc bind to protein A.

[0026] As used herein, “amino acid sequence identity percentage (%)” and “homology” with respect to nucleic acid molecules or polypeptide sequences mean the percentage of nucleic acid or amino acid residues in a reference sequence that are identical to the nucleic acid or amino acid residues in a particular nucleic acid molecule or polypeptide sequence after the sequences have been aligned and gaps introduced as necessary to obtain the maximum sequence identity percentage, and no conservative substitutions are considered part of sequence identity. Alignment for determining the sequence identity percentage can be performed by various methods within the scope of the art, e.g., BLAST, BLAST-2, ALIGN, or MEGALINE TMThis can be achieved using publicly available computer software such as DNASTAR software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to obtain the maximum alignment over the entire length of the sequences being compared.

[0027] In some embodiments, the variant has at least about 50% sequence identity with a reference nucleic acid molecule or polypeptide, after aligning the sequences and introducing gaps as necessary to obtain the maximum sequence identity percentage, and after disregarding any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N or C terminus of the polypeptide. In some embodiments, the 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 a reference nucleic acid or polypeptide.

[0028] A "point mutation" is a mutation that involves a single amino acid residue. Mutations can include the loss of an amino acid, the substitution of one amino acid residue with another, or the insertion of an additional amino acid residue.

[0029] "Amino acid substitution" refers to replacing one amino acid in a polypeptide with another. In some embodiments, amino acid substitutions are conservative substitutions. Non-limiting and exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions can be introduced into the target molecule, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, improved ADCC or CDC, or improved pharmacokinetics.

[0030] Table 2 TIFF2026048634000045.tif246170

[0031] Amino acids can be grouped according to their common side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0032] Non-conservative substitutions inevitably involve exchanging an element from one class with an element from another class.

[0033] As used herein, “mutant IgG Fc” is an IgG Fc polypeptide that differs from a reference IgG Fc polypeptide by one or more mino acid substitutions, deletions, and / or additions, and substantially retains at least one biological activity of the reference IgG Fc polypeptide.

[0034] As used herein, “amino acid derivative” means 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 pyrrolidine found in some microorganisms) and non-natural amino acids. Exemplary amino acid derivatives include, but are not limited to, those commercially available from chemical manufacturers (e.g., sigmaaldrich.com / chemistry / chemistry-products.html?TablePage=16274965, accessed May 6, 2017, incorporated herein by reference). One or more amino acid derivatives can be incorporated into a polypeptide at specific positions using translation systems that utilize host cells, orthogonal aminoacyl-tRNA synthetases derived from bacterial synthetases, orthogonal tRNAs, and amino acid derivatives. For further explanation, see, for example, U.S. Patent No. 9624485.

[0035] In some embodiments, the mutant IgG Fc polypeptide includes amino acid substitution with an amino acid derivative. In some embodiments, the amino acid derivative is an alanine derivative, cysteine ​​derivative, aspartic acid derivative, glutamic acid derivative, phenylalanine derivative, glycine derivative, histidine derivative, isoleucine derivative, lysine derivative, leucine derivative, methionine derivative, asparagine derivative, proline derivative, glutamine derivative, arginine derivative, serine derivative, threonine derivative, valine derivative, tryptophan derivative, or tyrosine derivative.

[0036] In some embodiments, the mutant IgG Fc polypeptide includes a mutant IgG Fc polypeptide from a companion animal species. In some embodiments, the mutant IgG Fc polypeptide includes a mutant canine IgG Fc polypeptide, a mutant horse IgG Fc polypeptide, or a feline IgG Fc polypeptide.

[0037] Exemplary variant IgG Fc polypeptide with modified protein A binding In some embodiments, the mutant IgG Fc polypeptide has a modified protein A binding affinity. In some embodiments, the mutant IgG Fc polypeptide has an increased binding affinity to protein A. In some embodiments, the mutant IgG Fc polypeptide can be purified using protein A column chromatography.

[0038] In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 21, 23, 25, 80, 205, and / or 207 of SEQ ID NO: 1. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 21, 23, and / or 24 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 21, 23, 25, 80, and / or 207 of SEQ ID NO: 4.

[0039] In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 15 and / or 203 of SEQ ID NO: 64. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 199 and / or 200 of SEQ ID NO: 67. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 199, 200, 201 and / or 202 of SEQ ID NO: 68.

[0040] In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions 21, 23, 25, 80, 205, and / or 207 of SEQ ID NO: 1. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions 21, 23, and / or 24 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions 21, 23, 25, 80, and / or 207 of SEQ ID NO: 3.

[0041] In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 15 and / or 203 of SEQ ID NO: 64. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 199, 23 and / or 200 of SEQ ID NO: 67. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 199, 200, 201 and / or 202 of SEQ ID NO: 68.

[0042] In some embodiments, the mutant IgG Fc polypeptide contains 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 mutant IgG Fc polypeptide contains threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 3, and / or histidine at position 207 of SEQ ID NO: 4.

[0043] In some embodiments, the mutant IgG Fc polypeptide contains threonine or valine at the position corresponding to position 15 of SEQ ID NO: 64, and / or tyrosine or valine at the position corresponding to position 203 of SEQ ID NO: 64. In some embodiments, the mutant IgG Fc polypeptide contains leucine at the position corresponding to position 199 of SEQ ID NO: 67, and / or histidine at the position corresponding to position 200 of SEQ ID NO: 67. In some embodiments, the mutant IgG Fc polypeptide contains isoleucine at the position corresponding to position 199 of SEQ ID NO: 68, histidine at the position corresponding to position 200 of SEQ ID NO: 68, asparagine at the position corresponding to position 201 of SEQ ID NO: 68, and / or histidine at the position corresponding to position 202 of SEQ ID NO: 68.

[0044] In some embodiments, the mutant IgG Fc polypeptide contains 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 mutant IgG Fc polypeptide contains threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4.

[0045] In some embodiments, the mutant IgG Fc polypeptide contains threonine or valine at position 15 of SEQ ID NO: 64, and / or tyrosine or valine at position 203 of SEQ ID NO: 64. In some embodiments, the mutant IgG Fc polypeptide contains leucine at position 199 of SEQ ID NO: 67, and / or histidine at position 200 of SEQ ID NO: 67. In some embodiments, the mutant IgG Fc polypeptide contains isoleucine at position 199 of SEQ ID NO: 68, histidine at position 200 of SEQ ID NO: 68, asparagine at position 201 of SEQ ID NO: 68, and / or histidine at position 202 of SEQ ID NO: 68.

[0046] In some embodiments, the mutant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 84. In some embodiments, the mutant IgG Fc polypeptide comprises SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQ ID NO: 76.

[0047] Exemplary variant IgG Fc polypeptide with modified CD16 binding In some embodiments, the mutant IgG Fc polypeptide has a modified CD16 binding affinity. In some embodiments, the mutant IgG Fc polypeptide has a reduced binding affinity to CD16. In some embodiments, the mutant IgG Fc may have a reduced ADCC immune response.

[0048] In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 3.

[0049] In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains amino acid substitutions at positions 5, 38, 39, 97, and / or 98 of SEQ ID NO: 3.

[0050] In some embodiments, the mutant IgG Fc polypeptide contains proline at position 5 of SEQ ID NO: 2, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98. In some embodiments, the mutant IgG Fc polypeptide contains proline at position 5 of SEQ ID NO: 3, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98.

[0051] In some embodiments, the mutant IgG Fc polypeptide contains proline at position 5 of SEQ ID NO: 2, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98. In some embodiments, the mutant IgG Fc polypeptide contains proline at position 5 of SEQ ID NO: 3, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98.

[0052] In some embodiments, the mutant IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 157.

[0053] Exemplary mutant IgG Fc polypeptide with modified C1q binding In some embodiments, the mutant IgG Fc polypeptide has a modified C1q binding affinity. In some embodiments, the mutant IgG Fc polypeptide has a reduced binding affinity to C1q. In some embodiments, the mutant IgG Fc polypeptide may have reduced complement binding. In some embodiments, the mutant IgG Fc may have a reduced complement-mediated immune response.

[0054] In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 63. In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 65. In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 66. In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 198 of SEQ ID NO: 80. In some embodiments, the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 198 of SEQ ID NO: 81.

[0055] In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 93 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 93 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 87 of SEQ ID NO: 63. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 87 of SEQ ID NO: 65. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 87 of SEQ ID NO: 66. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 198 of SEQ ID NO: 80. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 198 of SEQ ID NO: 81.

[0056] In some embodiments, the mutant IgG Fc polypeptide contains arginine at the position corresponding to position 93 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains arginine at the position corresponding to position 93 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 87 of SEQ ID NO: 63. In some embodiments, the mutant IgG Fc polypeptide contains a serine substitution at the position corresponding to position 87 of SEQ ID NO: 65. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 87 of SEQ ID NO: 66. In some embodiments, the mutant IgG Fc polypeptide contains alanine at the position corresponding to position 198 of SEQ ID NO: 80. In some embodiments, the mutant IgG Fc polypeptide contains alanine at the position corresponding to position 198 of SEQ ID NO: 81.

[0057] In some embodiments, the mutant IgG Fc polypeptide contains arginine at position 93 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 93 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains serine corresponding to position 87 of SEQ ID NO: 63. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 87 of SEQ ID NO: 65. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 87 of SEQ ID NO: 66. In some embodiments, the mutant IgG Fc polypeptide contains alanine at position 198 of SEQ ID NO: 80. In some embodiments, the mutant IgG Fc polypeptide contains alanine at position 198 of SEQ ID NO: 81.

[0058] In some embodiments, the mutant IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 84. In some embodiments, the mutant IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 77. In some embodiments, the mutant IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83.

[0059] Exemplary mutant IgG Fc polypeptide with modified interchain disulfide bonds In some embodiments, the mutant cat IgG Fc polypeptide has at least one additional interchain disulfide linkage compared to the wild-type cat IgG Fc polypeptide. In some embodiments, the mutant cat IgG Fc polypeptide has at least one additional interchain disulfide linkage in the hinge region. In some embodiments, the mutant cat IgG Fc polypeptide having at least one additional interchain disulfide linkage has increased interchain stability compared to the wild-type cat IgG Fc polypeptide. In some embodiments, the mutant IgG Fc polypeptide has at least one amino acid modification to the hinge region compared to the wild-type IgG Fc polypeptide. In some embodiments, the wild-type IgG Fc polypeptide is a wild-type cat or horse IgG Fc polypeptide. In some embodiments, the mutant IgG Fc polypeptide contains the hinge region or a portion of the hinge region of a different isotype of IgG Fc polypeptide. In some embodiments, the mutant IgG Fc polypeptide contains the hinge region of a wild-type cat IgG-1a Fc polypeptide, a wild-type cat IgG-1b Fc polypeptide, or a wild-type horse IgG1 Fc polypeptide. In some embodiments, the mutant IgG2 Fc polypeptide exhibits increased recombinant production and / or increased hinged disulfide formation compared to the wild-type IgG Fc polypeptide. In some embodiments, the increased recombinant production and / or increased hinged disulfide formation can be determined by SDS-PAGE analysis under reducing and / or non-reducing conditions.

[0060] In some embodiments, the mutant IgG Fc polypeptide contains cysteine ​​at positions 8, 9, 10, 11, 12, 13, 14, 15, or 16 of SEQ ID NO: 16. In some embodiments, the mutant IgG Fc polypeptide contains cysteine ​​at positions 8, 9, 10, 11, 12, 13, 14, 15, or 16 of SEQ ID NO: 16. In some embodiments, the mutant IgG Fc polypeptide contains SEQ ID NO: 17.

[0061] In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 3 and / or position 20 of SEQ ID NO: 129.

[0062] In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. In some embodiments, the mutant IgG Fc polypeptide contains an amino acid substitution at position 3 and / or corresponding to position 20 of SEQ ID NO: 129.

[0063] In some embodiments, the mutant IgG Fc polypeptide contains proline at the position corresponding to position 16 in SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 3 in SEQ ID NO: 129, and / or proline at the position corresponding to position 20.

[0064] In some embodiments, the mutant IgG Fc polypeptide contains proline at position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 3 and / or proline at position 20 of SEQ ID NO: 129.

[0065] In some embodiments, the variant IgG Fc polypeptide includes SEQ ID NO: 19, SEQ ID NO: 125 or SEQ ID NO: 126, SEQ ID NO: 127, 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.

[0066] Exemplary heterodimer polypeptide variant IgG Fc polypeptide In some embodiments, the heterodimer polypeptide provided herein is a bispecific antibody. A bispecific antibody has binding specificity to two different epitopes or target molecules. In some embodiments, a bispecific antibody binds to two different epitopes of the same target molecule. A bispecific antibody may be a full-length antibody or an antibody fragment.

[0067] In some embodiments, the heterodimer polypeptide comprises a first mutant IgG Fc polypeptide containing a “knob” mutation and a second mutant IgG Fc polypeptide containing a “hole” mutation. Non-limiting and exemplary knob and hole mutations are described, for example, in Merchant, AM et al. An efficient route to human bispecific IgG. Nat Biotechnol, 16(7):677-81 (1998).

[0068] In some embodiments, the mutant canine or mutant cat IgG Fc polypeptide contains a knob mutation. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at the position corresponding to position 138 of SEQ ID NO: 1. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at the position corresponding to position 137 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at the position corresponding to position 137 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at the position corresponding to position 138 of SEQ ID NO: 4. In some embodiments, the mutant IgG Fc polypeptide contains tryptophan at the position corresponding to position 154 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.

[0069] In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at position 138 of SEQ ID NO: 1. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at position 137 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at position 137 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains tyrosine or tryptophan at position 138 of SEQ ID NO: 4. In some embodiments, the mutant IgG Fc polypeptide contains tryptophan at position 154 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.

[0070] In some embodiments, the mutant IgG Fc polypeptide containing a knob mutation comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123.

[0071] In some embodiments, the mutant dog or mutant cat IgG Fc polypeptide contains a whole mutation. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 138 of SEQ ID NO: 1, alanine at the position corresponding to position 140, and / or threonine at the position corresponding to position 181. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 137 of SEQ ID NO: 2, alanine at the position corresponding to position 139, and / or threonine at the position corresponding to position 180. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 137 of SEQ ID NO: 3, alanine at the position corresponding to position 139, and / or threonine at the position corresponding to position 180. In some embodiments, the mutant IgG Fc polypeptide contains serine at the position corresponding to position 138 of SEQ ID NO: 4, alanine at the position corresponding to position 140, and / or threonine at the position corresponding to position 181. In some embodiments, the variant IgG Fc polypeptide contains serine at the position corresponding to position 154 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118, alanine at the position corresponding to position 156, and / or threonine at the position corresponding to position 197.

[0072] In some embodiments, the mutant IgG Fc polypeptide contains serine at position 138, alanine at position 140, and / or threonine at position 181 of SEQ ID NO: 1. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 137, alanine at position 139, and / or threonine at position 181 of SEQ ID NO: 2. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 137, alanine at position 139, and / or threonine at position 181 of SEQ ID NO: 3. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 138, alanine at position 140, and / or threonine at position 181 of SEQ ID NO: 4. In some embodiments, the mutant IgG Fc polypeptide contains serine at position 154, alanine at position 156, and / or threonine at position 197 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.

[0073] In some embodiments, the mutant IgG Fc polypeptide containing a whole mutation includes the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 124.

[0074] In some embodiments, the continuous polypeptide comprises a GLP1 polypeptide and a mutant canine or feline IgG Fc polypeptide containing a knob mutation. In some embodiments, the continuous polypeptide comprises a glucagon polypeptide and a mutant canine or feline IgG Fc polypeptide containing a knob mutation. In some embodiments, the continuous polypeptide comprises a GLP1 polypeptide and a mutant canine or feline IgG Fc polypeptide containing a hole mutation. In some embodiments, the continuous polypeptide comprises a glucagon polypeptide and a mutant canine or feline IgG Fc polypeptide containing a hole mutation.

[0075] In some embodiments, the heterodimer polypeptide comprises a first sequential polypeptide comprising a GLP1 polypeptide and a mutant canine or feline IgG Fc polypeptide containing a knob mutation, and a second sequential polypeptide comprising a glucagon polypeptide and a mutant canine or feline gG Fc polypeptide containing a hole mutation.

[0076] Exemplary GLP1 polypeptide and glucagon polypeptide As used herein, "GLP1" or "GLP1 polypeptide" is a polypeptide comprising the whole or a fragment of glucagon-like peptide 1 that binds to the glucagon-like peptide 1 receptor (GLP1R).

[0077] For example, "GLP1" refers to the GLP1 polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus macaques), rodents (e.g., mice, rats), and companion animals (e.g., dogs, cats, horses unless otherwise specified). In some embodiments, the GLP1 polypeptide is a wild-type GLP1 polypeptide such as GLP1(7-37)(SEQ ID NO: 85). In some embodiments, the GLP1 polypeptide is a mutant GLP1 polypeptide such as GLP1(7-36)(SEQ ID NO: 98), GLP1(7-35)(SEQ ID NO: 99), GLP1-S8(7-35)(SEQ ID NO: 86), or GLP1-G8(7-35)(SEQ ID NO: 87). In some embodiments, GLP1 contains the amino acid sequence of SEQ ID NO: 20.

[0078] As used herein, "GLP1R" is a polypeptide comprising the whole or a fragment of a glucagon-like peptide 1 receptor capable of binding to wild-type GLP1.

[0079] For example, "GLP1R" refers to the GLP1R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus macaques), rodents (e.g., mice, rats), and companion animals (e.g., dogs, cats, horses unless otherwise specified). In some embodiments, GLP1R is an extracellular domain fragment that binds to the wild-type GLP1 polypeptide. In some embodiments, canine GLP1R contains the amino acid sequence of SEQ ID NO: 49. In some embodiments, canine GLP1R contains the amino acid sequence of SEQ ID NO: 48.

[0080] An "extracellular domain" ("ECD") is the portion of a polypeptide that extends beyond the transmembrane domain into the extracellular space. As used herein, the term "extracellular domain" may include a complete extracellular domain or a cleaved extracellular domain lacking one or more amino acids that bind to its ligand. The composition of the extracellular domain depends on the algorithm used to determine which amino acids are intramembrane. Different algorithms may predict different extracellular domains for a particular protein, and different systems may express them.

[0081] As used herein, “glucagon” or “glucagon polypeptide” is a polypeptide that contains whole or fragments of glucagon that binds to a glucagon receptor.

[0082] For example, “glucagon” refers to glucagon polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus macaques), rodents (e.g., mice, rats), and companion animals (e.g., dogs, cats, horses unless otherwise specified). In some embodiments, the glucagon polypeptide is a wild-type glucagon polypeptide, such as SEQ ID NO: 21.

[0083] Exemplary mutant IgG Fc polypeptides and fusion molecules Polypeptides and other molecules may include mutant IgG Fc polypeptides. In some embodiments, the fusion molecule includes mutant IgG Fc polypeptides, for example, the mutant IgG Fc polypeptide described herein. In some embodiments, the antibody or antibody fragment includes mutant IgG Fc polypeptides, for example, the mutant IgG Fc polypeptide described herein.

[0084] As used herein, “fusion molecule” refers to a molecule comprising one or more “fusion partners.” In some embodiments, the fusion partners are covalently bonded (“fused”). If both fusion partners are polypeptides, the fusion partner polypeptides may be part of a continuous amino acid sequence (i.e., a continuous polypeptide). The first fusion partner polypeptide may be attached to either the N-terminus or C-terminus of the fusion partner. In some embodiments, the fusion partners are translated from coding sequences encoding both fusion partners into a single polypeptide. The fusion partners may be covalently bonded via other means, such as chemical bonds other than peptide bonds. Many known methods for covalently bonding a polypeptide to another molecule (e.g., a fusion partner) can be used. In other embodiments, the fusion partners are fused via a “linker” consisting of at least one amino acid or chemical moiety. In some embodiments, the fusion partners are non-covalently bonded. In some embodiments, the fusion partners may be bonded using, for example, bond pairs. Exemplary bond pairs include, but are not limited to, biotin and avidin or streptavidin, an antibody and its antigen, etc.

[0085] In some embodiments, the fusion partner includes an IgG Fc polypeptide and at least one GLP1 polypeptide. In some embodiments, the fusion partner includes an IgG Fc polypeptide, a GLP1 polypeptide, and a glucagon polypeptide. In some embodiments, the GLP1 polypeptide may be bound to either the N-terminus or the C-terminus of the IgG Fc polypeptide. In some embodiments, the glucagon polypeptide may be bound to either the N-terminus or the C-terminus of the IgG Fc polypeptide.

[0086] In this specification, the term “continuous polypeptide” is used to mean a continuous sequence of amino acids. A continuous polypeptide is typically translated from a single, continuous DNA sequence. It can be created by genetic engineering, for example, by removing a stop codon from the DNA sequence of a first protein and then in-frame adding the DNA sequence of a second protein, so that the DNA sequence is expressed as a single protein. This is usually achieved by cloning the cDNA in-frame into an expression vector using an existing gene.

[0087] A "linker" refers to one or more amino acid residues that connect the first polypeptide to the second polypeptide.

[0088] In some embodiments, the linker is a flexible, unstructured linker. In some embodiments, the linker is glycine-rich, serine-rich, or a glycine and serine-rich linker. In some embodiments, the linker contains 100%, at least 95%, at least 90%, or at least 85% serine and / or glycine amino acid residues.

[0089] As used herein, "extension" refers to one or more amino acid residues attached to the polypeptide at its C-terminus or N-terminus.

[0090] In some embodiments, the elongation portion is flexible. In some embodiments, the elongation portion adds flexibility to the polypeptide without interfering with the polypeptide's biological activity. In some embodiments, the elongation portion increases the solubility of the polypeptide. In some embodiments, the elongation portion comprises one or more glycine residues. In some embodiments, the elongation portion 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.

[0091] In some embodiments, the sequential polypeptide is sequence numbers 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, 8 0, 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, 13 6, 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, 1 It comprises an IgG Fc polypeptide containing any one of the amino acid sequences 71, 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 containing the amino acid sequence of SEQ ID NO: 85.In some embodiments, the sequential polypeptide is sequence numbers 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, 8 0, 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, 13 6, 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, 1 It comprises an IgG Fc polypeptide containing any one of the amino acid sequences 71, 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 containing the amino acid sequence of SEQ ID NO: 86.In some embodiments, the sequential polypeptide is sequence numbers 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, 8 0, 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, 13 6, 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, 1 It comprises an IgG Fc polypeptide containing any one of the amino acid sequences 71, 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 containing the amino acid sequence of SEQ ID NO: 87.In some embodiments, the sequential polypeptide is sequence numbers 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, 8 0, 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, 13 6, 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, 1 It comprises an IgG Fc polypeptide containing any one of the amino acid sequences 71, 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 containing the amino acid sequence of SEQ ID NO: 98.In some embodiments, the sequential polypeptide is sequence numbers 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, 8 0, 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, 13 6, 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, 1 It comprises an IgG Fc polypeptide containing any one of the amino acid sequences 71, 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 containing the amino acid sequence of SEQ ID NO: 99.

[0092] In some embodiments, the sequential polypeptide is sequence numbers 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, 8 0, 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, 13 6, 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, 1 It comprises an IgG Fc polypeptide containing any one of the amino acid sequences 71, 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 containing the amino acid sequence of SEQ ID NO: 21.

[0093] In some embodiments, the continuous polypeptide comprises a first GLP1 polypeptide (GLP1A) of the companion animal species, a first linker (L1), an Fc polypeptide, optionally a second linker (L2), and optionally a second GLP1 polypeptide (GLP1B). In some embodiments, the continuous polypeptide is Equation (I): GLP1A-L1-Fc; Formula (II): Fc-L1-GLP1A; or Equation (III): GLP1A-L1-Fc-L2-GLP1B Includes.

[0094] In some embodiments, the continuous polypeptide comprises a GLP1 polypeptide, a first linker (L1), an Fc polypeptide, a second linker (L2), and a glucagon polypeptide (Gluc). In some embodiments, the continuous polypeptide is Formula (IV): GLP1-L1-Fc-L2-Gluc; or Formula (V): Gluc-L1-Fc-L2-GLP1 Includes.

[0095] In some embodiments, the GLP1 fusion molecule has an extended serum half-life compared to the wild-type GLP1 polypeptide. The extended half-life of the GLP1 fusion molecule described herein may require lower doses and less frequent dosing regimens than those for the wild-type GLP1 polypeptide.

[0096] In some embodiments, GLP1B, if present, has the same amino acid sequence as GLP1A.

[0097] In some embodiments, GLP1, GLP1A, or GLP1B, if present, comprises the wild-type GLP1 polypeptide. In some embodiments, GLP1, GLP1A, or GLP1B, if present, comprises the mutant GLP1 polypeptide. In some embodiments, GLP1, GLP1A, or GLP1B, if present, comprises the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ ID NO: 99.

[0098] In some embodiments, the glucagon polypeptide comprises a wild-type glucagon polypeptide. In some embodiments, the glucagon polypeptide comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the glucagon polypeptide comprises a mutant glucagon polypeptide.

[0099] 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.

[0100] In some embodiments, the Fc polypeptide is IgG Fc from a companion animal. In some embodiments, the Fc polypeptide is canine IgG-A Fc, canine IgG-B Fc, canine IgG-C Fc, or 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.

[0101] In some embodiments, the Fc polypeptide is mutant IgG Fc. In some embodiments, the Fc polypeptide is mutant canine IgG-A Fc, mutant canine IgG-B Fc, mutant canine IgG-C Fc, or mutant canine IgG-D Fc. In some embodiments, Fc is mutant horse IgG1 Fc, mutant horse IgG2 Fc, mutant horse IgG3 Fc, mutant horse IgG4 Fc, mutant horse IgG5 Fc, mutant horse IgG6 Fc, or mutant horse IgG7 Fc. In some embodiments, Fc is mutant cat IgG1a Fc, mutant cat IgG1b Fc, or mutant cat IgG2 Fc.

[0102] In some embodiments, L1 and L2, if present, are independently flexible linkers. In some embodiments, the amino acid sequences of L1 and L2, if present, independently contain 100%, at least 95%, at least 90%, and at least 85% serine and / or glycine amino acid residues, respectively.

[0103] In some embodiments, the continuous polypeptide includes an extension at its C-terminus. In some embodiments, the continuous polypeptide includes 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 continuous polypeptide includes the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95 at its C-terminus.

[0104] In some embodiments, the continuous polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106.

[0105] A nucleotide sequence encoding a target polypeptide, such as the mutant IgG Fc polypeptide or other polypeptides described herein, can be inserted into an expression vector suitable for expression in selected host cells. The mutant IgG Fc polypeptide or other polypeptides described herein can be expressed by culturing host cells transfected with an expression vector containing the nucleotide sequence.

[0106] A "vector" is a plasmid that can be used to transfer DNA sequences from one organism to another or to express a target gene. Vectors typically contain a replication origin and regulatory sequences that control the expression of the target gene, and may or may not contain selection marker genes, such as antibiotic resistance genes. A vector is suitable for the host cell in which it is expressed. When the target gene is present in the vector, the vector is sometimes called a "recombinant vector."

[0107] "Host cell" refers to a cell that may or is a recipient of a vector or isolated polynucleotide. A host cell may be a prokaryotic or 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-exclusive and exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, as well as their derivatives, such as 293-6E, DG44, CHO-S, and CHO-K cells. Host cells include offspring of a single host cell, and due to spontaneous, accidental, or intentional mutations, the offspring may not be exactly identical (morphologically or in terms of genomic DNA complement) to the original parent. Host cells include cells transfected in vivo with polynucleotides encoding the amino acid sequences provided herein.

[0108] As used herein, the term “isolated” refers to a molecule that has been separated from at least some of the components that are normally found together or produced together in nature. For example, a polypeptide is “isolated” if it has been separated from at least some of the components of the cell in which it was produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered “isolation” of the polypeptide. Similarly, a polynucleotide is “isolated” if it is not part of a larger polynucleotide that is normally found in nature (e.g., in the case of a DNA polynucleotide, such as genomic DNA or mitochondrial DNA), or, for example, 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 said to be “isolated.”

[0109] A "signal sequence" refers to a sequence of amino acid residues or polynucleotides encoding them that promote the secretion of a target polypeptide and are normally cleaved during the polypeptide's efflux to the outside of the cell surface membrane.

[0110] In some embodiments, the mutant IgG Fc polypeptide or the serial polypeptide containing the mutant Fc polypeptide is isolated using chromatography such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0111] Labels can be attached to mutant IgG Fc polypeptides or serial polypeptides containing mutant Fc polypeptides. “Label” means a portion that attaches to a molecule and makes it detectable. In some embodiments, mutant IgG Fc polypeptides or serial polypeptides containing mutant Fc polypeptides are labeled with detectable portions 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 produce a signal detectable by visual or instrumental means, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl portion to the polypeptide (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); pigments, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorescent), 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 pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Typical examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, even if a moiety itself is not detectably labeled, it may become detectable through reaction with another moiety. The general techniques used when carrying out the various immunoassays described above are known to those skilled in the art.

[0112] Exemplary mutant IgG Fc polypeptide affinity for protein A and / or C1q and / or CD16 The mutant IgG Fc polypeptides described herein may have modified binding affinities to protein A and / or C1q and / or CD16. In some embodiments, the mutant IgG Fc polypeptide has increased binding affinity to protein A compared to the wild-type IgG Fc polypeptide. Such mutant IgG Fc polypeptides can be purified by protein A column chromatography. In some embodiments, the mutant IgG Fc polypeptide has decreased binding affinity to C1q compared to the wild-type IgG Fc polypeptide. Such mutant IgG Fc polypeptides may have a reduced complement-mediated immune response. In some embodiments, the mutant IgG Fc polypeptide has decreased binding affinity to CD16 compared to the wild-type IgG Fc polypeptide. Such mutant IgG Fc polypeptides may have a reduced ADCC immune response. In some embodiments, the mutant IgG Fc polypeptide has increased binding affinity to protein A compared to the wild-type IgG Fc polypeptide, and / or decreased binding affinity to C1q compared to the wild-type IgG Fc polypeptide, and / or decreased binding affinity to CD16 compared to the wild-type IgG Fc polypeptide.

[0113] "Protein A," as used herein, is a polypeptide comprising all or part of Protein A that can bind to wild-type canine IgG-B Fc, wild-type horse IgG1 Fc, wild-type horse IgG3 Fc, wild-type horse IgG4 Fc, wild-type horse IgG7 Fc, wild-type feline IgG1a Fc, wild-type feline IgG1b Fc, or wild-type feline IgG2 Fc.

[0114] "C1q" or "C1q complex" is used interchangeably to refer to a protein complex or part thereof involved in the complement system, and can bind to wild-type canine IgG-B Fc, wild-type canine IgG-C Fc, wild-type horse IgG1 Fc, wild-type horse IgG3 Fc, wild-type horse IgG4 Fc, wild-type horse IgG7 Fc, wild-type feline IgG1a Fc, or wild-type feline IgG1b Fc.

[0115] "CD16," as used herein, is a polypeptide comprising all or part of CD16 capable of binding to wild-type canine IgG-A Fc or wild-type canine IgG-D Fc. The term "binding" to a substance is a term well understood in the art, and methods for determining such binding are also well known in the art. A molecule is said to "bind" if it reacts with, associates with, or has affinity for a particular cell or substance, and this reaction, binding, or affinity is detectable by one or more methods known in the art, such as immunoblotting, ELISA, KinExA, biolayer interferometry (BLI), or surface plasmon resonance (SPL).

[0116] As used herein, "protein A+" means that the Fc polypeptide has protein A binding affinity. In some embodiments, the protein A+ Fc polypeptide includes at least one amino acid modification that increases protein A.

[0117] As used herein, "protein A-" means that the Fc polypeptide has low protein A binding affinity or no protein A binding affinity at all.

[0118] As used herein, "C1q+" means that the Fc polypeptide has C1q binding affinity.

[0119] As used herein, "C1q-" means that the Fc polypeptide has low C1q binding affinity or no C1q binding affinity at all. In some embodiments, the C1q-Fc polypeptide has at least one amino acid modification that reduces C1q binding affinity.

[0120] As used herein, "CD16+" means that the Fc polypeptide has CD16 binding affinity.

[0121] As used herein, "CD16-" means that the Fc polypeptide has low CD16 binding affinity or no CD16 binding affinity at all. In some embodiments, the CD16-Fc polypeptide has at least one amino acid modification that reduces CD16 binding affinity.

[0122] The term "affinity" refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Typically, the affinity of molecule X to its partner Y is expressed by the dissociation constant (K). D Affinity is expressed by ( ). Affinity can be measured by common methods known in the art, such as immunoblotting, ELISA, KinExA, biolayer interferometry (BLI), or surface plasmon resonance (SPL) spectroscopy.

[0123] "Surface plasmon resonance" is an example of BIAcore TM This refers to an optical phenomenon that enables real-time analysis of biomolecular-specific interactions by detecting changes in protein concentration within a biosensor matrix using BIAcore International AB (Uppsala, Sweden), a subsidiary of GE Healthcare, Piscataway, New Jersey. For further explanation, see Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26.

[0124] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from the tip of a biosensor and a layer of immobilized proteins on an internal reference layer. Changes in the number of molecules bound to the biosensor chip cause changes in the interference pattern that can be measured in real time. A non-exclusive and exemplary apparatus for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, for example, Abdiche et al., 2008, Anal. Biochem. 377: 209-277.

[0125] The term “K D "K" d "Kd" or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of receptor-ligand interactions or antibody-antigen interactions.

[0126] In some embodiments, the mutant IgG Fc polypeptide is measured by biolayer interferometry, and 5x10 -6 Less than M, 1x10 -6 Less than M, 5x10 -7 Less than M, 1x10 -7 Less than M, 5x10 -8 Less than M, 1x10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M, 5 x 10- 11 Less than M, 1 x 10 -11 Less than M, 5x10 -12 Less than M, or 1x10 -12 Dissociation constant less than M (K D It binds to protein A, which has )

[0127] In some embodiments, the mutant IgG Fc polypeptide is measured by biolayer interferometry, and 5x10 -6 Super M, 1x10 -5 Super M, 5x10 -5 Super M, 1x10 -4 Super M, 5x10 -4M or more, or 1x10 -3 Dissociation constant (K) for M D It binds to C1q or CD16 which has ).

[0128] In some embodiments, the K of IgG Fc polypeptides, such as mutant IgG Fc polypeptides, relative to protein A, C1q, or CD16. D This is measured using biolayer interferometry, employing biosensors such as the Octet® system (Pall ForteBio LLC, Fremont, California) according to the supplier's instructions. Specifically, biotinylated protein A, C1q, or CD16 is bound to the sensor chip, and the association of IgG Fc polypeptides is monitored for a specified time or until a steady state is reached. Dissociation can also be monitored for a specified time or until a steady state is reached. A buffer-only blank curve is subtracted to correct for drift. The data is then fitted to a 2:1 binding model using data analysis software, and the association rate constant (k) is determined. on ), dissociation rate constant (k off ) and K d Determine the equilibrium dissociation constant (K D ) is k off / k on It is calculated as the ratio of k. on " refers to the rate constant of association of molecule X with its partner Y, and the term "k" off This refers to the rate constant of dissociation of molecule X or partner Y from the molecule X / partner Y complex.

[0129] To “increase” or “stimulate” means to increase, improve, or enhance activity, function, or quantity compared to a baseline. In some embodiments, “increase” or “stimulate” means the ability to produce 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, or about 200% or more compared to a baseline. In some embodiments, “increase” or “stimulate” means the ability to produce an overall increase of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% compared to a baseline. In some embodiments, “increase” or “stimulate” means the ability to produce 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 amounts are stimulated or increased over the same period as a control dose (such as a placebo) over the same period.

[0130] In some embodiments, the mutant IgG Fc polypeptide can bind to protein A with an increased affinity of approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, and 200% or more compared to the reference IgG Fc polypeptide. In some embodiments, the mutant IgG Fc polypeptide can bind to protein A with an increased affinity of approximately 5% to 50%, 10% to 20%, 50% to 100%, and 25% to 70% compared to the reference IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is the wild-type IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a different mutant IgG Fc polypeptide.

[0131] To “reduce” or “inhibit” means to decrease, reduce, or prevent activity, function, or amount compared to a reference. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction 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 compared to a reference IgG Fc polypeptide. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction of about 5% to about 50%, about 10% to about 20%, about 50% to about 100%, or about 25% to about 70% compared to a reference value. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction of 50% or more. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amounts are inhibited or reduced over the same period as a control dose (such as a placebo) over a certain period.

[0132] In some embodiments, the mutant IgG Fc polypeptide can bind to C1q or CD16 with a reduced affinity of approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% or more compared to the reference IgG Fc polypeptide. In some embodiments, the mutant IgG Fc polypeptide can bind to C1q or CD16 with a reduced affinity of approximately 5% to 50%, 10% to 20%, 50% to 100%, and 25% to 70% compared to the reference IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is the wild-type IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a different mutant IgG Fc polypeptide.

[0133] As used herein, “reference” refers to any sample, standard, or level used for comparative purposes. A reference may be a wild-type reference or a variant reference. References may be obtained from healthy or unaffected samples. In some cases, references are obtained from unaffected or untreated samples of companion animals. In some cases, references are obtained from one or more healthy animals of a particular species, but these animals are not the animals being tested or treated.

[0134] Exemplary Bioactivity of Mutant GLP1 Fusion Molecules In some embodiments, the GLP1 fusion molecule binds to GLP1R and activates cAMP production. In some embodiments, the GLP1 fusion polypeptide increases cAMP production in cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to the signaling function in the absence of the GLP1 fusion polypeptide.

[0135] Exemplary pharmaceutical composition The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations that are in a form that enables the biological activity of the active ingredient and do not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0136] A "pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or carrier common in the art, intended for use with a therapeutic agent containing a "pharmaceutically acceptable composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine or HEPES 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, cellulosic substances; polyethylene glycol; sucrose; mannitol; or amino acids, including but not limited to arginine.

[0137] The pharmaceutical composition can be stored in lyophilized form. Therefore, in some embodiments, this preparation method includes a lyophilization step. The lyophilized composition can then be reconstituted as an aqueous composition, usually suitable for parenteral administration, before administration to dogs, cats, or horses. In other embodiments, particularly when the variant IgG Fc polypeptide or other polypeptides described herein are highly stable against thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., an aqueous composition, that can be administered directly to dogs, cats, or horses or with appropriate dilution. The lyophilized composition can be reconstituted with sterile water for injection (WFI). Bacteriostatic agents such as benzyl alcohol may be included. Therefore, the present invention provides pharmaceutical compositions in solid or liquid form.

[0138] The pH of the pharmaceutical composition may range from approximately pH 5 to approximately pH 8 at the time of administration. The compositions of the present invention are sterile when used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including filtration with a sterile filtration membrane (e.g., a 0.2 micron membrane). Sterility can be maintained with or without the presence of antimicrobial agents.

[0139] Specific uses of Fc polypeptides and pharmaceutical compositions Polypeptides containing the mutant Fc polypeptide of the present invention (e.g., mutant IgG Fc polypeptide) or pharmaceutical compositions containing the mutant Fc polypeptide of the present invention may be useful in extending the in vivo product half-life in companion animals including, but not limited to, dogs, cats, or horses.

[0140] As used herein, “treatment” is a method for obtaining a beneficial or desired clinical outcome. As used herein, “treatment” includes the administration or application of a therapeutic agent for a disease in mammals, including companion animals. In this disclosure, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms, reduction of the scope of the disease, prevention or delay of disease transmission, prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of the condition, inhibition of the disease or its progression, inhibition or slowing of the disease or its progression, prevention of its onset, and (partial or total) remission. “Treatment” also includes reduction of the pathological consequences of proliferative disorders. The methods provided herein are intended to achieve one or more of these aspects of treatment. As stated above, the term treatment does not require the complete elimination of all aspects of the disorder.

[0141] The "therapeutic effective dose" of a substance / molecule, agonist, or antagonist varies depending on factors such as the type of disease being treated, the condition, the severity and course of the disease, the type of therapeutic objective, previous treatments, medical history, response to previous treatments, the discretion of the attending veterinarian, the animal's age, sex, and weight, and the ability of the substance / molecule, agonist, or antagonist to induce a desirable response in the animal. Furthermore, a therapeutic effective dose is the amount in which the therapeutically beneficial effects of the substance / molecule, agonist, or antagonist outweigh the toxic or adverse effects. A therapeutic effective dose may be delivered in one or more doses. The therapeutic effective dose refers to the amount effective in obtaining the desired therapeutic or prophylactic outcome at the required dose and duration.

[0142] In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition containing the same, is administered parenterally by subcutaneous, intravenous, or intramuscular injection. In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition containing the same, is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition containing the same, is administered intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarterially, synovially, intrathecally, or by inhalation.

[0143] In some embodiments, a GLP1 fusion polypeptide or a pharmaceutical composition containing the same can be used to treat hyperglycemia-related conditions according to the methods described herein. In some embodiments, a GLP1 fusion polypeptide or a pharmaceutical composition is administered to a companion animal such as a dog, cat, or horse to treat hyperglycemia-related conditions.

[0144] In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition containing the same, is administered in an amount ranging from 0.0001 mg / kg body weight to 100 mg / kg body weight per dose. In some embodiments, the GLP1 analog is administered in an amount ranging from 0.005 mg / kg body weight to 20 mg / kg body weight per dose. In some embodiments, the GLP1 analog is administered in an amount ranging from 1 mg / kg body weight to 10 mg / kg body weight per dose. In some embodiments, the GLP1 analog may be administered in amounts ranging from 0.5 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 100 mg / kg body weight, 5 mg / kg body weight to 100 mg / kg body weight, 10 mg / kg body weight to 100 mg / kg body weight, 20 mg / kg body weight to 100 mg / kg body weight, 50 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 10 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, 0.5 mg / kg body weight to 10 mg / kg body weight, or 5 mg / kg body weight to 50 mg / kg body weight.

[0145] In some embodiments, the variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition containing the same, is administered to a companion animal in a single dose or a series of treatments. In some embodiments, the dose is administered once weekly for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, with an optional period of one week or more without treatment. In other embodiments, a therapeutically effective dose is administered once daily for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, with an optional period of one day or more or several weeks without treatment.

[0146] Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and sequential or sequential administration in any order. The term “simultaneously” is used herein to mean the administration of two or more therapeutic agents where at least part of the administrations overlaps in time or the administration of one therapeutic agent occurs within a shorter period than the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered at time intervals of approximately no more than specified minutes. The term “sequential” is used herein to mean the administration of two or more therapeutic agents where the administration of one or more agents continues after the discontinuation of the administration of one or more other agents, or the administration of one or more agents begins before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is administered at time intervals of no more than specified minutes. As used herein, “in combination with” refers to the administration of one therapeutic agent in addition to another therapeutic agent. That is, “in combination with” refers to the administration of one therapeutic agent to an animal before, during, or after the administration of another therapeutic agent.

[0147] In some embodiments, the dose 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 an optional period of one week or more without treatment. In other embodiments, the therapeutically effective dose is administered once daily for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, with an optional period of one day or more or several weeks without treatment.

[0148] Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and sequential or sequential administration in any order. The term “simultaneously” is used herein to mean the administration of two or more therapeutic agents where at least part of the administrations overlaps in time or the administration of one therapeutic agent occurs within a shorter period than the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered at time intervals of approximately no more than specified minutes. The term “sequential” is used herein to mean the administration of two or more therapeutic agents where the administration of one or more agents continues after the discontinuation of the administration of one or more other agents, or where the administration of one or more agents begins before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is administered at time intervals of no more than specified minutes. Where used herein, “in combination with” means the administration of one therapeutic agent in addition to another therapeutic agent. That is, “in combination with” means the administration of one therapeutic agent to an animal before, during, or after the administration of another therapeutic agent.

[0149] In some embodiments, the method involves administering a combination of GLP1 fusion polypeptide insulin, DPP4 inhibitors, SGLT2 inhibitors, biguanides, sulfonylureas, meglitinide derivatives, alpha-glucosidase inhibitors, thiazolidinediones (TZDs), amirinomimetics, bile acid scavengers, and dopamine agonists.

[0150] The following examples illustrate specific aspects of the Disclosure and are not intended to limit the Disclosure in any way. [Examples]

[0151] Example 1 Mutant canine IgG Fc polypeptide to increase protein A binding and / or decrease complement binding and / or decrease CD16 binding. Purification of antibodies using protein A affinity is a well-developed method. However, among the four subtypes of canine IgG, only IgG-B Fc (e.g., SEQ ID NO: 2 or SEQ ID NO: 107) has protein A binding affinity. Canine IgG-A Fc (e.g., SEQ ID NO: 1), IgG-C Fc (e.g., SEQ ID NO: 3 or SEQ ID NO: 108), and IgG-D Fc (e.g., SEQ ID NO: 4) have weak or undetectable protein A binding affinity. Mutant canine IgG-A Fc, IgG-C Fc, and gG-D Fc polypeptides were designed for modified protein A binding.

[0152] Furthermore, canine IgG-B Fc and IgG-C Fc have complement activity and bind to C1q, while canine IgG-A Fc and IgG-D Fc have weak or undetectable binding affinity to C1q. Mutant canine IgG-B Fc and IgG-C Fc polypeptides were designed to potentially reduce C1q binding and / or potentially reduce the complement-mediated immune response.

[0153] Furthermore, canine IgG-B Fc and IgG-C Fc have CD16 binding activity. Mutant 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.

[0154] Table 3 below summarizes the protein A and C1q binding characteristics of canine IgG Fc subtypes. In particular, none of the wild-type canine IgG Fc subtypes lack C1q binding and do not bind to protein A.

[0155] Table 3 TIFF2026048634000046.tif65170

[0156] Using three-dimensional protein modeling and protein sequence analysis, sequences of canine IgG-B Fc that are likely to be in contact with protein A were identified. Figure 1 shows the alignment of canine IgG-A, B, C, and D Fc sequences. The boxes indicate regions likely to be in contact with protein A.

[0157] Two methods were used to design mutant canine IgG-A, IgG-C, and IgG-D Fc polypeptides to increase protein A binding. In the first method, mutant canine IgG-A, IgG-C, and IgG-D Fc polypeptides were designed to have the same protein A binding motif sequence as canine IgG-B Fc (e.g., SEQ ID NOs. 5, 6, and 7, respectively). In the second method, mutant canine IgG-A FcI(21)T / Q(207)H (SEQ ID NOs. 60), mutant canine IgG-C FcI(21)T (SEQ ID NOs. 61), and mutant canine IgG-D FcI(21)T / Q(207)H (SEQ ID NOs. 62) were designed, each having one or more amino acid substitutions in the protein A binding region corresponding to the canine IgG-B Fc sequence.

[0158] Furthermore, mutant canine IgG-A Fc, IgG-C Fc, and IgG-D Fc polypeptides with increased protein A binding, having one or more amino acid substitutions listed in Table 4, can be prepared.

[0159] Table 4 TIFF2026048634000047.tif79170

[0160] To potentially reduce the binding of C1q to canine IgG-B Fc and IgG-C Fc, and / or potentially reduce the complement-mediated immune response, mutant canine IgG-B Fc and IgG-C Fc polypeptides can be prepared that have an amino acid substitution of Lys by any amino acid other than Lys at the amino acid position corresponding to position 93 of SEQ ID NO: 2 or SEQ ID NO: 3, respectively. These amino acid substitutions were identified after protein sequence analysis and 3D structural modeling of canine IgG-B Fc and IgG-C Fc compared to canine IgG-A Fc and IgG-D Fc, which are known to be non-complemental. For example, mutant canine IgG-B Fc K(93)R (SEQ ID NO: 78) and mutant canine IgG-C Fc K(93)R (SEQ ID NO: 79) can be prepared. When comparing fusion proteins containing wild-type canine IgG-B Fc, a decrease in binding between human C1q and fusion proteins containing mutant canine IgG-B Fc K(93)R was observed.

[0161] To potentially reduce the binding of CD16 to IgG-B Fc and IgG-C Fc, and / or to potentially reduce ADCC activity, mutant 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. These amino acid substitutions were identified after protein sequence analysis and 3D structural modeling of canine IgG-B and IgG-C compared to IgG-A and IgG-D, which are known to be non-ADCC active.

[0162] Table 5 TIFF2026048634000048.tif99170

[0163] Wild-type canine IgG-C Fc lacks protein A binding and possesses C1q binding. Therefore, by combining one or more amino acid substitutions listed in Table 4 with K(93)R substitutions or K(93)X substitutions (where X is any amino acid except Lys), a double mutant canine IgG-C Fc with reduced binding to protein A and reduced binding to C1q can be prepared. By combining one or more amino acid substitutions listed in Table 5 with K(93)R substitutions or K(93)X substitutions (where X is any amino acid except Lys), a double mutant canine IgG-B Fc or double mutant canine IgG-C Fc with reduced binding to both C1q and CD16 can be prepared. By combining one or more amino acid substitutions listed in Table 4 and one or more amino acid substitutions listed in Table 5 with K(93)R substitutions or K(93)X substitutions (where X is any amino acid other than Lys), triple mutant canine-IgG-C Fc that binds to protein A and has reduced binding to C1q and CD16 can be prepared.

[0164] The binding of any mutant canine IgG Fc to protein A, CD16, and / or C1q can be determined and compared to the binding of another IgG Fc to protein A, CD16, and / or C1q (e.g., the corresponding wild-type canine IgG Fc, another wild-type or mutant canine IgG Fc, or a wild-type or mutant IgG Fc from another companion animal, etc.).

[0165] Binding analysis can be performed using an octet biosensor. Specifically, target molecules (e.g., protein A, C1q, CD16, etc.) can be biotinylated, and unreacted free biotin can be removed (e.g., by dialysis). The biotinylated target molecule is captured on a streptavidin sensor chip. The association of the target molecule with 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 specific time or until a steady state is reached. A buffer-only blank curve can be subtracted to correct for drift. The data is then processed using ForteBio. TM We used data analysis software to fit a 1:1 coupled model, k on , k off and K d To decide.

[0166] Example 2 To increase protein A binding and / or decrease complement binding mutant horse IgG Fc polypeptide Of the seven subtypes of equine IgG, IgG1 Fc (e.g., SEQ ID NO: 63), IgG3 Fc (e.g., SEQ ID NO: 65), IgG4 Fc (e.g., SEQ ID NO: 66), and IgG7 Fc (e.g., SEQ ID NO: 69) possess protein A binding affinity. Equivalent equine IgG2 Fc (e.g., SEQ ID NO: 18, SEQ ID NO: 64), IgG5 Fc (e.g., SEQ ID NO: 67), and IgG6 Fc (e.g., SEQ ID NO: 68) have weak or unmeasurable protein A binding affinity. Mutant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides were designed for modified protein A binding.

[0167] Furthermore, while equine IgG2 Fc, IgG5 Fc, and IgG6 Fc have weak or unmeasurable binding affinity to C1q, equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc do bind to C1q. To potentially reduce C1q binding and / or potentially reduce the complement-mediated immune response, mutant equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc polypeptides were designed.

[0168] Table 6 below summarizes the protein A and C1q binding characteristics of horse IgG Fc subtypes. In particular, none of the wild-type horse IgG Fc subtypes lack C1q binding and do not bind to protein A.

[0169] Table 6 TIFF2026048634000049.tif96170

[0170] Using three-dimensional protein modeling and protein sequence analysis, sequences of equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc, which are likely to be in contact with protein A, were identified. Furthermore, mutant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides with increased protein A binding, having one or more amino acid substitutions listed in Table 7, can be prepared.

[0171] Table 7 TIFF2026048634000050.tif63170

[0172] For example, mutant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides having one or more amino acid substitutions in the Protein A binding region were designed to correspond to the sequence of wild-type equine IgG Fc that binds to Protein A. Mutants with increased Protein A binding can be prepared: equine IgG2 Fc F(203)Y (SEQ ID NO: 71); equine IgG2 Fc A(15)T / F(203)Y (SEQ ID NO: 72); equine IgG5 Fc V(199)L / E(200)Y (SEQ ID NO: 75); and equine IgG6 Fc I(199)L / R(200)H / H(201)N / T(202)H (SEQ ID NO: 76).

[0173] Mutant canine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc polypeptides having an amino acid substitution with any amino acid other than Lys at the amino acid position corresponding to position 87 of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 69, respectively, can be prepared 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 response. These amino acid substitutions were identified after protein sequence analysis and 3D structure modeling 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, mutant equine IgG1 Fc K(87)S (SEQ ID NO: 70), mutant equine IgG 3 Fc K(87)S (SEQ ID NO: 73), mutant equine IgG4 Fc K(87)S (SEQ ID NO: 74), and mutant equine IgG7 Fc K(87)S (SEQ ID NO: 77) can be prepared.

[0174] The binding of any mutant horse IgG Fc to protein A and / or C1q can be determined and compared to the binding of another IgG Fc to protein A and / or C1q (e.g., the corresponding wild-type horse IgG Fc, another wild-type or mutant horse IgG Fc, or a wild-type or mutant IgG Fc from another companion animal). The binding assay described in Example 1 can be used.

[0175] Example 3 Mutant cat IgG Fc polypeptide for reducing complement binding The three subtypes of feline IgG, IgG1a Fc (SEQ ID NO: 80 or 117), IgG1b Fc (SEQ ID NO: 81 or 118), and IgG2 Fc (SEQ ID NO: 16), each possess protein A binding affinity. However, only feline IgG2 Fc has weak or unmeasurable binding affinity to C1q, while feline IgG1a Fc and IgG1b Fc bind to C1q. To potentially reduce C1q binding and / or potentially reduce complement-mediated immune responses, mutant feline IgG1a Fc and IgG1b Fc polypeptides were designed.

[0176] Table 8 below summarizes the protein A and C1q binding characteristics of feline IgG Fc subtypes. In particular, none of the wild-type horse IgG Fc subtypes lack C1q binding and do not bind to protein A.

[0177] Table 8 TIFF2026048634000051.tif55170

[0178] To potentially reduce the binding of C1q to feline IgG1a Fc and IgG1b Fc, and / or potentially reduce the complement-mediated immune response, mutant feline IgG1a Fc and IgG1b Fc polypeptides can be prepared that have 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: 80 or SEQ ID NO: 81, respectively. These amino acid substitutions were identified after protein sequence analysis and 3D structural modeling of feline IgG1a Fc and IgG1b Fc compared to feline IgG2 Fc, which is known to be non-complemental. For example, mutant feline IgG1a Fc P(198)A (SEQ ID NO: 82) and mutant feline IgG1b Fc P(198)A (SEQ ID NO: 83) can be prepared.

[0179] The binding of any mutant cat IgG Fc to C1q can be determined and compared to the binding of another IgG Fc to C1q (e.g., the corresponding wild-type cat IgG Fc, another wild-type or mutant cat IgG Fc, or a wild-type or mutant IgG Fc from another companion animal). The binding assay described in Example 1 can be used.

[0180] Example 4 Heterodimeric protein variants in canine and feline IgG Fc polypeptides Using a knob-in-hole heterodimerization technique, we investigated pairing between mutant canine IgG Fc polypeptides and mutant feline IgG Fc polypeptides to enable the preparation of bispecific canine or feline antibodies or bifunctional canine or feline Fc fusion proteins.

[0181] An amino acid substitution from threonine to tyrosine (T138Y or T137Y) can be introduced into a single Fc chain (heterodimer chain 1) at the position corresponding to position 138 of canine IgG-A (sequence number 1), position corresponding to position 137 of canine IgG-B Fc (sequence number 2), position corresponding to position 137 of canine IgG-C Fc (sequence number 3), or position corresponding to position 138 of canine IgG-D Fc (sequence number 4). Examples of amino acid sequences for mutant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 1 are sequence numbers 8, 10, 12, and 14, respectively.

[0182] An amino acid substitution from tyrosine to threonine (Y181T or Y180T) can be introduced into the second Fc chain (heterodimer chain 2) at the position corresponding to position 181 of canine IgG-A (sequence number 1), position corresponding to position 180 of canine IgG-B Fc (sequence number 2), position corresponding to position 180 of canine IgG-C Fc (sequence number 3), or position corresponding to position 181 of canine IgG-D Fc (sequence number 4). Examples of amino acid sequences for mutant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 2 are sequence numbers 9, 11, 13, and 15, respectively.

[0183] The formation of a second pair between mutant canine IgG Fc heterodimer chains 3 and 4 was also investigated. An amino acid substitution (T138W) (T137W) from threonine to tryptophan at the position corresponding to position 138 of canine IgG-A (SEQ ID NO: 1) or canine IgG-D (SEQ ID NO: 4), or at the position corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 2) or canine IgG-C Fc (SEQ ID NO: 3), can be introduced into one Fc chain (heterodimer chain 3). Examples of amino acid sequences for mutant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 3 are SEQ ID NOs: 109, 111, 113, and 115.

[0184] The amino acid substitutions (T138S, L140A, Y181T) of canine IgG-A (SEQ ID NO: 1) or IgG-D (SEQ ID NO: 4) can be introduced into the second Fc chain (heterodimer chain 4) by changing threonine to serine at position 138, leucine to alanine at position 140, and tyrosine to threonine at position 181, or by changing threonine to serine at position 137, leucine to alanine at position 139, and tyrosine to threonine at position 180 of canine IgG-B Fc (SEQ ID NO: 2) or IgG-C (SEQ ID NO: 3) by changing threonine to serine at position 137, leucine to alanine at position 139, and tyrosine to threonine at position 180. Examples of amino acid sequences for mutant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 4 are SEQ ID NOs. 110, SEQ ID NOs. 112, SEQ ID NOs. 114, and SEQ ID NOs. 116.

[0185] An amino acid substitution (T154W) from threonine to tryptophan at the position corresponding to position 154 of feline IgG2 (sequence number 16), feline IgG1a Fc (sequence number 80 or 117), or feline IgG1b Fc (sequence number 81 or 118) can be introduced into a single Fc chain (heterodimer chain 1). Examples of amino acid sequences for mutant feline IgG2 Fc, IgG1a Fc, and IgG1b Fc heterodimer chain 1 are sequence numbers 119, 121, and 123, respectively.

[0186] Amino acid substitutions (T154S, L156A, Y197T) can be introduced into the second Fc chain (heterodimer chain 4) of feline IgG2 Fc (SEQ ID NO: 16), IgG-1a (SEQ ID NO: 80 or 117), or IgG-1b Fc (SEQ ID NO: 81 or 118) at positions 154 (threonine to serine), 156 (leucine to alanine), and 197 (tyrosine to threonine). Examples of amino acid sequences for mutant feline IgG2 Fc, IgG1a Fc, and IgG1b Fc heterodimer chain 4 are SEQ ID NOs: 120, 122, and 124.

[0187] Pairing of mutant canine IgG Fc heterodimer chains 1 and 2, mutant canine IgG Fc heterodimer chains 3 and 4, or mutant feline 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 may be paired with heterodimer chain 2 of the same or a different canine IgG subtype. Heterodimer chain 3 of one canine IgG subtype may be paired with heterodimer chain 4 of the same or a different canine IgG subtype. Heterodimer chain 1 of one feline IgG subtype may be paired with heterodimer chain 2 of the same or a different feline IgG subtype. This design enables dimerization of bispecific canine or feline antibodies. Furthermore, two different peptides or proteins, or combinations of different proteins, can be fused to the heterodimer Fc chain. For example, a GLP1 and glucagon dual molecule can be created using mutant canine IgG-D Fc heterodimer chains or mutant cat IgG-Fc heterodimer chains, such as a GLP1 polypeptide fused to mutant canine IgG-D Fc heterodimer chain 1 (e.g., SEQ ID NO: 14) (e.g., SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, or SEQ ID NO: 91) and a glucagon polypeptide fused to mutant canine IgG-D Fc heterodimer chain 2 (e.g., SEQ ID NO: 15) (e.g., SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95).

[0188] Example 5 GLP1 fusion protein The amino acid sequence of GLP1 is conserved among humans, cats, dogs, and horses, among other species. GLP1 protein degradation products (e.g., GLP1 (amino acids 7-37) (SEQ ID NO: 85)) are understood to be rapidly degraded by dipeptidyl peptidase-4 (DPP-4) and have a short serum half-life of several minutes.

[0189] To generate long-acting GLP1 fusion proteins, we investigated the design of sequential polypeptides containing at least one GLP1 polypeptide and a cat, canine, or equine IgG Fc polypeptide. The following constructs were designed. Equation (I): GLP1A-L1-Fc; Formula (II): Fc-L1-GLP1A; or Equation (III): GLP1A-L1-Fc-L2-GLP1B In the formula, GLP1A is the first GLP1 polypeptide, GLP1B is the second GLP1 polypeptide, L1 and L2 are linkers, and Fc is the IgG Fc polypeptide of the companion animal species.

[0190] GLP1 was modified to be DPP-4 resistant by replacing alanine with either glycine or serine at the position corresponding to position 8 of wild-type GLP1 (7-37) (SEQ ID NO: 85). The minimal sequence of wild-type GLP1 (7-37) (SEQ ID NO: 87) for binding to the N-terminal domain of mature cat GLP1R (SEQ ID NO: 49) was analyzed by three-dimensional protein modeling of the complex. Based on this modeling, two C-terminal amino acids were removed from DPP-4 resistant GLP1 to generate the GLP1-S8 (7-35) (SEQ ID NO: 86) and GLP1-G8 (7-35) (SEQ ID NO: 87) polypeptides.

[0191] GLP1 polypeptides are less susceptible to DPP-4 degradation when located at the C-terminus of constructs such as formulas II and III. Therefore, substitution from alanine to glycine or serine is not necessary for GLP1 polypeptides located at the C-terminus. Consequently, wild-type GLP1(7-37)(SEQ ID NO: 85) can be used at the C-terminus.

[0192] The linker may be a flexible, unstructured linker, such as a glycine and serine-rich linker. A flexible extension can be added to the C-terminus of a continuous polypeptide. The extension may contain 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.

[0193] Example 6 GLP1 and feline IgG Fc fusion protein (1) Nucleotide sequences encoding formula I sequential polypeptide (ssGLP1-G8_I_WTfeIgG2; SEQ ID NO: 96) having a signal sequence, GLP1-G8(7-35)(SEQ ID NO: 87), a flexible linker, and wild-type feline IgG2 Fc were synthesized and cloned into separate mammalian expression vectors. Wild-type feline IgG2 Fc (SEQ ID NO: 16) was selected based on its weak or absent C1Q binding for reduced complement activity and its protein A binding for easy purification.

[0194] The obtained vectors were transfected separately into CHO cells. The supernatant containing the serial polypeptides without the signal peptides (SEQ ID NOs. 24 and 23) was collected and filtered. Both proteins were affinity-purified using a Protein A column (Repligen CaptivA® Protein Affinity). The proteins were evaluated by HPLC gel filtration and determined to be monomers. However, SDS-PAGE analysis showed that percentages of both serial polypeptides migrated to the same position in the gel both in the absence and in the presence of a reducing agent (DTT) (Figure 2A). This result suggests that the hinge of wild-type feline IgG2 Fc, which has only one pair of cysteine ​​residues, may be insufficient for forming an effective disulfide bond.

[0195] Using three-dimensional protein modeling analysis of several orthologous hinge structures, we determined the approximate locations for modifying the feline IgG2 hinge to increase disulfide formation. To increase disulfide formation at the feline IgG2 hinge, the hinge sequence can be modified by substituting amino acids with cysteine. For example, a mutant feline IgG2 Fc (SEQ ID NO: 17) with a modified hinge was prepared by substituting Gly with Cys at the amino acid position corresponding to position 14 of SEQ ID NO: 16. Corresponding serial polypeptides containing the mutant feline IgG Fc of SEQ ID NO: 17, ssGLP1-G8_I_VARfeIgG2 (SEQ ID NO: 39) and ssGLP1-G8 / GLP1-2G_III_WTfeIgG2 (SEQ ID NO: 38), were designed, 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 NOs: 26 and 25, respectively. SDS-PAGE analysis of the mutant feline IgG2 construct showed a decrease in the amount of low molecular weight band protein compared to the wild-type feline IgG2 construct in the absence of a reducing agent (compare Figure 2B with Figure 2A). These results suggest that Fc covalent pair formation was improved in both mutant feline IgG2 constructs.

[0196] Furthermore, differential scanning fluorescence analysis was used to evaluate the stability of the serial polypeptides at various pH levels, expressed as the mean melting temperature (n=3) (Table 9 below). The improved stability of the mutant cat IgG2 hinge was most pronounced at pH 6. For example, constructs containing mutant cat IgG2 (SEQ ID NOs. 25 and 26) showed higher Tm (56.9°C and 59.7°C, respectively) at pH 6 than their corresponding constructs containing wild-type cat IgG2 (SEQ ID NOs. 23 and 24).

[0197] Table 9 TIFF2026048634000052.tif83170

[0198] Sequential polypeptides of formulas I, II, and III, including GLP1-S8(7-35)(SEQ ID NO: 86) instead of GLP1-G8(7-35)(SEQ ID NO: 87), can be similarly designed and prepared. For example, ssGLP1-S8_I_WTfeIgG2(SEQ ID NO: 40), GLP1-S8_I_WTfeIgG2(SEQ ID NO: 28), ssGLP1-S8 / GLP1-3G_III_WTfeIgG2(SEQ ID NO: 37), and GLP1-S8 / GLP1-3G_III_WTfeIgG2(SEQ ID NO: 27) can be prepared. Similar constructs with variants such as GLP1-S8_I_VARfeIgG2, GLP1-S8_I_VARfeIgG2, GLP1-S8 / GLP1-2G_III_VARfeIgG2, and GLP1-S8 / GLP1-3G_III_VARfeIgG2 can also be prepared instead of wild-type feline IgG Fc.

[0199] In this example, feline IgG2 Fc was used, but sequential polypeptides of formulas I, II, and III can be designed and prepared, containing feline IgG1a Fc or IgG1b Fc instead of IgG2. For example, similar sequential polypeptides can be designed and prepared, having wild-type feline IgG1a Fc, wild-type feline IgG1b Fc, mutant feline IgG1a Fc, or mutant feline IgG1b Fc (sequences 80 to 83, respectively).

[0200] Example 7 GLP1 and canine IgG Fc fusion protein Various sequential polypeptides of formulas I, II, and III, containing mutant GLP1 and canine IgG Fc, can be designed and prepared. For example, mutant canine IgGD Fc (SEQ ID NO: 7 or SEQ ID NO: 62) can be selected based on the weak or complete absence of C1q binding for reduced complement activity and on protein A binding for easy purification. Furthermore, flexible non-structural linkers, such as glycine and serine-rich linkers, can be used.

[0201] GLP1-G8_I_VARcaIgGD (SEQ ID NO: 30) and GLP1-S8_I_VARcaIgGD (SEQ ID NO: 32) are examples of formula I sequence polypeptides comprising (1) either GLP1-G8(7-35) or GLP1-S8(7-35), (2) a flexible linker, and (3) a mutant canine IgGD Fc (e.g., SEQ ID NO: 7). GLP1-G8 / GLP1-3G_III_VARcaIgGD (SEQ ID NO: 29) and GLP1-S8 / GLP1-3G_III_VARcaIgGD (SEQ ID NO: 31) are examples of formula III sequence polypeptides comprising (1) either GLP1-G8 (SEQ ID NO: 87) or GLP1-S8 (SEQ ID NO: 86), (2) GLP1(7-35) (SEQ ID NO: 61), (3) two flexible linkers, (4) a 3G C-terminal extension, and (5) a mutant canine IgGD Fc.

[0202] Sequential polypeptides can be designed with various modifications to the signal sequence, different GLP1 polypeptides (e.g., SEQ ID NOs. 85, 86, 87, 98, or 99), or canine IgG Fc. Examples of such mutations include ssGLP1-S8 / GLP1-2G_III_VARcaIgGD (SEQ ID NOs. 41), ssGLP1-G8 / GLP1-2G_III_VARcaIgGD (SEQ ID NOs. 42), ssGLP1-S8 / GLP1-3G_III_VARcaIgGD (SEQ ID NOs. 43), GLP1-S8 / GLP1-2G_III_VARcaIgGD (SEQ ID NOs. 105), and GLP1-G8 / GLP1-2G_III_VARcaIgGD (SEQ ID NOs. 106).

[0203] Furthermore, the sequential polypeptides of formulas I, II, and III may contain wild-type canine IgGD or wild-type or mutant canine IgGA, IgGB, or IgGC instead of mutant canine IgGD. For example, similar sequential polypeptides having wild-type canine IgGA Fc, IgGB Fc, IgGC Fc, or IgGD Fc (e.g., SEQ ID NOs: 1, 2, 3, or 4, respectively) can be designed and prepared. Further examples include sequential polypeptides containing mutant canine IgGA Fc (e.g., SEQ ID NOs: 5 or 60), mutant canine IgGB Fc (e.g., SEQ ID NOs: 78), or mutant canine IgGC Fc (e.g., SEQ ID NOs: 6, 61, 79, or 84).

[0204] GLP1-G8 / GLP1-3G_III_VARcaIgGD (SEQ ID NO: 29) and GLP1-G8_I_VARcaIgGD (SEQ ID NO: 30) were expressed separately in CHO cells, and the supernatant containing the proteins was collected and filtered. Both sequential polypeptides were affinity-purified by protein A chromatography. The SDS-PAGE profiles of the two sequential polypeptides were compared in the absence and presence of a reducing agent (DTT), and the results suggested that Fc disulfide bonds were effectively formed for both polypeptides (data not shown).

[0205] Example 8 GLP1 and horse IgG Fc fusion protein Various sequential polypeptides of formulas I, II, and III, containing mutant GLP1 and equine IgG Fc, can be designed and prepared. For example, mutant canine IgG2 Fc (e.g., SEQ ID NOs: 19, 71, or 72) can be selected based on the weak or absent C1q binding for reduced complement activity and on protein A binding for easy purification. Furthermore, flexible non-structural linkers, such as glycine and serine-rich linkers, can be used.

[0206] GLP1-G8_I_VAReqIgG2 (SEQ ID NO: 34) and GLP1-S8_I_VAReqIgG2 (SEQ ID NO: 36) are examples of formula I sequence polypeptides comprising (1) either GLP1-G8 or GLP1-S8, (2) a flexible linker, and (3) a mutant horse IgG2 Fc (e.g., SEQ ID NO: 19). GLP1-G8 / GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 33) and GLP1-S8 / GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 35) are examples of formula III sequence polypeptides comprising (1) either GLP1-G8(7-35)(SEQ ID NO: 87) or GLP1-S8(7-35)(SEQ ID NO: 86), (2) GLP1(7-35)(SEQ ID NO: 61), (3) two flexible linkers, (4) a 3G C-terminal extension, and (5) a mutant horse IgGD Fc.

[0207] Sequential polypeptides can be designed with additional modifications to the signal sequence, different GLP1 analogs (e.g., SEQ ID NOs. 86, 87, 98, or 99), glycine extensions (e.g., SEQ ID NOs. 88 through 95), or equine IgG Fc. Examples of such mutations include ssGLP1-G8 / GLP1-3G_III_VAReqIgG2 (SEQ ID NOs. 44), ssGLP1-G8_I_VAReqIgG2 (SEQ ID NOs. 45), ssGLP1-S8 / GLP1-3G_III_VAReqIgG2 (SEQ ID NOs. 46), and ssGLP1-S8_I_VAReqIgG2 (SEQ ID NOs. 47).

[0208] Furthermore, the sequential polypeptides of formulas I, II, and III may contain wild-type horse IgG2 or wild-type or mutant horse IgG1, IgG3, IgG4, IgG5, IgG6, or IgG7 instead of mutant horse IgG2. For example, similar sequential polypeptides having wild-type horse IgG1 Fc (e.g., SEQ ID NO: 63), IgG2 Fc (e.g., SEQ ID NO: 18 or 64), IgG3 Fc (e.g., SEQ ID NO: 65), IgG4 Fc (e.g., SEQ ID NO: 66), IgG5 Fc (e.g., SEQ ID NO: 67), IgG6 Fc (e.g., SEQ ID NO: 68), or IgG7 Fc (e.g., SEQ ID NO: 69) can be designed and prepared. Further examples include sequential polypeptides containing mutant horse IgG1Fc (e.g., SEQ ID NO: 70), mutant horse IgG3Fc (e.g., SEQ ID NO: 73), mutant horse IgG4Fc (e.g., SEQ ID NO: 74), mutant horse IgG5Fc (e.g., SEQ ID NO: 75), mutant horse IgG6Fc (e.g., SEQ ID NO: 76), or mutant horse IgG7Gc (e.g., SEQ ID NO: 77).

[0209] The nucleotide sequences encoding ssGLP1-G8 / GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 44) and ssGLP1-G8_I_VAReqIgG2 (SEQ ID NO: 45) were synthesized and cloned into separate mammalian expression vectors. The resulting vectors were transfected separately into CHO cells. The supernatant containing the serial polypeptides after cleavage of the signal peptides (SEQ ID NOs: 103 and 104) was collected and filtered. Both proteins were affinity-purified using a Protein A column (Repligen's CaptivA® Protein Affinity Resin) and expressed separately in CHO cells. The supernatant containing the proteins was collected and filtered. Both serial polypeptides were affinity-purified by Protein A chromatography. The SDS-PAGE profiles of the two serial polypeptides were compared in the absence and presence of a reducing agent (DTT), and the results suggested that Fc disulfide bonds were effectively formed for both polypeptides (data not shown).

[0210] Example 9 Expression and purification of the feline GLP1R N-terminal soluble domain The N-terminal domain of mature feline GLP1R (SEQ ID NO: 49), which is involved in the binding of GLP1 to GLP1R, was identified from the full-length feline GLP1R amino acid sequence (SEQ ID NO: 48). (1) Nucleotide sequences encoding the signal sequence, feline N-terminal GLP1R, human Fc and polyHis tag (ssFeGLP1R-N-huFc_PolyHis; SEQ ID NO: 50), and (2) the signal sequence, feline N-terminal GLP1R and polyHis tag (ssFeGLP1R-N_polyHis; SEQ ID NO: 51) were synthesized and cloned into separate mammalian expression vectors. The resulting vectors were transfected separately into CHO cells. The supernatant containing the polypeptides was collected and filtered. For the huFc construct, the proteins were affinity-purified using a Ni-Sepharose column or a Protein A column (Repligen CaptivA® Protein A Affinity). Both proteins were used for GLP1 functional binding and ELISA.

[0211] Example 10 GLP1 fusion protein binding kinetics The binding affinity of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) to FeGLP1R-N-huFc_PolyHis was evaluated using biolayer interferometry (octet). Specifically, FeGLP1R-N-huFc_PolyHis was biotinylated, unreacted free biotin was removed, and the biotinylated protein was captured on a streptavidin sensor chip. The association of GLP1-G8_I_VARfeIgG2 at various concentrations was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was subtracted to correct for drift. The data was then processed using ForteBio. TM We used data analysis software to fit a 1:1 coupled model, k on , k off and K dThe following was determined. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. The K between the feline GLP1R N-terminal domain and GLP1-G8_I_VARfeIgG2 was determined. d is 8.0x10 -9 and 16x10 -9 It was during the M period.

[0212] Example 11 Bioactivity of GLP1 fusion proteins in cell-based assays GLP1 cell activity was measured using the cAMP Hunter Bioassay Kit (Discoverx, catalog #95-0062Y2) with the CHOK-1-GL1R cell line (Discoverx, catalog #95-0062C2), a hamster ovary cell line overexpressing Gs-coupled human glucagon ligand peptide-1 receptor (GLP1R) on its cell surface. Cells were seeded in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, cells were treated in 3-fold serial dilutions with either a control agonist—GLP1 human (37a.a.) (Prospec, catalog # HOR-236) or Extendin-4 (Discoverx, catalog # 92-1115)—or a serial polypeptide containing mutant GLP1, and then 37 o The samples were incubated in 1C for 30 minutes. The serial polypeptides tested were GLP1-G8 / GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) and GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26).

[0213] When GLP1 binds to the Gs-coupled GLP1R receptor, Gs stimulates adenylate cyclase to produce cAMP. At the end of incubation, cells were added a cAMP Working Detection Solution containing cAMP Antibody Reagent, lysis buffer, cAMP conjugated with small fragments of β-galactosidase (β-gal), and substrates. Cells were incubated in the dark at room temperature for 1 hour to induce an immune competition response for cAMP antibody binding between endogenously produced cAMP and cAMP conjugated with β-gal fragments.

[0214] At the end of a 1-hour incubation, cAMP Solution A containing large β-gal fragments, which could be supplemented with free (antibody-unbound) β-gal small fragments cAMP to form a functional enzyme, was added to the cell lysates. This lysate was incubated in the dark at room temperature for 3 to 6 hours to allow β-gal to hydrolyze the substrate and generate a luminescence signal.

[0215] The more unbound, free β-gal fragments cAMP that remain, the more functional the β-gal enzyme is formed. Therefore, the amount of signal generated is directly proportional to the amount of cAMP in the cell lysate. At the end of incubation, luminescence was read using a Synergy HT microplate reader (Biotek, Winuski, Vermont). EC50 was calculated using GraphPad Prism software (GraphPad Software, La Jolla, USA).

[0216] Figure 3 shows plots of relative light intensity (RLU) and concentration for each sample tested. The EC50 values ​​are listed in Table 10 below. Both GLP1-G8 / GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) and GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) are more active than GLP1(7-37) and Extendin-4. GLP1-G8 / GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) is more potent than GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26), suggesting that C-terminal GLP1 may contribute to additional activity.

[0217] Table 10 TIFF2026048634000053.tif58170

[0218] Example 12 Long-term stability of GLP1 fusion protein GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored at a concentration of 1.3 mg / mL in PBS at pH 7.2 in a 1.5 mL Eppendorf tube and stored at 2-8°C for 1 year (Lot 2-29-2016). Additionally, GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored at a concentration of 10 mg / mL in PBS at pH 7.2 in a 1.5 mL Eppendorf tube and stored at 2-8°C for 1 day (Lot 2-2-2017). To evaluate stability, the stored samples were analyzed using a cell-based assay with the same CHOK-1-GL1R cell line (Discoverx, catalog #95-0062C2) as described in Example 11. Cell activity was measured using cAMP-Glo TM Evaluation was performed using Max Assay (Promega, catalog #PAV1682).

[0219] CHOK-1-GL1R cells were seeded at a density of 20,000 cells per well in F-12K medium (ATCC, catalog #ATCC(registered trademark)30-2004) supplemented with heat-inactivated 10% fetal bovine serum (Sigma, catalog #2868) in 96-well plates (Corning, catalog #3610) and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, cells were stimulated with the control agonist GLP1 human (37a.a.) (Prospec, catalog #HOR-236), lot 2-20-2016 or lot 2-2-2017, serially diluted three-fold in serum-free medium. Then, Complete Induction Buffer containing MgCl2 up to a final concentration of 20 mM, isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog #I7018) up to a final concentration of 500 μM, and Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)imidazolidone] (Sigma-Aldrich, catalog #B8279) up to a final concentration of 100 μM was added.

[0220] Cells were incubated at room temperature for approximately 30 minutes. During this process, when GLP1 binds to the Gs-coupled GLP1R receptor, Gs stimulates adenylate cyclase to produce cAMP. At the end of incubation, cAMP Detection Solution containing inactive protein kinase A holoenzyme, protein kinase A substrate, and lysis buffer was added to the cells. The plate was placed in an orbital shaker for 1-2 minutes, and then incubated at room temperature (23°C) for 20 minutes. Cellular cAMP activates protein kinase A by binding to its regulatory inhibitory subunit and releasing a catalytic subunit. The released catalytic subunit catalyzes the transfer of the terminal phosphate of ATP to the protein kinase A substrate, consuming cellular ATP in this process. At the end of incubation, luciferase-based Kinase-Glo® Reagent was added to the cell lysates, the plate was shaken in an orbital shaker for 2 minutes, and then incubated in the dark at room temperature for 10 minutes.

[0221] The monooxygenation of luciferin occurs when Mg is present in the cell lysate. 2+ Luciferase catalyzed the luminescence in the presence of ATP, yielding a luminescence signal proportional to the amount of ATP in the cell. At the end of a 10-minute incubation, the plate was read using a Synergy HT microplate reader (Biotek, Winuski, Vermont). The luminescence was proportional to the ATP level but inversely proportional to the cAMP level. Therefore, as the cAMP concentration increased, the luminescence decreased.

[0222] Figure 4 shows the results of a cell-based assay, plotting the luminescence pair concentration of lot 2-2-2017 and lot 2-29-2016 compared to GLP(31 aa). The GLP1-G8_I_VARfeIgG2 sample maintained cellular activity even after being stored in PBS at 2-8°C for one year.

[0223] Example 13 GLP1 Fusion Protein Serum Stability G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored in pH 7.2 PBS containing cat serum at 37°C for 24 hours, and serum stability was tested in vitro. This cell-based assay was performed as described in Example 12, and the results suggested that activity was maintained (data not shown). Furthermore, no visible degradation was observed by Western blot analysis (Figure 5).

[0224] Example 14 GLP1 fusion protein in vivo pharmacokinetics GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was administered as a single subcutaneous injection (2 mg / kg) to five cats. Serum samples were collected before administration (0 hours) and at 4, 8, 12, 24, 48, 72, and 168 hours. The concentration of GLP1-G8_I_VARfeIgG2 in the serum samples was measured quantitatively by ELISA. GLP1-G8_I_VARfeIgG2 polypeptide with a detection limit of 4 ng / mL was used as the reference. The serum concentration of GLP1-G8_I_VARfeIgG2 was plotted against time (Figure 6). The mean serum half-life (t) of GLP1-G8_I_VARfeIgG2 was plotted. 1 / 2 The average time was 39 hours. max It was 22 hours, average C max The concentration was 12 μg / mL, and the mean area under the curve (AUC) was approximately 950 μg(h) / mL.

[0225] For quantitative ELISA, an anti-GLP1 antibody (4F3, Novus Biologicals, catalog No. NBP1-97413) and a goat anti-feline IgG-Fc, HRP conjugate antibody (Bethyl Laboratories, Inc., catalog No. A20-117P) were used to quantify GLP1-G8_I_VARfeIgG2 in feline serum samples for in vivo pharmacokinetic studies. 96-well plates were coated with the anti-GLP1 antibody (5 μg / mL in coating buffer, 100 μl / well). The plates were sealed and incubated overnight at 4°C. The plates were washed three times with 1XTBST (10XTBST, Teknova, catalog No. T9511) and blocking buffer was added. After removing the blocking buffer, serial dilutions of standard samples and samples in blocking buffer were added (100 μl / well), and the plates were incubated at room temperature for 2 hours. The plates were washed three times with 1XTBST, and goat anti-feline IgG Fc antibody was added (0.1 μg / mL in blocking buffer; 100 μl / well). After incubation at room temperature for 1 hour, the plates were washed five times with 1XTBST. TMB substrate (ScyTek, catalog no. TM1999) was added (100 μl / well), and incubated at room temperature for 1 minute. The reaction was stopped by adding 2 M H2SO4 (50 μl / well). The absorbance at 50 nm was measured, and the concentration of GLP1-G8_I_VARfeIgG2 in the serum samples was calculated.

[0226] Furthermore, the wGLP1-G8_I_VARfeIgG2 concentration in the same serum samples (without the addition of a DPP-4 inhibitor) was evaluated using a cell-based activity assay. The same CHOK-1-GL1R cell line (Discoverx, catalog #95-0062C2) as described in Example 12 and cAMP-Glo TMA Max Assay (Promega, catalog #PAV1682) was used. However, in this example, cells were stimulated with the control agonist GLP1 human (37 aa) (Prospec, catalog #HOR-236) or with cat serum samples collected before administration (0 hours) and at 4, 8, 12, 24, 48, 72, and 168 hours, diluted in serum-free medium (5%, 0.5%, and 0.05% dilutions). The concentration of GLP1-G8_I_VARfeIgG2 in each sample was calculated using SoftMax pro 7 (Molecular Devices, Sunnyvale, California). The mean concentration of GLP1-G8_I_VARfeIgG2 in five cats was plotted against time (Figure 7). The mean AUC was approximately 840 μg(h) / mL, and the mean t 1 / 2 The incubation period was 36 hours. The concentrations calculated from the cell-based activity assay were consistent with those obtained from the ELISA, suggesting that the mutant GLP1 detected by ELISA is biologically active.

[0227] Example 15 GLP1, glucagon, and IgG Fc fusion protein To investigate long-acting GLP1 receptor and glucagon receptor dual agonists, we have constructed a construct containing a GLP1 polypeptide, a glucagon polypeptide, and an IgG Fc polypeptide, as follows: Formula (IV): GLP1-L1-Fc-L2-Gluc; and Formula (V): Gluc-L1-Fc-L2-GLP1 A series of polypeptides having the following characteristics has been designed: In the above formula, GLP1 is a GLP1 polypeptide, Gluc is a glucagon polypeptide, L1 and L2 are linkers, and Fc is an Fc polypeptide.

[0228] As described above, GLP1 was modified to be DPP-4 resistant by replacing alanine with either glycine or serine at the position corresponding to position 8 of wild-type GLP1(7-37)(SEQ ID NO: 85). Furthermore, DPP-4 resistant GLP1 was further modified by removing two C-terminal amino acids to generate mutant GLP1-S8(7-35)(SEQ ID NO: 86) and mutant GLP1-G8(7-35)(SEQ ID NO: 87) polypeptides.

[0229] GLP1 polypeptides are less susceptible to DPP-4 degradation when located at the C-terminus of constructs such as formula (V). Therefore, substitution from alanine to glycine or serine is not necessary for C-terminal GLP1 polypeptides. Consequently, wild-type GLP1(7-37) (SEQ ID NO: 85) can be used at the C-terminus.

[0230] The linker may be a flexible, unstructured linker, such as a glycine and serine-rich linker. A flexible extension can be added to the C-terminus of a continuous polypeptide. The extension may contain 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.

[0231] The serial polypeptide may include wild-type glucagon polypeptide (e.g., SEQ ID NO: 21) or mutant glucagon polypeptide.

[0232] The serial polypeptide may contain human IgG Fc or IgG Fc from a companion animal species such as dog, cat, or horse. The subtype of IgG Fc used is based on low or no C1q binding activity and / or protein A binding ability. For example, wild-type or mutant human, dog, horse, or cat IgG Fc with low or no C1q binding affinity and / or protein A binding ability may be used (e.g., SEQ ID NOs: 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, or 84).

[0233] Exemplary serial polypeptides, including GLP1 polypeptide, glucagon polypeptide, and feline IgG Fc polypeptide, include GLP1-G8 / Gluc-3G_IV_WTfeIgG2 (SEQ ID NO: 52) and Gluc / GLP1-2G_V_WTfeIgG2 (SEQ ID NO: 53).

[0234] Exemplary serial polypeptides containing GLP1 polypeptide, glucagon polypeptide, and canine IgG Fc polypeptide include GLP1-G8 / Gluc-4G_IV_VARcaIgGD (SEQ ID NO: 54) and Gluc / GLP1-3G_V_VARcaIgGD (SEQ ID NO: 55).

[0235] Exemplary serial polypeptides, including GLP1 polypeptide, glucagon polypeptide, and horse IgG Fc polypeptide, include GLP1-G8 / Gluc-4G_IV_VAReqIgGD (SEQ ID NO: 56) and Gluc / GLP1-3G_V_VAReqIgG2 (SEQ ID NO: 57).

[0236] Exemplary serial polypeptides, including GLP1 polypeptides, glucagon polypeptides, and human IgG Fc polypeptides, include GLP1-G8 / Gluc-4G_IV_huIgG4 (SEQ ID NO: 8) and Gluc / GLP1-3G_V_huIgG4 (SEQ ID NO: 59).

[0237] Example 16 Mutant IgG Fc polypeptide for enhancing hinged disulfide formation We modified feline and equine IgG hinges using additional three-dimensional protein modeling analysis of several orthologous hinge structures. To enhance disulfide formation in the feline IgG hinge, the hinge sequence can be modified by substituting lysine with proline at the position corresponding to position 16 of feline IgG2 (SEQ ID NO: 16), feline IgG1a (SEQ ID NO: 80 or 117), or feline IgG1b (SEQ ID NO: 81 or 118) (e.g., K16P). Examples of amino acid sequences of mutant feline IgG polypeptides with modified hinges include SEQ ID NOs: 125, 126, and 127.

[0238] To enhance disulfide formation at the equine IgG hinge, the hinge sequence can be modified by substituting cysteine ​​with serine at the position corresponding to position 3 of equine IgG (e.g., IgG2 Fc (SEQ ID NO: 129)) and / or by substituting glutamine with proline at the position corresponding to position 20 of equine IgG (e.g., IgG2 Fc (SEQ ID NO: 129) (e.g., C3S, Q20P)). Examples of amino acid sequences of mutant equine IgG polypeptides with modified hinges include SEQ ID NOs: 130, 131, 132, 134, and 135.

[0239] Example 17 Mutant IgG Fc polypeptides for enhanced recombinant production and / or enhanced disulfide formation Using three-dimensional protein modeling, feline and equine mutant IgG Fc polypeptides containing sequences from the hinge regions of different IgG isotypes were designed to enhance recombinant production and improve hinged disulfide formation. Mutant feline IgG2 Fc polypeptides can be prepared containing sequences from the hinge regions of feline IgG1a or IgG1b (e.g., SEQ ID NO: 125). Furthermore, mutant equine IgG2 Fc polypeptides can be prepared containing sequences from the hinge region of equine IgG1 (e.g., SEQ ID NO: 19).

[0240] SDS-PAGE analysis under reducing and non-reducing conditions allows for the determination of the level of recombinant production and / or hinged disulfide formation of mutant IgG Fc polypeptides and their comparison to levels of other IgG Fc (e.g., the corresponding wild-type IgG Fc of the same or different isotype, or wild-type or mutant IgG Fc from another companion animal).

Claims

1. A polypeptide comprising a mutant IgGFc polypeptide having at least one amino acid modification to the wild-type IgGFc polypeptide of a companion animal species, wherein the mutant IgGFc polypeptide has increased binding affinity to protein A compared to the wild-type IgGFc polypeptide.

2. A polypeptide comprising a mutant IgGFc polypeptide having at least one amino acid modification to the wild-type IgGFc polypeptide of a companion animal species, wherein the mutant IgGFc polypeptide has a reduced binding affinity to C1q and / or CD16 compared to the wild-type IgGFc polypeptide.

3. The mutant IgG Fc polypeptide was measured by biolayer interferometry and measured 5 x 10⁻¹⁰ －６ Super M, 1x10 －５ Super M, 5x10 －５ Super M, 1x10 －４ Super M, 5x10 －４ M or 1 x 10 －３ Dissociation constant (K) above M ｄ The polypeptide according to claim 1 or claim 2, wherein the polypeptide is bonded to C1q and / or CD16 at )

4. The polypeptide according to any one of claims 1 to 3, wherein the mutant IgG Fc polypeptide has increased binding affinity to protein A compared to the wild-type IgG Fc polypeptide.

5. 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 －６ M, less than 5×10 －７ M, less than 1×10 －７ M, less than 5×10 －８ M, less than 1×10 －８ M, less than 5×10 －９ M, less than 1×10 －９ M, less than 5×10 －１０ M, less than 1×10 －１０ M, less than 5×10 －１１ M, less than 1×10 －１１ M, less than 5×10 －１２ [[ID=ABC]] M, or less than 1×10 －１２ M, as measured by biolayer interferometry. The polypeptide according to any one of claims 1 to 4.

6. The polypeptide according to any one of claims 1 to 5, wherein the companion animal species is a dog, a cat, or a horse.

7. Wild-type IgG Fc polypeptide a) Canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc; b) Horse 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 The polypeptide according to any one of claims 1 to 6.

8. A polypeptide comprising a mutant IgGFc polypeptide having at least one amino acid modification to the hinge region of a wild-type feline or equine IgGFc polypeptide, wherein the mutant IgGFc polypeptide has increased recombinant production and / or increased hinge disulfide formation compared to the wild-type IgGFc polypeptide, as determined by SDS-PAGE analysis under reducing and / or non-reducing conditions.

9. The mutant IgG Fc polypeptide, a) At least one amino acid substitution for a wild-type cat IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 3 of SEQ ID NO: 129; and / or c) At least one amino acid substitution relative to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide includes an amino acid substitution at the position corresponding to position 20 of SEQ ID NO:

129. A polypeptide according to any one of claims 1 to 8, comprising:

10. The mutant IgG Fc polypeptide, a) At least one amino acid substitution for wild-type feline IgGFc polypeptide, wherein the mutant IgGFc polypeptide includes an amino acid substitution at position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide includes an amino acid substitution at position 3 of SEQ ID NO: 129; and / or c) At least one amino acid substitution to the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains an amino acid substitution at position 20 of SEQ ID NO:

129. A polypeptide according to any one of claims 1 to 9, comprising:

11. The mutant IgG Fc polypeptide, a) At least one amino acid substitution for wild-type feline IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains proline at the position corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118, or at position 16; b) At least one amino acid substitution in wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide has a serine molecule at the position corresponding to position 3 of SEQ ID NO: 129 or at position 3; and / or c) At least one amino acid substitution for the wild-type horse IgG Fc polypeptide, wherein the mutant IgG Fc polypeptide contains proline at the position corresponding to position 20 of SEQ ID NO: 129 or at position 20. A polypeptide according to any one of claims 1 to 10, comprising:

12. The polypeptide according to any one of claims 1 to 11, wherein the mutant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region of a different isotype of IgG Fc polypeptide.

13. The polypeptide according to any one of claims 1 to 12, wherein the mutant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region of a wild-type feline IgG-1a Fc polypeptide, a wild-type feline IgG-1b Fc polypeptide, or a wild-type horse IgG1 Fc polypeptide.

14. The polypeptide according to any one of claims 1 to 13, wherein the mutant IgG Fc polypeptide comprises SEQ ID NO: 19, SEQ ID NO: 125 or SEQ ID NO: 126, SEQ ID NO: 127, 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.

15. A polypeptide containing the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 125, or SEQ ID NO: 126, SEQ ID NO: 127, 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.

16. A polypeptide comprising a mutant IgG2 Fc polypeptide having at least one amino acid substitution compared to wild-type feline IgG2 Fc polypeptide, wherein at least one amino acid substitution is cysteine, and the mutant IgG2 Fc polypeptide can form at least one additional interchain disulfide bond compared to wild-type feline IgG2 Fc polypeptide.

17. The polypeptide according to any one of claims 1 to 16, wherein the mutant IgGFc polypeptide comprises at least one amino acid substitution relative to the wild-type feline IgGFc polypeptide, the at least one amino acid substitution being cysteine, and the mutant IgGFc polypeptide can form at least one additional interchain disulfide bond relative to the wild-type feline IgGFc polypeptide.

18. The polypeptide according to any one of claims 1 to 17, wherein the mutant IgG Fc polypeptide contains cysteine ​​at positions corresponding to the 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or 16th positions of SEQ ID NO:

16.

19. The polypeptide according to any one of claims 1 to 18, wherein the mutant IgG Fc polypeptide contains cysteine ​​at the position corresponding to position 14 of SEQ ID NO:

16.

20. The polypeptide according to any one of claims 1 to 19, wherein the mutant IgG Fc polypeptide contains cysteine ​​at position 14 of SEQ ID NO:

16.

21. The mutant IgG Fc polypeptide is represented by SEQ ID NOs: 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, SEQ ID NOs: The polypeptide according to any one of claims 1 to 20, which is at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to the amino acid sequence of 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, 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: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO:

157.

22. The polypeptide according to any one of claims 1 to 21, wherein the mutant IgG Fc polypeptide comprises SEQ ID NO:

17.

23. A polypeptide containing the amino acid sequence of SEQ ID NO:

17.

24. The mutant IgG Fc polypeptide, a) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 205 of SEQ ID NO: 1, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 1; b) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 3, and / or amino acid substitutions at the position corresponding to position 24 of SEQ ID NO: 3; c) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 4, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 4; d) Amino acid substitution at the position corresponding to position 15 of SEQ ID NO: 64, and / or amino acid substitution at the position corresponding to position 203 of SEQ ID NO: 64; e) an amino acid substitution at the position corresponding to position 199 of SEQ ID NO: 67, and / or an amino acid substitution at the position corresponding to position 200 of SEQ ID NO: 67; and / or f) Amino acid substitutions at the position corresponding to position 199 of SEQ ID NO: 68, amino acid substitutions at the position corresponding to position 200 of SEQ ID NO: 68, amino acid substitutions at the position corresponding to position 201 of SEQ ID NO: 68, and / or amino acid substitutions at the position corresponding to position 202 of SEQ ID NO: 68 A polypeptide according to any one of claims 1 to 23, comprising:

25. The mutant IgG Fc polypeptide, a) Amino acid substitutions at position 21 of SEQ ID NO: 1, position 23 of SEQ ID NO: 1, position 25 of SEQ ID NO: 1, position 80 of SEQ ID NO: 1, position 205 of SEQ ID NO: 1, and / or position 207 of SEQ ID NO: 1; b) Amino acid substitution at position 21 of SEQ ID NO: 3, amino acid substitution at position 23 of SEQ ID NO: 3, and / or amino acid substitution at position 24 of SEQ ID NO: 3; c) Amino acid substitution at position 21 of SEQ ID NO: 4, amino acid substitution at position 23 of SEQ ID NO: 4, amino acid substitution at position 25 of SEQ ID NO: 4, amino acid substitution at position 80 of SEQ ID NO: 4, and / or amino acid substitution at position 207 of SEQ ID NO: 4; d) Amino acid substitution at position 15 of SEQ ID NO: 64, and / or amino acid substitution at position 203 of SEQ ID NO: 64; e) an amino acid substitution at position 199 of SEQ ID NO: 67, and / or an amino acid substitution at position 200 of SEQ ID NO: 67; and / or f) Amino acid substitution at position 199 of SEQ ID NO: 68, amino acid substitution at position 200 of SEQ ID NO: 68, amino acid substitution at position 201 of SEQ ID NO: 68, and / or amino acid substitution at position 202 of SEQ ID NO: 68 A polypeptide according to any one of claims 1 to 24, comprising:

26. The mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4; d) Threonine or valine at position 15 of sequence number 64, and / or tyrosine or valine at position 203 of sequence number 64; e) Leucine at the position corresponding to position 199 of SEQ ID NO: 67, and / or histidine at the position corresponding to position 200 of SEQ ID NO: 67; and / or f) Leucine at position 199 of SEQ ID NO: 68, histidine at position 200 of SEQ ID NO: 68, asparagine at position 201 of SEQ ID NO: 68, and / or histidine at position 202 of SEQ ID NO:

68. A polypeptide according to any one of claims 1 to 25, comprising:

27. The mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4; d) Threonine or valine at position 15 of sequence number 64, and / or tyrosine or valine at position 203 of sequence number 64; e) Leucine at position 199 of sequence number 67, and / or histidine at position 200 of sequence number 67; and / or f) Leucine at position 199 of SEQ ID NO: 68, histidine at position 200 of SEQ ID NO: 68, asparagine at position 201 of SEQ ID NO: 68, and / or histidine at position 202 of SEQ ID NO: 68 A polypeptide according to any one of claims 1 to 26, comprising:

28. The mutant IgG Fc polypeptide, a) Sequence ID 5, Sequence ID 6, Sequence ID 7, Sequence ID 60, Sequence ID 61, Sequence ID 62 or Sequence ID 84; or b) Sequence ID 19, Sequence ID 71, Sequence ID 72, Sequence ID 75, or Sequence ID 76 A polypeptide according to any one of claims 1 to 27, comprising the amino acid sequence.

29. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 84, SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQ ID NO:

76.

30. The mutant IgG Fc polypeptide, a) Amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 2, or amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 3; b) An amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 63, an amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 65, an amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 66, or an amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 69; or c) Amino acid substitution at the position corresponding to position 198 of SEQ ID NO: 80, or amino acid substitution at the position corresponding to position 198 of SEQ ID NO: 81 A polypeptide according to any one of claims 1 to 29, comprising:

31. The mutant IgG Fc polypeptide, a) Amino acid substitution at position 93 of SEQ ID NO: 2, or amino acid substitution at position 93 of SEQ ID NO: 3; b) An amino acid substitution at position 87 of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 69; or c) Amino acid substitution at position 198 of SEQ ID NO: 80, or amino acid substitution at position 198 of SEQ ID NO: 81 A polypeptide according to any one of claims 1 to 30, comprising:

32. The mutant IgG Fc polypeptide, a) Arginine at position 93 of sequence number 2, or arginine at position 93 of sequence number 3; b) Serine at the position corresponding to position 87 of SEQ ID NO: 63, serine substitution at the position corresponding to position 87 of SEQ ID NO: 65, serine at the position corresponding to position 87 of SEQ ID NO: 66, or serine at the position corresponding to position 87 of SEQ ID NO: 69; or c) Alanine at position 198 of sequence number 80, or alanine at position 198 of sequence number 81. A polypeptide according to any one of claims 1 to 31, comprising:

33. The mutant IgG Fc polypeptide, a) Arginine at position 93 of sequence number 2, or arginine at position 93 of sequence number 3; b) Serine at position 87 of SEQ ID NO: 63, serine at position 87 of SEQ ID NO: 65, serine at position 87 of SEQ ID NO: 66, or serine at position 87 of SEQ ID NO: 69; or c) Alanine at position 198 of sequence number 80, or alanine at position 198 of sequence number 81 A polypeptide according to any one of claims 1 to 32, comprising:

34. The mutant IgG Fc polypeptide, a) Sequence ID 78, Sequence ID 79, or Sequence ID 84; or b) Sequence ID 70, Sequence ID 73, Sequence ID 74 or Sequence ID 77; or c) Sequence ID 82 or Sequence ID 83 A polypeptide according to any one of claims 1 to 33, comprising the amino acid sequence.

35. A polypeptide comprising the amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 82, or SEQ ID NO:

83.

36. The mutant IgG Fc polypeptide, a) an amino acid substitution at the position corresponding to position 5 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution at the position corresponding to position 98 of SEQ ID NO: 2; or b) Amino acid substitutions at the position corresponding to position 5 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 38 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 39 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 97 of SEQ ID NO: 3, and / or amino acid substitutions at the position corresponding to position 98 of SEQ ID NO: 3 A polypeptide according to any one of claims 1 to 35, comprising:

37. The mutant IgG Fc polypeptide, a) Amino acid substitution at position 5 of SEQ ID NO: 2, amino acid substitution at position 38 of SEQ ID NO: 2, amino acid substitution at position 39 of SEQ ID NO: 2, amino acid substitution at position 97 of SEQ ID NO: 2, and / or amino acid substitution at position 98 of SEQ ID NO: 2; or b) Amino acid substitution at position 5 of SEQ ID NO: 3, position 38 of SEQ ID NO: 3, position 39 of SEQ ID NO: 3, position 97 of SEQ ID NO: 3, and / or position 98 of SEQ ID NO: 3 A polypeptide according to any one of claims 1 to 36, comprising:

38. The mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO:

3. A polypeptide according to any one of claims 1 to 37, comprising:

39. The mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO: 3 A polypeptide according to any one of claims 1 to 38, comprising:

40. The mutant IgG Fc polypeptide, a) Sequence ID 139, Sequence ID 140, Sequence ID 141, Sequence ID 142, Sequence ID 143, Sequence ID 144, Sequence ID 145, Sequence ID 146 or Sequence ID 147; or b) Sequence IDs 148, 149, 150, 151, 152, 154, 155, 156, or 157 A polypeptide according to any one of claims 1 to 39, comprising the amino acid sequence.

41. A polypeptide containing the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO:

157.

42. 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: 3, or tyrosine or tryptophan at position 138 of SEQ ID NO: 4; or b) Tyrosine or tryptophan at position 154 of sequence number 16, tyrosine or tryptophan at position 154 of sequence number 80 or sequence number 117, or tyrosine or tryptophan at position 154 of sequence number 81 or sequence number 118 A polypeptide containing a mutant IgG Fc polypeptide.

43. The mutant IgG Fc polypeptide, 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: 3, or tyrosine or tryptophan at position 138 of SEQ ID NO: 4; or b) Tyrosine or tryptophan at position 154 of sequence number 16, tyrosine or tryptophan at position 154 of sequence number 80 or sequence number 117, or tyrosine or tryptophan at the position corresponding to position 154 of sequence number 81 or sequence number 118 A polypeptide according to any one of claims 1 to 42, comprising:

44. The polypeptide according to any one of claims 1 to 43, wherein the mutant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO:

123.

45. A polypeptide containing the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO:

123.

46. A continuous polypeptide comprising the polypeptide according to any one of claims 1 to 45 and a glucagon-like peptide 1 (GLP1) polypeptide.

47. A continuous polypeptide comprising the polypeptide according to any one of claims 1 to 46 and a glucagon polypeptide.

48. 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: 3, serine at position 138 of SEQ ID NO: 4, serine at position 154 of SEQ ID NO: 16, serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; 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: 3, alanine at position 140 of SEQ ID NO: 4, alanine at position 156 of SEQ ID NO: 16, alanine at position 156 of SEQ ID NO: 80 or 117, or alanine at position 156 of SEQ ID NO: 81 or 118; and / or c) Threonine at position 181 of SEQ ID NO: 1, threonine at position 180 of SEQ ID NO: 2, threonine at position 180 of SEQ ID NO: 3, threonine at position 181 of SEQ ID NO: 4, threonine at position 197 of SEQ ID NO: 16, threonine at position 197 of SEQ ID NO: 80 or 117, or threonine at position 197 of SEQ ID NO: 81 or 118 A polypeptide containing a mutant IgG Fc polypeptide.

49. The mutant 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: 3, serine at position 138 of SEQ ID NO: 4, serine at position 154 of SEQ ID NO: 16, serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; 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: 3, alanine at position 140 of SEQ ID NO: 4, alanine at position 156 of SEQ ID NO: 16, alanine at position 156 of SEQ ID NO: 80 or SEQ ID NO: 117, or alanine at position 156 of SEQ ID NO: 81 or SEQ ID NO: 118; 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: 3, threonine at position 181 of SEQ ID NO: 4, threonine at position 197 of SEQ ID NO: 16, threonine at position 197 of SEQ ID NO: 80 or SEQ ID NO: 117, or threonine at position 197 of SEQ ID NO: 81 or SEQ ID NO: 118 A polypeptide according to any one of claims 1 to 48, comprising:

50. The polypeptide according to any one of Embodiments 1 to 49, wherein the mutant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO:

124.

51. A polypeptide containing the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO:

124.

52. The polypeptide according to any one of claims 1 to 51, wherein the polypeptide is glycosylated.

53. The polypeptide according to any one of claims 1 to 51, wherein the polypeptide is nonglycosylated.

54. A continuous polypeptide comprising the polypeptide according to any one of claims 48 to 53 and a glucagon-like peptide 1 (GLP1) polypeptide.

55. A continuous polypeptide comprising the polypeptide according to any one of claims 48 to 53 and a glucagon polypeptide.

56. A heterodimer protein comprising the continuous polypeptide described in claim 46 and the continuous polypeptide described in claim 54.

57. A heterodimer protein comprising the continuous polypeptide described in claim 47 and the continuous polypeptide described in claim 55.

58. The continuous polypeptide or heterodimer protein according to any one of claims 46, 47, or 54 to 57, wherein the GLP1 polypeptide is a wild-type GLP1 polypeptide and optionally comprises the amino acid sequence of SEQ ID NO:

85.

59. The sequential polypeptide or heterodimer protein according to any one of claims 46, 47, or 54 to 58, wherein the GLP1 polypeptide is a mutant GLP1 polypeptide.

60. The continuous polypeptide or heterodimer protein according to any one of claims 46, 47, or 54 to 59, wherein the GLP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ ID NO:

99.

61. The continuous polypeptide or heterodimer protein according to any one of claims 46, 47, or 54 to 60, wherein the glucagon polypeptide is a wild-type glucagon polypeptide and optionally comprises the amino acid sequence of SEQ ID NO:

21.

62. The sequential polypeptide or heterodimer protein according to any one of claims 46, 47, or 54 to 61, wherein the glucagon polypeptide is a mutant glucagon polypeptide.

63. i) A first mutant canine IgG Fc polypeptide comprising at least one amino acid modification to a first wild-type canine IgG Fc polypeptide, and a second mutant canine IgG Fc polypeptide comprising at least one amino acid modification to a second wild-type canine IgG Fc polypeptide; or ii) A first mutant cat IgG Fc polypeptide having at least one amino acid modification compared to the first wild-type cat IgG Fc polypeptide, and a second mutant cat IgG Fc polypeptide having at least one amino acid modification compared to the second wild-type cat IgG Fc polypeptide. A heterodimeric protein containing, a) The first mutant canine IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 138 of SEQ ID NO: 1, position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position 138 of SEQ ID NO: 4; b) The second mutant canine IgG Fc polypeptide contains amino acid substitutions at positions corresponding to positions 138, 140 and / or 181 of SEQ ID NO: 1, 137, 139 and / or 180 of SEQ ID NO: 2, 137, 139 and / or 180 of SEQ ID NO: 3, and 138, 140 and / or 181 of SEQ ID NO: 4; c) The first mutant cat IgG Fc polypeptide contains an amino acid substitution at the position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; and / or d) The second mutant cat IgG Fc polypeptide contains amino acid substitutions at positions 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 Heterodimeric protein.

64. The heterodimer protein according to claim 63, wherein the first wild-type canine IgG Fc polypeptide and the second wild-type canine IgG Fc polypeptide are of the same IgG subtype, and / or the first wild-type feline IgG Fc polypeptide and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype.

65. The heterodimer protein according to claim 63, wherein the first wild-type canine IgG Fc polypeptide and the second wild-type canine IgG Fc polypeptide are of different IgG subtypes, and / or the first wild-type feline IgG Fc polypeptide and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype.

66. a) The first mutant canine IgG Fc polypeptide contains tyrosine or tryptophan at the position corresponding to position 138 of SEQ ID NO: 1, position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position 138 of SEQ ID NO: 4; and / or b) The first mutant cat IgG Fc polypeptide contains tryptophan at the position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO:

118. A heterodimer protein according to any one of claims 63 to 65.

67. a) The second mutant canine IgG Fc polypeptide contains serine at the position corresponding to position 138 of SEQ ID NO: 1, alanine at the position corresponding to position 140, and / or threonine at the position corresponding to position 181; serine at the position corresponding to position 137 of SEQ ID NO: 2, alanine at the position corresponding to position 139, and / or threonine at the position corresponding to position 180; serine at the position corresponding to position 137 of SEQ ID NO: 3, alanine at the position corresponding to position 139, and / or threonine at the position corresponding to position 180; and / or serine at the position corresponding to position 138 of SEQ ID NO: 4, alanine at the position corresponding to position 140, and / or threonine at the position corresponding to position 181; and / or b) The second mutant cat IgG Fc polypeptide contains serine at the position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117 or SEQ ID NO: 118, alanine at the position corresponding to position 156, and / or threonine at the position corresponding to position 197. A heterodimer protein according to any one of claims 63 to 66.

68. a) The first mutant canine IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113 or SEQ ID NO: 115; and / or b) The first mutant cat IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123, A heterodimer protein according to any one of claims 63 to 67.

69. a) The second mutant canine IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114 or SEQ ID NO: 116; and / or b) The second mutant cat IgG Fc polypeptide contains the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO:

123. A heterodimer protein according to any one of claims 63 to 68.

70. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of claims 42 to 69, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises at least one additional amino acid modification compared to the wild-type IgG Fc polypeptide and has increased binding affinity to protein A compared to the wild-type IgG Fc polypeptide.

71. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 1, amino acid substitutions at the position corresponding to position 205 of SEQ ID NO: 1, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 1; b) an amino acid substitution at the position corresponding to position 21 of SEQ ID NO: 3, an amino acid substitution at the position corresponding to position 23 of SEQ ID NO: 3, and / or an amino acid substitution at the position corresponding to position 24 of SEQ ID NO: 3; or c) Amino acid substitutions at the position corresponding to position 21 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 23 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 25 of SEQ ID NO: 4, amino acid substitutions at the position corresponding to position 80 of SEQ ID NO: 4, and / or amino acid substitutions at the position corresponding to position 207 of SEQ ID NO: 4 A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 70, comprising:

72. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) Amino acid substitutions at position 21 of SEQ ID NO: 1, position 23 of SEQ ID NO: 1, position 25 of SEQ ID NO: 1, position 80 of SEQ ID NO: 1, position 205 of SEQ ID NO: 1, and / or position 207 of SEQ ID NO: 1; b) an amino acid substitution at position 21 of SEQ ID NO: 3, an amino acid substitution at position 23 of SEQ ID NO: 3, and / or an amino acid substitution at position 24 of SEQ ID NO: 3; or c) Amino acid substitution at position 21 of SEQ ID NO: 4, amino acid substitution at position 23 of SEQ ID NO: 4, amino acid substitution at position 25 of SEQ ID NO: 4, amino acid substitution at position 80 of SEQ ID NO: 4, and / or amino acid substitution at position 207 of SEQ ID NO: 4 A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 71, comprising:

73. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; or c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO:

4. A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 72, comprising:

74. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) 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; b) Threonine at position 21 of SEQ ID NO: 3, leucine at position 23 of SEQ ID NO: 3, and / or isoleucine at position 24 of SEQ ID NO: 3; or c) Threonine at position 21 of SEQ ID NO: 4, leucine at position 23 of SEQ ID NO: 4, alanine at position 25 of SEQ ID NO: 4, glycine at position 80 of SEQ ID NO: 4, and / or histidine at position 207 of SEQ ID NO: 4 A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 73, comprising:

75. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of claims 42 to 74, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises at least one additional amino acid modification compared to the wild-type IgG Fc polypeptide and has a reduced binding affinity to CD16 compared to the wild-type IgG Fc polypeptide.

76. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) an amino acid substitution at the position corresponding to position 5 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution at the position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution at the position corresponding to position 98 of SEQ ID NO: 2; or b) Amino acid substitutions at the position corresponding to position 5 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 38 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 39 of SEQ ID NO: 3, amino acid substitutions at the position corresponding to position 97 of SEQ ID NO: 3, and / or amino acid substitutions at the position corresponding to position 98 of SEQ ID NO: 3 A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 75, comprising the above.

77. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) Amino acid substitution at position 5 of SEQ ID NO: 2, amino acid substitution at position 38 of SEQ ID NO: 2, amino acid substitution at position 39 of SEQ ID NO: 2, amino acid substitution at position 97 of SEQ ID NO: 2, and / or amino acid substitution at position 98 of SEQ ID NO: 2; or b) Amino acid substitution at position 5 of SEQ ID NO: 3, position 38 of SEQ ID NO: 3, position 39 of SEQ ID NO: 3, position 97 of SEQ ID NO: 3, and / or position 98 of SEQ ID NO: 3 A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 76, comprising:

78. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO:

3. A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 77, comprising:

79. The mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide and / or the second mutant IgG Fc polypeptide, a) Proline at position 5 of SEQ ID NO: 2, glycine at position 38 of SEQ ID NO: 2, arginine at position 39 of SEQ ID NO: 2, isoleucine at position 97 of SEQ ID NO: 2, and / or glycine at position 98 of SEQ ID NO: 2; or b) Proline at position 5 of SEQ ID NO: 3, glycine at position 38 of SEQ ID NO: 3, arginine at position 39 of SEQ ID NO: 3, isoleucine at position 97 of SEQ ID NO: 3, and / or glycine at position 98 of SEQ ID NO: 3 A polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 42 to 78, comprising:

80. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of claims 42 to 79, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises at least one additional amino acid modification to the wild-type canine IgG Fc polypeptide and has a reduced binding affinity to C1q for the wild-type canine IgG Fc polypeptide.

81. A polypeptide, sequential polypeptide, or heterodimeric protein according to any one of claims 42 to 80, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises an amino acid substitution at the position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution at the position corresponding to position 93 of SEQ ID NO:

3.

82. A polypeptide, sequential polypeptide, or heterodimeric protein according to any one of claims 42 to 81, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises an amino acid substitution at position 93 of SEQ ID NO: 2 or at position 93 of SEQ ID NO:

3.

83. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of claims 42 to 82, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide each contain 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:

3.

84. A polypeptide, sequential polypeptide, or heterodimer protein according to any one of claims 42 to 83, wherein the mutant IgG Fc polypeptide, the first mutant IgG Fc polypeptide, and / or the second mutant IgG Fc polypeptide comprises arginine at position 93 of SEQ ID NO: 2, or arginine at position 93 of SEQ ID NO:

3.

85. The polypeptide, serial polypeptide, or heterodimer protein according to any one of claims 1 to 84, wherein the polypeptide is an antibody, an antibody fusion, or a fusion polypeptide.

86. a) The first glucagon-like peptide 1 (GLP1) polypeptide (GLP1A); b) First linker (L1); c) Fc polypeptide (Fc) of companion animal species; d) A second linker (L2) if applicable; and e) In some cases, a second GLP1 polypeptide (GLP1B) A continuous polypeptide containing [a specific component].

87. Formula (I): GLP1A-L1-Fc; or Formula (II): Fc-L1-GLP1A The continuous polypeptide according to claim 65, comprising:

88. Formula (III): GLP1A-L1-Fc-L2-GLP1B The continuous polypeptide according to claim 65, comprising:

89. The continuous polypeptide according to any one of claims 86 to 88, wherein, if GLP1B is present, it comprises the same amino acid sequence as GLP1A.

90. a) Glucagon-like peptide 1 (GLP1) polypeptide; b) First linker (L1); c) Fc polypeptide (Fc); d) Second linker (L2); and e) Glucagon polypeptide (Gluc) A continuous polypeptide containing [a specific component].

91. Formula (IV): GLP1-L1-Fc-L2-Gluc; or Formula (V): Gluc-L1-Fc-L2-GLP1 The continuous polypeptide according to claim 90, comprising:

92. A continuous polypeptide according to any one of claims 86 to 91, comprising GLP1A, GLP1 and / or GLP1B, if present, a wild-type GLP1 polypeptide.

93. A sequential polypeptide according to any one of claims 86 to 92, comprising, if present, a variant GLP1 polypeptide, GLP1A, GLP1, and / or GLP1B.

94. A continuous polypeptide according to any one of claims 86 to 93, wherein GLP1A, GLP1 and / or GLP1B, if present, comprises the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ ID NO:

99.

95. The continuous polypeptide according to any one of claims 86 to 94, wherein the glucagon polypeptide comprises a wild-type glucagon polypeptide and optionally comprises the amino acid sequence of SEQ ID NO:

21.

96. The continuous polypeptide according to any one of claims 86 to 95, wherein the glucagon polypeptide is a mutant glucagon polypeptide.

97. The continuous polypeptide according to any one of claims 86 to 96, wherein the Fc polypeptide is human IgGFc.

98. The continuous polypeptide according to any one of claims 86 to 97, wherein the Fc polypeptide is human IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc.

99. The continuous polypeptide according to any one of claims 86 to 98, wherein the Fc polypeptide is the Fc of a companion animal species.

100. Fc polypeptide, a) Canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc; b) Horse 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 A continuous polypeptide according to any one of claims 86 to 97 or 99, comprising:

101. The continuous polypeptide according to any one of claims 86 to 100, wherein the Fc polypeptide is a wild-type IgG Fc polypeptide.

102. The continuous polypeptide according to any one of claims 86 to 100, wherein the Fc polypeptide is a mutant IgG Fc polypeptide.

103. A continuous polypeptide according to any one of claims 86 to 102, wherein the Fc polypeptide comprises a polypeptide, a continuous polypeptide, or a heterodimer protein according to any one of claims 1 to 84.

104. A continuous polypeptide according to any one of claims 85 to 102, having a longer serum half-life than the wild-type GLP1 polypeptide.

105. The continuous polypeptide according to any one of claims 86 to 104, wherein L1 and L2, if present, are each independently flexible linkers.

106. The continuous polypeptide according to any one of claims 86 to 105, wherein the L1 and L2 amino acid sequences, if present, independently comprise 100%, at least 95%, at least 90%, and at least 85% serine and / or glycine amino acid residues, respectively.

107. A continuous polypeptide according to any one of claims 86 to 106, comprising an elongated portion at the C-terminus.

108. A continuous polypeptide according to any one of claims 86 to 107, comprising 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.

109. A continuous polypeptide according to any one of claims 86 to 108, comprising the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95 at its C-terminus.

110. a) The amino acid sequences of SEQ ID NOs. 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, 96, 97, 103, 104, 105, or 106; or b) Amino acid sequences of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59 A continuous polypeptide according to any one of claims 86 to 109, comprising:

111. A polypeptide containing the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO:

106.

112. A polypeptide, heterodimer protein, or continuous polypeptide according to any one of claims 1 to 111, wherein at least one amino acid modification or substitution comprises an amino acid substitution by an amino acid derivative.

113. An isolated nucleic acid encoding a polypeptide, heterodimer protein, or sequential polypeptide according to any one of claims 1 to 112.

114. A host cell containing the nucleic acid described in claim 113.

115. A method for producing a polypeptide, comprising culturing the host cells described in claim 114 and isolating the polypeptide.

116. A pharmaceutical composition comprising a polypeptide, heterodimer protein, or continuous polypeptide according to any one of claims 1 to 112 and a pharmaceutically acceptable carrier.

117. A method for increasing cAMP production in cells, comprising exposing cells to a polypeptide, heterodimer protein, serial polypeptide, or pharmaceutical composition according to any one of claims 1 to 112 or 116, under conditions that allow binding of the polypeptide, heterodimer protein, or serial polypeptide to GLP1R.

118. The method according to claim 117, wherein cells are exposed ex vivo to a polypeptide, heterodimeric protein, serial polypeptide, or pharmaceutical composition.

119. The method according to claim 117, wherein cells are exposed in vivo to a polypeptide, heterodimeric protein, serial polypeptide, or pharmaceutical composition.

120. The method according to claim 118 or 119, wherein the cells are human cells, canine cells, feline cells, or equine cells.

121. A method for delivering a polypeptide, comprising parenterally administering a polypeptide, heterodimer protein, continuous polypeptide, or pharmaceutical composition according to any one of claims 1 to 112 or 116.

122. A method for delivering a polypeptide, comprising administering the polypeptide, heterodimer protein, continuous polypeptide, or pharmaceutical composition according to any one of claims 1 to 112 or 116 via an intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation route.

123. A method for treating a subject having diabetes or obesity, comprising administering a therapeutically effective amount of a polypeptide, heterodimer protein, continuous polypeptide, or pharmaceutical composition according to any one of claims 1 to 112 or 116 to the subject.

124. The method according to claim 123, comprising administering insulin, a DPP4 inhibitor, an SGLT2 inhibitor, biguanides, sulfonylurea, a meglitinide derivative, an alpha-glucosidase inhibitor, a thiazolidinedione (TZD), an amirinomimetic, a bile acid chelating agent, or a dopamine agonist.

125. The method according to any one of claims 121 to 124, wherein the subject is a human subject.

126. The method according to any one of claims 121 to 124, wherein the subject is a companion animal species.

127. The method according to claim 126, wherein the companion animal species is a dog, a horse, or a cat.

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