fc igg variants for veterinary use

专利摘要:
The present invention relates to various modalities which refer to variant IgG Fc polypeptides of companion animals that have increased protein A binding for ease of purification, decreased C1q binding for reduced complement-mediated immune responses, decreased CD16 binding (for example, to induce reduced antibody-dependent cell cytotoxicity (ADCC), increased stability and / or the ability to form heterodimeric proteins. In addition, several modalities referring to antibodies and fusion proteins comprising said IgG Fc polypeptides Various modalities referring to contiguous polypeptides comprising one or more GLP1 polypeptide (s) variant having improved serum half-life are also provided. GLP1 polypeptide and a glucagon polypeptide as an agonist GLP1 receptor and double glucagon receptor. In various embodiments, said polypeptides can be used to treat, for example, diabetes, obesity, or related indications, in companion animals, such as canines, felines and horses.
公开号:BR112020002871A2
申请号:R112020002871-1
申请日:2018-08-15
公开日:2020-07-28
发明作者:Hangjun Zhan；Lam Nguyen；Yongzhong Li；Fawn Qian；Shyr Jiann Li
申请人:Kindred Biosciences, Inc.；
IPC主号:

专利说明:

[001] [001] This application claims the priority benefit of US provisional patent application No. 62 / 545,858, filed on August 15, 2017, which is incorporated by reference in its entirety for any purpose. FIELD
[002] [002] The present invention relates to IgG Fc polypeptides from companion animals with improved characteristics, including improved protein A binding (eg, for ease of purification), reduced C1q binding (eg, for responses reduced complement-mediated immune responses), reduced CD16 binding (for example, to induce reduced antibody-dependent cell cytotoxicity (ADCC), increased stability and / or the ability to form homeric proteins. The IgG Fc polypeptides variant of the present disclosure may have wide applicability in the treatment of companion animals, for example, variant IgG Fc polypeptides can be used in the design and production of long-lasting GLP1 polypeptides to treat, for example, diabetes, obesity or related indications , in companion animals, such as canines, felines and horses. In addition, variant IgG Fc polypeptides can be used in the design and production of antibodies or pro fusion proteins to treat various disorders in pets. BACKGROUND
[003] [003] IgG Fc plays an important role in Fc-mediated functions through interactions with FcRn, Fc receptor and C1q. In companion animals, several IgG subtypes have differences in these functions, which are often considered when choosing a specific IgG antibody or Fc fusion protein from
[004] [004] However, most IgG Fc subtypes in dogs, cats and horses do not have protein A binding properties, weak or unmeasurable binding affinity to CD16, and weak or unmeasurable 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 appreciable affinity for protein A. However, only Fc of Canine IgG-A and IgG-D have weak or no binding to C1q or CD16. Antibodies and Fc fusion proteins comprising variant IgG Fc polypeptides that have reduced binding to C1q and / or CD16, and / or that are capable of binding protein A are desirable.
[005] [005] Glucagon-like peptide 1 (GLP1) is a potent anti-hyperglycemic hormone, which plays an important role in regulating the blood glucose level. 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 lipids, treat conditions that would benefit from reduced blood glucose levels, suppress gastric or bowel movement, delay emptying gastric, and / or decrease food intake. There is still a need for long-acting GLP1 polypeptides to treat high blood glucose or conditions induced by uncontrollable blood glucose in pets, such as canines, felines and horses. SUMMARY OF THE INVENTION
[006] [006] Modality 1. A polypeptide comprising a variant IgG Fc polypeptide comprising at least one amino acid modification relative to a wild IgG Fc polypeptide of a species of companion animal, wherein the polypeptide - variant IgG Fc peptide has increased binding affinity to protein A relative to the wild-type IgG Fc polypeptide.
[007] [007] Modality 2. A polypeptide comprising a variant IgG Fc polypeptide comprising at least one amino acid modification relative to a wild IgG Fc polypeptide of a species of companion animal, wherein the polypeptide - variant IgG Fc peptide has reduced binding affinity to C1q and / or CD16 relative to the wild type IgG Fc polypeptide.
[008] [008] Mode 3. The polypeptide of mode 1 or mode 2, in which the variant IgG Fc polypeptide binds to C1q and / or CD16 with a dissociation constant (Kd) greater than 5 x 10-6 M , greater than 1 x 10-5 M, greater than 5 x 10-5 M, greater than 1 x 10-4 M, greater than 5 x 10-4 M, or greater than 1 x 10-3 M, as measured by bi-layer interferometry.
[009] [009] Mode 4. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide has increased binding affinity to protein A relative to the wild type IgG Fc polypeptide.
[0010] [0010] Mode 5. The polypeptide according to any of the preceding modalities, in which the variant IgG Fc polypeptide binds to protein A with a dissociation constant (Kd) less than 5 x 10-6 M, less than 1 x 10-6 M, less than 5 x 10-7 M, less than 1 x 10-7 M, less than 5 x 10-8 M, less than 1 x 10-8 M, less than 5 x 10-9 M, less than 1 x 10-9 M, less than 5 x 10-10 M, less than 1 x 10-10 M, less than 5 x 10-11 M, less than 1 x 10-11 M, less than 5 x 10-12 M, or less than 1 x 10-12 M, as measured by bi-layer interferometry.
[0011] [0011] Modality 6. The polypeptide according to any of the preceding modalities, in which the species of companion animal is canine, feline or equine.
[0012] [0012] Mode 7. The polypeptide according to any of the preceding modalities, wherein the wild-type IgG Fc polypeptide is a) an IgG-A Fc, IgG-B Fc, IgG-C Fc or Fc canine IgG-D; b) an IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc or equine IgG7 Fc; or c) an IgG1a Fc, IgG1b Fc, feline IgG2 Fc.
[0013] [0013] Mode 8. A polypeptide comprising a variant IgG Fc polypeptide comprising at least one amino acid modification to a hinge region relative to a wild-type feline or equine IgG Fc polypeptide, wherein the polypeptide - variant IgG Fc deo has increased recombinant production and / or increased hinge disulfide formation relative to the wild type IgG Fc polypeptide, as determined by the SDS-PAGE analysis under conditions of reduction and / or non-reduction.
[0014] [0014] Mode 9. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a substitution on the amino acid at a 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;
[0015] [0015] Mode 10. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, in that the variant IgG Fc polypeptide comprises 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 relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 3 of SEQ ID NO: 129; and / or c) at least one amino acid substitution relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 20 of SEQ ID NO: 129.
[0016] [0016] Mode 11. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a proline in a position corresponding to position 16 or 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 relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a serine in a position corresponding to position 3 or position 3 of SEQ ID NO: 129; and / or c) at least one amino acid substitution relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a proline in a position corresponding to position 20 or position 20 of SEQ ID NO: 129.
[0017] [0017] Mode 12. The polypeptide according to any of the preceding embodiments, wherein the variant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region from an isotype IgG Fc polypeptide. different.
[0018] [0018] Mode 13. The polypeptide according to any of the preceding embodiments, wherein the variant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region from a feline IgG-1a Fc polypeptide wild-type, a wild-type feline IgG-1b Fc polypeptide, or a wild-type equine IgG1 Fc polypeptide.
[0019] [0019] Modality 14. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises SEQ ID NO: 19, SEQ ID NO: 125 or SEQ ID NO:
[0020] [0020] Mode 15. A polypeptide comprising an 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: 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.
[0021] [0021] Mode 16. A polypeptide comprising a variant IgG2 Fc polypeptide comprising at least one amino acid substitution relative to a wild-type feline IgG2 Fc polypeptide, wherein at least one amino acid substitution is a cysteine, and wherein the variant IgG2 Fc polypeptide is capable of forming at least one additional, inter-chain disulfide bond relative to the wild-type feline IgG2 polypeptide.
[0022] [0022] Mode 17. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, wherein at least at least one amino acid substitution is a cysteine, and wherein the variant IgG Fc polypeptide is capable of forming at least one additional and relative interchain disulfide bond to the wild-type feline IgG Fc polypeptide.
[0023] [0023] Mode 18. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises a cysteine in a position corresponding to position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15 or position 16 of SEQ ID NO: 16.
[0024] [0024] Mode 19. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises a cysteine in a position corresponding to position 14 of SEQ ID NO: 16.
[0025] [0025] Mode 20. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises a cysteine at position 14 of SEQ ID NO: 16.
[0026] [0026] Mode 21. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide is at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO : 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO : 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SE Q ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, 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,
[0027] [0027] Mode 22. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises SEQ ID NO: 17.
[0028] [0028] Mode 23. A polypeptide comprising an amino acid sequence of SEQ ID NO: 17.
[0029] [0029] Mode 24. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 1, a amino acid substitution in one position corresponding to position 23 of SEQ ID NO: 1, an amino acid substitution in one position corresponding to position 25 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 80 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 205 of SEQ ID NO: 1, and / or an amino acid substitution in a position corresponding to position 207 of SEQ ID NO: 1; b) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 3, and / or an amino acid substitution in a position corresponding to position 24 of SEQ ID NO: 3; c) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 23 of SEQ ID
[0030] [0030] Mode 25. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acid substitution at position 23 of SEQ ID NO: 1, an amino acid substitution at position 25 of SEQ ID NO: 1, an amino acid substitution at position 80 of SEQ ID NO: 1, an amino acid substitution at position 205 of SEQ ID NO: 1, and / or an amino acid substitution at position 207 of SEQ ID NO: 1;
[0031] [0031] Modality 26. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) a threonine in a position corresponding to position 21 of SEQ ID NO: 1, a leucine in a position corresponding to position 23 of SEQ ID NO: 1, an alanine in a position corresponding to position 25 of SEQ ID NO: 1, a glycine in a position corresponding to position 80 of SEQ ID NO: 1, an alanine in a position corresponding to position 205 of SEQ ID
[0032] [0032] Mode 27. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) a threonine at position 21 of SEQ ID NO: 1, a leukemia
[0033] [0033] Mode 28. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises an amino acid sequence of: a) 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; or b) SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQ ID NO: 76.
[0034] [0034] Mode 29. A polypeptide comprising an amino acid sequence 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,
[0035] [0035] Mode 30. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an amino acid substitution in a position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 3; b) an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 63, an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 65, an amino acid substitution in a correct position - corresponding to position 87 of SEQ ID NO: 66, or an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 69; or c) an amino acid substitution in a position corresponding to position 198 of SEQ ID NO: 80, or an amino acid substitution in a position corresponding to position 198 of SEQ ID NO: 81.
[0036] [0036] Mode 31. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an amino acid substitution at position 93 of SEQ ID NO: 2, or an amino acid substitution at position 93 of SEQ ID NO: 3; b) an amino acid substitution at position 87 of SEQ ID NO: 63, an amino acid substitution at position 87 of SEQ ID NO: 65, an amino acid substitution at position 87 of SEQ ID NO: 66, or an amino acid substitution at SEQ ID 87
[0037] [0037] Mode 32. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an arginine in a position corresponding to position 93 of SEQ ID NO: 2, or an arginine in a position corresponding to position 93 of SEQ ID NO: 3; b) a serine in a position corresponding to position 87 of SEQ ID NO: 63, a serine substitution in a position corresponding to position 87 of SEQ ID NO: 65, a serine in a position corresponding to position 87 of SEQ ID NO: 65 : 66, or a serine in a position corresponding to position 87 of SEQ ID NO: 69; or c) an alanine in a position corresponding to position 198 of SEQ ID NO: 80, or an alanine in a position corresponding to position198 of SEQ ID NO: 81.
[0038] [0038] Mode 33. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an arginine at position 93 of SEQ ID NO: 2, or an arginine at position 93 of SEQ ID NO: 3; b) a serine at position 87 of SEQ ID NO: 63, a serine at position 87 of SEQ ID NO: 65, a serine at position 87 of SEQ ID NO: 66, or a serine at position 87 of SEQ ID NO: 69 ; or c) an alanine at position 198 of SEQ ID NO: 80, or alanine at position 198 of SEQ ID NO: 81.
[0039] [0039] Modality 34. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises the amino acid sequence of: a) SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO : 84; or b) SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 77; or c) SEQ ID NO: 82 or SEQ ID NO: 83.
[0040] [0040] Mode 35. A polypeptide comprising an amino 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.
[0041] [0041] Mode 36. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an amino acid substitution in a position corresponding to position 5 of SEQ ID NO: 2, a amino acid substitution in one position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution in one position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution in a position corresponding to position 98 of SEQ ID NO: 2; or b) an amino acid substitution in one position corresponding to position 5 of SEQ ID NO: 3, an amino acid substitution in one position corresponding to position 38 of SEQ ID NO: 3, an amino acid substitution in one position corresponding to position 39 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 97 of SEQ ID NO: 3, and / or an amino acid substitution in a position corresponding to position 98 of SEQ ID NO: 3.
[0042] [0042] Mode 37. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) an amino acid substitution at position 5 of SEQ ID NO: 2, an amino acid substitution at position 38 of SEQ ID NO: 2, an amino acid substitution at position 39 of SEQ ID NO: 2, an amino acid substitution at position 97 of SEQ ID NO: 2, and / or an amino acid substitution at position 98 of SEQ ID NO: 2; or b) an amino acid substitution at position 5 of SEQ ID NO: 3, an amino acid substitution at position 38 of SEQ ID NO: 3, an amino acid substitution at position 39 of SEQ ID NO: 3, an amino acid substitution at position 97 of SEQ ID NO: 3, and / or an amino acid substitution in position 98 of SEQ ID NO: 3.
[0043] [0043] Mode 38. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) a proline in a position corresponding to position 5 of SEQ ID NO: 2, a glycine in a position corresponding to position 38 of SEQ ID NO: 2, an arginine in a position corresponding to position 39 of SEQ ID NO: 2, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 2, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 2; or b) a proline in a position corresponding to position 5 of SEQ ID NO: 3, a glycine in a position corresponding to position 38 of SEQ ID NO: 3, an arginine in a position corresponding to position 39 of SEQ ID NO: : 3, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 3, and / or a glycine in a position corresponding to position 98 of SEQ ID NO:
[0044] [0044] Mode 39. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) a proline at position 5 of SEQ ID NO: 2, a glycine at position 38 of SEQ ID NO : 2, an arginine at position 39 of SEQ ID NO: 2, an isoleucine at position 97 of SEQ ID NO: 2, and / or a glycine at position 98 of SEQ ID NO: 2; or b) a proline at position 5 of SEQ ID NO: 3, a glycine at position 38 of SEQ ID NO: 3, an arginine at position 39 of SEQ ID NO: 3, an isoleucine at position 97 of SEQ ID NO: 3 , and / or a glycine at position 98 of SEQ ID NO: 3.
[0045] [0045] Mode 40. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises an amino acid sequence of: a) 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, or SEQ ID NO: 147; or b) 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.
[0046] [0046] Mode 41. A polypeptide comprising an amino 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.
[0047] [0047] Mode 42. A polypeptide comprising a variant IgG Fc polypeptide comprising: a) a tyrosine or a tryptophan in a position corresponding to position 138 of SEQ ID NO: 1, a tyrosine or a triphosphate
[0048] [0048] Mode 43. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) a tyrosine or a tryptophan at position 138 of SEQ ID NO: 1, a tyrosine or a tryptophan in position 137 of SEQ ID NO: 2, a tyrosine or a tryptophan in position 137 of SEQ ID NO: 3, or a tyrosine or a tryptophan in position 138 of SEQ ID NO: 4; or b) a tyrosine or a tryptophan at position 154 of SEQ ID NO: 16, a tyrosine or a tryptophan at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a tyrosine or a tryptophan in a position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO: 118.
[0049] [0049] Mode 44. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises an 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.
[0050] [0050] Mode 45. A polypeptide comprising an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
[0051] [0051] Mode 46. A contiguous polypeptide comprising the polypeptide according to any of the foregoing modalities and a glucagon-like peptide-1 polypeptide (GLP1).
[0052] [0052] Mode 47. A contiguous polypeptide comprising the polypeptide according to any of the foregoing modalities and a glucagon polypeptide.
[0053] [0053] Mode 48. A polypeptide comprising a variant IgG Fc polypeptide comprising: a) a serine in a position corresponding to position 138 of SEQ ID NO: 1, a serine in a position corresponding to position 137 of SEQ ID NO: 2, a serine in a position corresponding to position 137 of SEQ ID NO: 3, a serine in a position corresponding to position 138 of SEQ ID NO: 4, a serine in a position corresponding to position 154 of SEQ ID NO: 16, a serine in a position corresponding to position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a serine in a position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; b) an alanine in a position corresponding to position 140 of SEQ ID NO: 1, an alanine in a position corresponding to position 139 of SEQ ID NO: 2, an alanine in a position corresponding to position 139 of SEQ ID NO: 3, an alanine in a position corresponding to position 140 of SEQ ID NO: 4, an alanine in a position corresponding to position 156 of SEQ ID NO: 16, an alanine in a position corresponding to position 156 of SEQ ID NO: 80 or SEQ ID NO: 117, or an alanine in a position corresponding to position 156 of SEQ ID NO: 81 or SEQ ID NO: 118; and / or c) a threonine in a position corresponding to position 181 of SEQ ID NO: 1, a threonine in a position corresponding to position 180 of SEQ ID NO: 2, a threonine in a position corresponding to position 180 of SEQ ID NO: 3, a threonine in a position corresponding to position 181 of SEQ ID NO: 4, a threonine in a position corresponding to position 197 of SEQ ID NO: 16, a threonine in a position corresponding to position 197 of SEQ ID NO: 80 or SEQ ID NO: 117, or a threonine in a position corresponding to position 197 of SEQ ID NO: 81 or SEQ ID NO: 118.
[0054] [0054] Mode 49. The polypeptide according to any of the preceding modalities, wherein the variant IgG Fc polypeptide comprises: a) a serine at position 138 of SEQ ID NO: 1, a serine at position 137 of SEQ ID NO : 2, a serine at position 137 of SEQ ID NO: 3, a serine at position 138 of SEQ ID NO: 4, a serine at position 154 of SEQ ID NO: 16, a serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; b) an alanine at position 140 of SEQ ID NO: 1, an alanine at position 139 of SEQ ID NO: 2, an alanine at position 139 of SEQ ID NO: 3, an alanine at position 140 of SEQ ID NO: 4, an alanine at position 156 of SEQ ID NO: 16, an alanine at position 156 of SEQ ID NO: 80 or SEQ ID NO: 117, or an alanine at position 156 of SEQ ID NO: 81 or SEQ ID NO: 118; and / or; c) a threonine at position 181 of SEQ ID NO: 1, a threonine at position 181 of SEQ ID NO: 2, a threonine at position 181 of SEQ ID NO: 3, a threonine at position 181 of SEQ ID NO: 4, a threonine at position 197 of SEQ ID NO: 16, a threonine at position 197 of SEQ ID NO: 80 or SEQ ID NO: 117, or a threonine at position 197 of SEQ ID NO: 81 or SEQ ID NO: 118.
[0055] [0055] Mode 50. The polypeptide according to any of the foregoing modalities, wherein the variant IgG Fc polypeptide comprises an 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.
[0056] [0056] Mode 51. A polypeptide comprising an 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.
[0057] [0057] Mode 52. The polypeptide according to any of the preceding modalities, in which the polypeptide is glycosylated.
[0058] [0058] Mode 53. The polypeptide of any of the types 1 to 51, in which the polypeptide is aglycosylated.
[0059] [0059] Mode 54. A contiguous polypeptide comprising the polypeptide of any one of modalities 48 to 53 and a glucagon-like peptide-1 polypeptide (GLP1).
[0060] [0060] Mode 55. A contiguous polypeptide comprising the polypeptide of any one of modalities 48 to 53 and a glucagon polypeptide.
[0061] [0061] Mode 56. A heterodimeric protein comprising the contiguous polypeptide of modality 46 and the contiguous polypeptide of modality 54.
[0062] [0062] Mode 57. A heterodimeric protein comprising the contiguous polypeptide of modality 47 and the contiguous polypeptide of modality 55.
[0063] [0063] Mode 58. The contiguous polypeptide or heterogeneous protein
[0064] [0064] Modality 59. The contiguous polypeptide or heterodimeric protein of any of the modalities 46, 47, or 54 to 58, wherein the GLP1 polypeptide is a variant GLP1 polypeptide.
[0065] [0065] Mode 60. The contiguous polypeptide or heterodimeric protein of any of the modalities 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.
[0066] [0066] Mode 61. The contiguous polypeptide or heterodimeric protein of any of the modalities 46, 47, or 54 to 60, wherein the glucagon polypeptide is a wild-type glucagon polypeptide, optionally comprising the amino acid sequence of SEQ ID NO: 21.
[0067] [0067] Mode 62. The contiguous polypeptide or heterodimeric protein of any of the modalities 46, 47, or 54 to 61, wherein the glucagon polypeptide is a variant glucagon polypeptide.
[0068] [0068] Mode 63. A heterodimeric protein comprising: i) a first variant canine IgG Fc polypeptide comprising at least one amino acid modification relative to a first canine IgG Fc polypeptide of the wild type and a second canine IgG Fc polypeptide variant comprising at least one modification in the amino acid relative to a second canine wild-type IgG Fc polypeptide; or ii) a first variant feline IgG Fc polypeptide comprising at least one modification in the amino acid relative to a first feline IgG Fc polypeptide of the wild type and a second variant feline IgG Fc polypeptide comprising at least one modification in the amino acid relative to a second wild-type feline IgG Fc polypeptide, in which: a) the first variant canine IgG Fc polypeptide comprises a substitution at the amino acid in a 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 variant canine IgG Fc polypeptide comprises a substitution of the amino acid in a position corresponding to position 138, position 140, and / or position 181 of SEQ ID NO: 1, position 137, position 139, and / or position 180 of SEQ ID NO: 2, position 137, position 139, and / or position 180 of SEQ ID NO: 3, or position 138, position 140, and / or position 181 of SEQ ID NO: 4; c) the first variant feline IgG Fc polypeptide comprises a substitution of the amino acid in a 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 variant feline IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 154, position 156, and / or position 197 of SEQ ID NO: 6, of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.
[0069] [0069] Mode 64. The heterodimeric protein of mode 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 Fc polypeptide of Feline wild-type IgG and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype.
[0070] [0070] Mode 65. The heterodimeric protein of mode 63, wherein the first wild-type canine IgG Fc polypeptide and the second wild-type canine IgG Fc polypeptide are from a different IgG subtype and / or the first Fc polypeptide of wild-type female IgG and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype.
[0071] [0071] Mode 66. The heterodimeric protein of any of the modalities 63 to 65, in which: a) the first variant canine IgG Fc polypeptide comprises a tyrosine or tryptophan in a 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 variant feline IgG Fc polypeptide comprises a tryptophan in a position corresponding to position 154 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO : 117, or from SEQ ID NO: 118.
[0072] [0072] Mode 67. The heterodimeric protein of any of the modalities 63 to 66, in which: a) the second variant canine IgG Fc polypeptide comprises a serine in a position corresponding to position 138, an alanine in a position corresponding to position 140, and / or a threonine in a position corresponding to position 181 of SEQ ID NO: 1, a serine in a position corresponding to position 137, an alanine in a position corresponding to position 139, and / or a threonine in a position corresponding to position 180 of SEQ ID NO: 2, a serine in a position corresponding to position 137, an alanine in a position corresponding to position 139, and / or a threonine in a position corresponding to position 180 of SEQ ID NO: 3, and / or a serine in a position corresponding to position 138, an alanine in a position corresponding to position
[0073] [0073] Mode 68. The heterodimeric protein of any of the modalities 63 to 67, in which: a) the first variant canine IgG Fc polypeptide comprises an 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 variant feline IgG Fc polypeptide comprises an amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123.
[0074] [0074] Mode 69. The heterodimeric protein of any of the modalities 63 to 68, in which: a) the second variant canine IgG Fc polypeptide comprises an 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 variant feline IgG Fc polypeptide comprises an amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 123.
[0075] [0075] Mode 70. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 69, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of IgG variant com-
[0076] [0076] Mode 71. The polypeptide, contiguous polypeptide or heterodimeric protein of any of the modalities 42 to 70, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 25 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 80 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 205 of SEQ ID NO: 1, and / or an amino acid substitution in a position corresponding to position 207 of SEQ ID NO: 1; b) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 3, and / or an amino acid substitution in a position corresponding to position 24 of SEQ ID NO: 3; or c) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 25 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 80 of SEQ ID
[0077] [0077] Mode 72. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 71, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acid substitution at position 23 of SEQ ID NO: 1, an amino acid substitution at position 25 of SEQ ID NO: 1, an amino acid substitution at position 80 of SEQ ID NO: 1, an amino acid substitution at position 205 of SEQ ID NO: 1, and / or an amino acid substitution at position 207 of SEQ ID NO: 1; b) an amino acid substitution at position 21 of SEQ ID NO: 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) an amino acid substitution at position 21 of SEQ ID NO: 4, an amino acid substitution at position 23 of SEQ ID NO: 4, an amino acid substitution at position 25 of SEQ ID NO: 4, an amino acid substitution at position 80 of SEQ ID NO: 4, and / or an amino acid substitution in position 207 of SEQ ID NO:
[0078] [0078] Mode 73. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 72, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) a threonine in a position corresponding to position 21 of SEQ ID NO: 1, a leucine in a position corresponding to position 23 of SEQ ID NO: 1, an alanine in a position corresponding to the position 25 of SEQ ID NO: 1, a glycine in a position corresponding to position 80 of SEQ ID NO: 1, an alanine in a position corresponding to position 205 of SEQ ID NO: 1, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 1; b) a threonine in a position corresponding to position 21 of SEQ ID NO: 3, a leucine in a position corresponding to position 23 of SEQ ID NO: 3, and / or an isoleucine in a position corresponding to position 24 of SEQ ID NO: 3; or c) a threonine in a position corresponding to position 21 of SEQ ID NO: 4, a leucine in a position corresponding to position 23 of SEQ ID NO: 4, an alanine in a position corresponding to position 25 of SEQ ID NO: 4 : 4, a glycine in a position corresponding to position 80 of SEQ ID NO: 4, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 4.
[0079] [0079] Mode 74. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 73, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) a threonine at position 21 of SEQ ID NO: 1, a leukin at position 23 of SEQ ID NO: 1, an alanine at position 25 of SEQ ID NO: 1, a glycine at position 80 of SEQ ID NO: 1, an aline at position 205 of SEQ ID NO: 1, and / or a histidine at position 207 of SEQ ID NO: 1; b) a threonine at position 21 of SEQ ID NO: 3, a leukin at position 23 of SEQ ID NO: 3, and / or an isoleucine at position
[0080] [0080] Mode 75. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 74, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises at least one additional amino acid modification relative to a wild type IgG Fc polypeptide and has reduced binding affinity to CD16 relative to the wild type IgG Fc polypeptide.
[0081] Modality 76. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 75, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) an amino acid substitution in a position corresponding to position 5 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position 38 of SEQ ID NO: 2, a substitution amino acid in a position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution in a correct position corresponding to position 98 of SEQ ID NO: 2; or b) an amino acid substitution in one position corresponding to position 5 of SEQ ID NO: 3, an amino acid substitution in one position corresponding to position 38 of SEQ ID NO: 3, an amino acid substitution in one position run-
[0082] Modality 77. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 76, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) an amino acid substitution at position 5 of SEQ ID NO: 2, an amino acid substitution at position 38 of SEQ ID NO: 2, an amino acid substitution at position 39 of SEQ ID NO: 2, an amino acid substitution at position 97 of SEQ ID NO: 2, and / or an amino acid substitution at position 98 of SEQ ID NO: 2; or b) an amino acid substitution at position 5 of SEQ ID NO: 3, an amino acid substitution at position 38 of SEQ ID NO: 3, an amino acid substitution at position 39 of SEQ ID NO: 3, an amino acid substitution at position 97 of SEQ ID NO: 3, and / or an amino acid substitution in position 98 of SEQ ID NO: 3.
[0083] [0083] Mode 78. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 77, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) a proline in a position corresponding to position 5 of SEQ ID NO: 2, a glycine in a position corresponding to position 38 of SEQ ID NO: 2, an arginine in a position corresponding to the position 39 of SEQ ID NO: 2, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 2, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 2; or b) a proline in a position corresponding to position 5 of SEQ ID NO: 3, a glycine in a position corresponding to position 38 of SEQ ID NO: 3, an arginine in a position corresponding to position 39 of SEQ ID NO: : 3, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 3, and / or a glycine in a position corresponding to position 98 of SEQ ID NO:
[0084] [0084] Mode 79. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 78, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) a proline at position 5 of SEQ ID NO: 2, a glycine at position 38 of SEQ ID NO: 2, an arginine at position 39 of SEQ ID NO: 2, an isoleucine at position 97 of SEQ ID NO: 2, and / or a glycine at position 98 of SEQ ID NO: 2; or b) a proline at position 5 of SEQ ID NO: 3, a glycine at position 38 of SEQ ID NO: 3, an arginine at position 39 of SEQ ID NO: 3, an isoleucine at position 97 of SEQ ID NO: 3 , and / or a glycine at position 98 of SEQ ID NO: 3.
[0085] [0085] Mode 80. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 79, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises at least one additional amino acid modification relative to a canine wild type IgG Fc polypeptide and has reduced affinity for binding to C1q relative to the canine wild type IgG Fc polypeptide.
[0086] [0086] Mode 81. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 80, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises a substitution of the amino acid in a position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution in a position corresponding to position 93 of SEQ ID NO: 3.
[0087] [0087] Mode 82. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 81, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises an amino acid substitution at position 93 of SEQ ID NO: 2, or an amino acid substitution at position 93 of SEQ ID NO: 3.
[0088] [0088] Mode 83. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any one of modalities 42 to 82, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises an arginine in a position corresponding to position 93 of SEQ ID NO: 2, or an arginine in a position corresponding to position 93 of SEQ ID NO: 3.
[0089] [0089] Mode 84. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any of the modalities 42 to 83, wherein the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises an arginine at position 93 of SEQ ID NO: 2, or an arginine at position 93 of SEQ ID NO: 3.
[0090] [0090] Mode 85. The polypeptide, the contiguous polypeptide or the heterodimeric protein of any one of embodiments 1 to 84, wherein the polypeptide is an antibody, an antibody fusion or a fusion polypeptide.
[0091] [0091] Mode 86. A contiguous polypeptide comprising: a) a first peptide-1 polypeptide of the glucagon type (GLP1) (GLP1A); b) a first linker (L1); c) an Fc polypeptide (Fc) from a species of pet animal; d) optionally, a second linker (L2); and e) optionally, a second GLP1 polypeptide (GLP1B).
[0092] [0092] Mode 87. The contiguous polypeptide of modality 65 comprising: formula (I): GLP1A — L1 — Fc; or formula (II): Fc — L1 — GLP1A.
[0093] [0093] Mode 88. The contiguous polypeptide of mode 65 comprising: formula (III): GLP1A — L1 — Fc — L2 — GLP1B.
[0094] [0094] Mode 89. The contiguous polypeptide of any one of modalities 86 to 88, where GLP1B, if present, comprises the same amino acid sequence as GLP1A.
[0095] [0095] Mode 90. A contiguous polypeptide comprising: a) a glucagon-like peptide-1 polypeptide (GLP1); b) a first linker (L1); c) an Fc (Fc) polypeptide; d) a second linker (L2); and e) a glucagon polypeptide (Gluc).
[0096] [0096] Mode 91. The contiguous polypeptide of mode 90 comprising: Formula (IV): GLP1 — L1 — Fc — L2 — Gluc; or
[0097] [0097] Mode 92. The contiguous polypeptide of any of modes 86 to 91, wherein GLP1A, GLP1, and / or GLP1B, if present, comprise a wild-type GLP1 polypeptide.
[0098] [0098] Mode 93. The contiguous polypeptide of any one of modalities 86 to 92, wherein GLP1A, GLP1, and / or GLP1B, if present, comprise a variant GLP1 polypeptide.
[0099] [0099] Mode 94. The contiguous polypeptide of any one of modalities 86 to 93, wherein GLP1A, GLP1, and / or GLP1B, if present, comprise an 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.
[00100] [00100] Mode 95. The contiguous polypeptide of any one of modalities 86 to 94, wherein the glucagon polypeptide comprises a wild type glucagon polypeptide, optionally comprising the amino acid sequence of SEQ ID NO: 21.
[00101] [00101] Mode 96. The contiguous polypeptide of any of the modalities 86 to 95, wherein the glucagon polypeptide is a variant glucagon polypeptide.
[00102] [00102] Mode 97. The contiguous polypeptide of any of the modalities 86 to 96, wherein the Fc polypeptide is a human IgG Fc.
[00103] [00103] Mode 98. The contiguous polypeptide of any of the modalities 86 to 97, wherein the Fc polypeptide is an IgG1 Fc, IgG2 Fc, IgG3 Fc or human IgG4 Fc.
[00104] [00104] Mode 99. The contiguous polypeptide of any of the modalities 86 to 98, wherein the Fc polypeptide is a Fc of a species of pet animal.
[00105] [00105] Mode 100. The contiguous polypeptide of any of the 86 to 97 or 99 modalities, wherein the Fc polypeptide comprises
[00106] [00106] Mode 101. The contiguous polypeptide of any of the modalities 86 to 100, wherein the Fc polypeptide is a wild-type IgG Fc polypeptide.
[00107] [00107] Mode 102. The contiguous polypeptide of any of the modalities 86 to 100, wherein the Fc polypeptide is a variant IgG Fc polypeptide.
[00108] [00108] Mode 103. The contiguous polypeptide of any one of modalities 86 to 102, wherein the Fc polypeptide comprises the polypeptide, the contiguous polypeptide or the heterodimeric protein of any one of modalities 1 to 84.
[00109] [00109] Mode 104. The contiguous polypeptide of any of the modalities 85 to 102, wherein the contiguous polypeptide has a longer serum half-life than a wild-type GLP1 polypeptide.
[00110] [00110] Mode 105. The contiguous polypeptide of any of the modes 86 to 104, in which L1 to L2, if present, each independently is a flexible ligand.
[00111] [00111] Mode 106. The contiguous polypeptide of any of the modalities 86 to 105, wherein the amino acid sequence of L1 and L2, if present, each independently comprises 100%, at least 95%, at least 90%, at least minus 85% of serine and / or glycine amino acid residues.
[00112] [00112] Modality 107. The contiguous polypeptide of any of the modalities 86 to 106, wherein the contiguous polypeptide comprises
[00113] [00113] Mode 108. The contiguous polypeptide of any of the modalities 86 to 107, in which the contiguous polypeptide comprises a glycine residue, two glycine residues, three glycine residues, four glycine residues, five residues glycine residues, six glycine residues, seven glycine residues, eight glycine residues or more than eight glycine residues at its C-terminal.
[00114] [00114] Mode 109. The contiguous polypeptide of any of the modalities 86 to 108, wherein the contiguous polypeptide comprises an 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 your C-terminal.
[00115] [00115] Mode 110. The contiguous polypeptide of any of the modalities 86 to 109, wherein the contiguous polypeptide comprises: 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) the amino acid sequence 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.
[00116] [00116] Mode 111. A polypeptide comprising an 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:
[00117] [00117] Mode 112. The polypeptide, the heterodimeric protein, or the contiguous polypeptide of any of the foregoing modalities, in which at least one modification or substitution of amino acid comprises an amino acid substitution with an amino acid derivative .
[00118] [00118] Mode 113. An isolated nucleic acid encoding the polypeptide, the heterodimeric protein or the contiguous polypeptide of any of the preceding modalities.
[00119] [00119] Mode 114. A host cell comprising nucleic acid of mode 113.
[00120] [00120] Mode 115. A method of producing a polypeptide comprising cultivating the host cell of modality 114 and isolating the polypeptide.
[00121] [00121] Mode 116. A pharmaceutical composition comprising the polypeptide, the heterodimeric protein, or the contiguous polypeptide of any one of modalities 1 to 112, and a pharmaceutically acceptable carrier.
[00122] [00122] Mode 117. A method of increasing the production of cAMP in a cell, the method comprising exposing the cell to the polypeptide, the heterodimeric protein, the contiguous polypeptide or the pharmaceutical composition of any of the modalities 1 to 112 or 116 under permissive conditions for binding the polypeptide, heterodimeric protein or contiguous polypeptide to GLP1R.
[00123] [00123] Mode 118. The method of mode 117, wherein the cell is exposed to the polypeptide, heterodimeric protein, contiguous polypeptide or the pharmaceutical composition ex vivo.
[00124] [00124] Mode 119. The method of mode 117, wherein the cell is exposed to the polypeptide, heterodimeric protein, contiguous polypeptide or the pharmaceutical composition in vivo.
[00125] [00125] Mode 120. The method of any of the modalities 118 to 119, in which the cell is a human cell, a kain cell, a feline cell or an equine cell.
[00126] [00126] Mode 121. A method of releasing a polypeptide to an individual comprising administering the polypeptide, the heterodimeric protein, the contiguous polypeptide, or the pharmaceutical composition of any one of modalities 1 to 112 or 116 of parenteral form.
[00127] [00127] Mode 122. A method of releasing a polypeptide to an individual comprising administering the polypeptide, the heterodimeric protein, the contiguous polypeptide, or the pharmaceutical composition of any of modalities 1 to 112 or 116 via an intramuscular route, an intraperitoneal route, an intracerebrospinal route, a subcutaneous route, an intra-arterial route, an intrasynovial route, an intrathecal route or through inhalation.
[00128] [00128] Mode 123. A method of treating an individual who has diabetes or obesity, the method comprising administering to the individual a therapeutically effective amount of the polypeptide, the heterodimeric protein, the contiguous polypeptide, or the pharmaceutical composition of any of the modalities 1 to 112 or 116.
[00129] [00129] Method 124. The method of method 123, comprising administering insulin, a DPP4 inhibitor, an inhibitor of
[00130] [00130] Modality 125. The method of any of modalities 121 to 124, in which the individual is a human individual.
[00131] [00131] Mode 126. The method of any of the modalities 121 to 124, in which the individual is a kind of companion animal.
[00132] [00132] Mode 127. The method of mode 126, in which the species of pet animal is canine, equine or feline. BRIEF DESCRIPTION OF THE DRAWINGS
[00133] [00133] Figure 1 shows an alignment of canine IgG-A, B, C and D Fc sequences. The tables indicate the likely regions in contact with protein A.
[00134] [00134] Figure 2A shows an 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; " variant of GLP1 B "in this figure) having a wild-type feline IgG2 hinge with a disulfide bond in the absence and presence of reducing agent (DTT).
[00135] [00135] Figure 2B shows an SDS-PAGE analysis of GLP1- G8 / GLP-2G_III_VARfeIgG2 (SEQ ID NO: 25; "GLP1 MA variant" in this figure) of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26; "variant of GLP1 MB "in this figure) having a variant feline IgG2 hinge with two disulfide bonds in the absence and presence of reducing agent (DTT).
[00136] [00136] Figure 3 shows a cAMP CHO-K1 GLP1R bioassay to assess GLP1-G8 / GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) and GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) activity compared to controls ( GLP1 (7-37) and Extendin-4).
[00137] [00137] Figure 4 shows a cell-based bioassay to assess GLP1-G8_I_VARfeIgG2 activity (SEQ ID NO: 26) ("GLP1-B" in this figure) after 1 year of storage with CHOK1-GLP1R and cAMP- cells glo (n = 2).
[00138] [00138] Figure 5 shows a Western Blot analysis of GLP1- G8_I_VARfeIgG2 (SEQ ID NO: 26) ("GLP1B" in this figure) after incubation in serum for 24 hours at 37 ° C (line 1), in PBS for 24 hours at 37 ° C (line 2), in PBS for 24 hours at 4 ° C (line 9). An anti-mouse GLP1 antibody was used.
[00139] [00139] Figure 6 is a graph of the concentration of GLP1- G8_I_VARfeIgG2 (SEQ ID NO: 26) in serum with time after subcutaneous administration to 5 cats, as measured by quantitative ELISA.
[00140] [00140] Figure 7 shows a graph of the concentration of GLP1- G8_I_VARfeIgG2 (SEQ ID NO: 26) in the serum with time after subcutaneous administration to 5 cats, as measured by the cell-based activity assay. The mean AUC from 0 to 168 hours was about 840 μg (h) / mL and the mean t1 / 2 was 36 hours. DESCRIPTION OF SEQUENCES
[00141] [00141] Table 1 provides a listing of exemplary sequences referenced in this document. Table 1: Description of the sequences
[00142] [00142] DESCRIPTION OF THE MODALITIES
[00143] [00143] IgG variant Fc polypeptides from companion animals, such as canines, horses and felines, are described. In some embodiments, variant IgG Fc polypeptides have increased protein A binding, reduced C1q binding, reduced CD16 binding, increased stability, increased recombinant production, increased hinge disulfide formation and / or heterodimeric polypeptide formation . In some embodiments, antibodies, antibody fragments or fusion proteins comprise a variant IgG Fc polypeptide. Methods of producing or purifying variant IgG Fc polypeptides and methods of administering variant IgG Fc polypeptides to companion animals are also provided in the assay.
[00144] [00144] Various modalities are also provided in relation to contiguous polypeptides and heterodimeric polypeptides comprising one or more variant GLP1 polypeptide (s) having improved serum half-life. In some embodiments, contiguous polypeptides or heterodimeric polypeptides comprise a GLP1 polypeptide and a glucagon polypeptide as a GLP1 receptor agonist and double glucagon receptor. In some modalities, said polypeptides can be used to treat, for example, diabetes, obesity, or related indications, in companion animals, such as canines, felines and horses.
[00145] [00145] For the convenience of the reader, the definitions that follow from the terms used in this document are provided.
[00146] [00146] As used in this document, numerical terms such as KD are calculated based on scientific measurements, and thus are subject to appropriate measurement error. In some cases, a numeric term may include numeric values that are rounded to the nearest significant number.
[00147] [00147] As used herein, "one" or "one" 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 a dependent multiple claim, the use of "or" when referring back to other claims refers to those claims only in the alternative.
[00148] [00148] Exemplifying variant IgG Fc polypeptides
[00149] [00149] New variant IgG Fc polypeptides are provided, for example, variant IgG Fc polypeptides for binding to increased protein A, for binding to reduced C1q, for binding to reduced CD16, for increased stability, for increased recombinant production, for formation of enlarged hinge disulfide and / or for formation of the heterodimeric protein assay.
[00150] [00150] "Amino acid sequence" means a sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Said polymers of amino acid residues may contain natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both proteins and full-length fragments are covered by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation and the like. In addition, for the purposes of the present disclosure, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, as the protein maintain the desired activity. These changes may be deliberate, through site-directed mutagenesis, or they may be accidental, such as through host mutations that produce proteins or errors due to PCR amplification.
[00151] [00151] A "crystallizable polypeptide fragment" or "Fc polypeptide" is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the C-terminal portions of the immunoglobulin heavy chains. As used herein, an Fc polypeptide includes fragments of the Fc domain having one or more biological activities of an entire Fc polypeptide. In some embodiments, a biological activity of an Fc polypeptide is the ability to bind FcRn. In some embodiments, a biological activity of an Fc polypeptide is the ability to bind C1q. In some embodiments, a biological activity of an Fc polypeptide is the ability to bind CD16. In some embodiments, a biological activity of an Fc polypeptide is the ability to bind protein A. An "effector function" of the Fc polypeptide is an action or activity performed in whole or in part by any antibody in response to a stimulus and may include complement fixation and / or ADCC induction (antibody-dependent cell cytotoxicity).
[00152] [00152] "IgX Fc" refers to an Fc polypeptide derived from a specific antibody isotype (for example, IgG, IgA, IgD, IgE, IgM, etc.), where "X" denotes the antibody isotype. Thus, "IgG Fc" denotes that the Fc polypeptide is derived from an γ chain, "IgA Fc" denotes that the Fc polypeptide is derived from an α chain, "IgD Fc" denotes that the Fc polypeptide is derived from an δ chain , "IgE Fc" denotes that the Fc polypeptide is derived from an ε chain, "IgM Fc" denotes that the Fc polypeptide is derived from a μ chain, etc. In some embodiments, the IgG Fc polypeptide comprises the hinge, CH2 and CH3, but does not comprise CH1 or CL. In some embodiments, the IgG Fc polypeptide comprises CH2 and CH3, but does not comprise CH1, the hinge or CL. In some modalities, the IgG Fc polypeptide comprises CH1, hinge, CH2, CH3, with or without CL. "IgX-N Fc" or "IgGXN Fc" denotes that the Fc polypeptide is derived from an antibody isotype specific subclass (such as canine IgG-A, IgG-B, IgG-C or IgG-D; feline IgG subclass IgG1a, IgG1b or IgG2; or subclass of
[00153] "Hinge" refers to any portion of an Fc polypeptide or variant Fc polypeptide that is rich in proline and comprises at least one cysteine residue located between CH1 and CH2 of a full-length heavy chain constant region.
[00154] [00154] In some embodiments, a hinge is able to form a disulfide bond within the same hinge region, within the same Fc polypeptide, with a hinge region of a separate Fc polypeptide, or with a separate Fc polypeptide. In some embodiments, a hinge comprises 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 .
[00155] [00155] The term "species of companion animal" refers to an animal suitable for companionship to humans. In some modalities, a species of pet is a canine (or dog), a feline (or cat), or an equine (or horse). In some embodiments, a species of pet is a small mammal, such as a canine, feline, dog, cat, rabbit, ferret, guinea pig, rodent, etc. In some embodiments, a species of pet is a farm animal, such as a horse, cow, pig, etc.
[00156] [00156] In some embodiments, an IgX Fc polypeptide or an IgX-N Fc polypeptide is derived from a companion animal, such as a dog, cat or horse. In some embodiments, IgG Fc polypeptides are isolated from canine γ heavy chains, such as IgG-A, IgG-B, IgG-C or IgG-D. In some cases, IgG Fc polypeptides are isolated from feline γ heavy chains, such as IgG1a, IgG1b or IgG2. In other cases, polypeptides
[00157] [00157] The terms "IgX Fc" and "IgX Fc polypeptide" include wild-type IgX Fc polypeptides and variant IgX Fc polypeptides, unless otherwise indicated.
[00158] [00158] "Wild type" refers to an unmuted version of a polypeptide that occurs in nature or a fragment of the same. A wild-type polypeptide can be produced recombinantly.
[00159] [00159] In some embodiments, a wild type IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 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, SEQ ID NO: 118.
[00160] [00160] A "variant" is a polypeptide that differs from a reference polypeptide in single or multiple non-native amino acid substitutions, deletions and / or additions. In some embodiments, a variant retains at least one biological activity of the reference polypeptide. In some embodiments, a variant (eg, a variant canine IgG-A Fc, a canine variant IgG-C Fc, a canine variant IgG-D Fc, a variant equine IgG2 Fc, IgG5 Fc variant equine or equine variant IgG6 Fc) has an activity in which the reference polypeptide is substantially lacking. For example, in some embodiments, a canine variant IgG-A Fc, a canine variant IgG-C Fc, a canine variant IgG-D Fc, a variant equine IgG2 Fc, a variant equine IgG5 Fc or IgG6 Fc from equine variant binds to protein A.
[00161] [00161] As used in this document, "percentage (%)
[00162] [00162] In some embodiments, a variant has at least about 50% of sequential identity with the nucleic acid molecule or reference polypeptide after the alignment of the sequences and introduction of gaps, if necessary, to reach the per - maximum percentage of sequential identity, and not considering any conservative substitution as part of the sequential identity. Said variants include, for example, polypeptides in which one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 50% sequential identity, at least about 60% sequential identity, at least about 65% sequential identity, at least about 70% sequential identity, at least least about 75% sequential identity,
[00163] [00163] A "point mutation" is a mutation that involves a single amino acid residue. The mutation can be the loss of an amino acid, replacement of one amino acid residue with another, or the insertion of an additional amino acid residue.
[00164] [00164] An "amino acid substitution" refers to the replacement of an amino acid in a polypeptide with another amino acid. In some modalities, an amino acid substitution is a conservative substitution. Exemplary non-limiting conservative amino acid substitutions are shown in Table 2. Amino acid substitutions can be introduced into a molecule of interest and products screened for a desired activity, for example, retained / improved antigen binding, re- immunogenicity. reduced, or improved ADCC or CDC or increased pharmacokinetics. Table 2. Original waste Exemplary substitutions Ala (A) Val; Read; Ile Arg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp; Lys; Arg Asp (D) Glu; Asn Cys (C) Ser; Gln (Q) Asn wing; Glu Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Allah; Phe; Norleucine Leu (L) Norleucine; Ile; Val; Met; Allah; Phe Lys (K) Arg; Gln; Asn Met (M) Leu; Phe; Ile Phe (F) Trp; Read; Val; Ile; Allah; Tyr
[00165] [00165] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence the orientation of the chain: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
[00166] [00166] Non-conservative substitutions imply exchanging a member of one of these classes with another class.
[00167] [00167] An "IgG Fc variant" as used in this document is an IgG Fc polypeptide that differs from a single or multiple IgG Fc polypeptide in amino acid substitutions, deletions and / or additions and substantially retains at least one biological activity of the reference IgG Fc polypeptide.
[00168] [00168] An "amino acid derivative", as used herein, refers to any amino acid, modified amino acid, and / or amino acid analog, which 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 (for example, selene cysteine and pyrrolysin, which can be found in some microorganisms) and unnatural amino acids. Exemplary amino acid derivatives include, but are not limited to, commercially available amino acid derivatives through chemical manufacturers (eg sigmaal-
[00169] [00169] In some embodiments, an IgG Fc polypeptide comprises an amino acid substitution with an amino acid derivative. In some embodiments, the amino acid derivative is an alanine derivative, a cysteine derivative, an aspartic acid derivative, a glutamic acid derivative, a phenylalanine derivative, a glycine derivative, a histidine derivative, an isoleucine derivative, a lysine derivative, a leucine derivative, a methionine derivative, an asparagine derivative, a proline derivative, a glutamine derivative, an arginine derivative, a serine derivative threonine, a valine derivative, a tryptophan derivative or a tyrosine derivative.
[00170] [00170] In some embodiments, a variant IgG Fc polypeptide comprises a variant IgG Fc polypeptide from a species of companion animal. In some embodiments, a variant IgG Fc polypeptide comprises a variant canine IgG Fc polypeptide, a variant equine IgG Fc polypeptide. Exemplifying variant IgG Fc polypeptides with binding to modified protein A
[00171] [00171] In some embodiments, a variant IgG Fc polypeptide has affinity for binding to the modified protein. In some modalities, a variant IgG Fc polypeptide has increased binding affinity to protein A. In some embodiments, a variant IgG Fc polypeptide can be purified using protein A column chromatography.
[00172] [00172] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 21, position 23, position 25, position 80, position 205, and / or position 207 of SEQ ID NO: 1. In some modalities, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 21, position 23, and / or position 24 of SEQ ID NO: 3. In some embodiments, a polypeptide Variant IgG Fc comprises a substitution of the amino acid in a position corresponding to position 21, position 23, position 25, position 80, and / or position 207 of SEQ ID NO: 4.
[00173] [00173] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 15, and / or position 203 of SEQ ID NO: 64. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 199 and / or position 200 of SEQ ID NO: 67. In some embodiments, a variant IgG Fc polypeptide comprises a substitution at the amino acid at a position corresponding to position 199 , position 200, position 201, and / or 202 of SEQ ID NO: 68.
[00174] [00174] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 21, position 23, position 25, position 80, position 205, and / or position 207 of SEQ ID NO: 1. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 21, position 23, and / or position 24 of SEQ ID NO: 3. In some modalities, a variant IgG Fc polypeptide comprises a substitution of amino acid at position 21, position 23, position 25, position 80,
[00175] [00175] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 15 and / or position 203 of SEQ ID NO: 64. In some embodiments, a variant IgG Fc polypeptide comprises a substitution of amino acid at position199 and / or position 200 of SEQ ID NO: 67. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 199, position 200, position 201, and / or position 202 of SEQ ID NO: 68.
[00176] [00176] In some embodiments, a variant IgG Fc polypeptide comprises a threonine in a position corresponding to position 21 of SEQ ID NO: 1, a leucine in a position corresponding to position 23 of SEQ ID NO: 1, an alanine in a position corresponding to position 25 of SEQ ID NO: 1, a glycine in a position corresponding to position 80 of SEQ ID NO: 1, an alanine in a position corresponding to position 205 of SEQ ID NO: 1, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 1. In some embodiments, a variant IgG Fc polypeptide comprises a threonine in a position corresponding to position 21 of SEQ ID NO: 3, a leucine in a position corresponding to position 23 of SEQ ID NO: 3, and / or an isoleucine in a position corresponding to position 24 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a threonine in a position corresponding to position 21 of SEQ ID NO: 4, a leucine in a position c corresponding to position 23 of SEQ ID NO: 4, an alanine in a position corresponding to position 25 of SEQ ID NO: 4, a glycine in a position corresponding to position 80 of SEQ ID NO: 3, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 4.
[00177] [00177] In some embodiments, a variant IgG Fc polypeptide
[00178] [00178] In some embodiments, a variant IgG Fc polypeptide comprises a threonine at position 21 of SEQ ID NO: 1, a leucine at position 23 of SEQ ID NO: 1, an alanine at position 25 of SEQ ID NO: 1, a glycine at position 80 of SEQ ID NO: 1, an aline at position 205 of SEQ ID NO: 1, and / or a histidine at position 207 of SEQ ID NO: 1. In some embodiments, an Fc polypeptide variant IgG comprises a threonine at position 21 of SEQ ID NO: 3, a leucine at position 23 of SEQ ID NO: 3, and / or an isoleucine at position 24 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a threonine at a position 21 of SEQ ID NO: 4, a leucine at position 23 of SEQ ID NO: 4, an alanine at position 25 of SEQ ID NO: 4, a glycine at position 80 of SEQ ID NO: 4, and / or a histidine at position 207 of SEQ ID NO: 4.
[00179] [00179] In some embodiments, a variant IgG Fc polypeptide comprises a threonine or a valine at position 15 of SEQ ID NO: 64, and / or a tyrosine or a valine at position 203 of SEQ ID
[00180] [00180] In some embodiments, a variant IgG Fc polypeptide comprises an 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, a variant 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. Exemplary variant IgG Fc polypeptides with modified CD16 binding
[00181] [00181] In some embodiments, a variant IgG Fc polypeptide has modified CD16 binding affinity. In some modalities, a variant IgG Fc polypeptide has reduced affinity for binding to CD16. In some embodiments, a variant IgG Fc may have a reduced ADCC immune response.
[00182] [00182] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 5, position 38, position 39, position 97, and / or position 98 of SEQ ID NO: 2. In in some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 5, position 38, position 39, position 97, and / or position 98 of SEQ ID NO: 3.
[00183] [00183] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 5, position 38, position 39, position 97, and / or position 98 of SEQ ID NO: 2. In some embodiments , a variant IgG Fc polypeptide comprising an amino acid substitution at position 5, position 38, position 39, position 97, and / or position 98 of SEQ ID NO: 3.
[00184] [00184] In some embodiments, a variant IgG Fc polypeptide comprises a proline in a position corresponding to position 5, a glycine in a position corresponding to position 38, an arginine in a position corresponding to position 39, an iso - leucine in a position corresponding to position 97, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises a proline in a position corresponding to at position 5, a glycine in a position corresponding to position 38, an argin in a position corresponding to position 39, an isoleucine in a position corresponding to position 97, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 3.
[00185] [00185] In some embodiments, a variant IgG Fc polypeptide comprises proline at position 5, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises proline at position 5, glycine at position 38, arginine at position 39, isoleucine at position 97, and / or glycine at position 98 SEQ ID NO: 3.
[00186] [00186] In some embodiments, a variant IgG Fc polypeptide comprises an 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.
[00187] [00187] In some embodiments, a variant IgG Fc polypeptide has modified C1q binding affinity. In some modalities, a variant IgG Fc polypeptide has reduced C1q binding affinity. In some embodiments, a variant IgG Fc polypeptide may have reduced complement fixation. In some modalities, a variant IgG Fc may have a reduced complement-mediated immune response.
[00188] [00188] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises a substitution amino acid in a position corresponding to position 93 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a substitution for the amino acid in a position corresponding to position 87 of SEQ ID NO: 63. In some modalities a variant IgG Fc polypeptide comprises a substitution for the amino acid in a position corresponding to position 87 of SEQ ID NO: 65. In some embodiments, a variant IgG Fc polypeptide comprises a substitution for the amino acid in a position corresponding to the position 87 of SEQ ID NO: 66. In some embodiments, a variant IgG Fc polypeptide comprises a substitution for the amino acid at a position corresponding to position 198 of SEQ ID NO: 80. In some but embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 198 of SEQ ID NO: 81.
[00189] [00189] In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 93 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 93 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 87 of SEQ ID NO: 63. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 87 of SEQ ID NO: 65. In some embodiments, a variant IgG Fc polypeptide comprises or an amino acid substitution at position 87 of SEQ ID NO: 66. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 198 of SEQ ID NO: 80. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 198 of SEQ ID NO: 81.
[00190] [00190] In some embodiments, a variant IgG Fc polypeptide comprises an arginine in a position corresponding to position 93 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises an arginine in a position corresponding to position 93 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a serine in a position corresponding to position 87 of SEQ ID NO: 63. In some embodiments, a variant IgG Fc polypeptide comprises a serine substitution in a position corresponding to position 87 of SEQ ID NO: 65. In some embodiments, a variant IgG Fc polypeptide comprises a serine in a position corresponding to position 87 of SEQ ID NO: 66. In some embodiments, a variant IgG Fc polypeptide comprises an alanine in a position corresponding to position 198 of SEQ ID NO: 80. In some embodiments, a variant IgG Fc polypeptide comprising an alanine in one position runs corresponding to position 198 of SEQ ID NO: 81.
[00191] [00191] In some embodiments, a variant IgG Fc polypeptide
[00192] [00192] In some embodiments, a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 84. In some embodiments, an Fc polypeptide of Variant IgG comprises 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, a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83. Exemplifying variant IgG Fc polypeptides with a modified interchain disulfide bond
[00193] [00193] In some embodiments, a variant feline IgG Fc polypeptide has at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide. In some embodiments, a variant feline IgG Fc polypeptide has at least one additional interchain disulfide bond in the hinge region. In some embodiments, a variant feline IgG2 IgG Fc polypeptide with at least one additional interchain disulfide bond has increased interchain stability relative to the wild type feline IgG Fc polypeptide. In some embodiments, a variant IgG polypeptide has at least one amino acid modification to a hinge region relative to a wild type IgG IgG polypeptide Fc polypeptide. In some embodiments, the wild-type IgG Fc polypeptide is a feline or equine wild-type IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region from an IgG Fc polypeptide of a different isotype. In some embodiments, the variant IgG Fc polypeptide comprises a hinge region from a wild type feline IgG-1a IgG polypeptide, a wild type feline IgG-1b IgG polypeptide or a wild-type equine IgG1 IgG Fc polypeptide. In some embodiments, a variant IgG2 IgG Fc polypeptide has increased recombinant production and / or increased hinge disulfide formation relative to the wild-type IgG Fc polypeptide. In some embodiments, the production of increased recombinant and / or formation of increased hinge disulfide can be determined by SDS-PAGE analysis under conditions of reduction and / or non-reduction.
[00194] [00194] In some embodiments, a variant IgG Fc polypeptide comprises a cysteine in a position corresponding to position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, or position 16 of SEQ ID NO: 16. In some embodiments, a variant IgG Fc polypeptide comprises a cysteine at position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, or SEQ ID NO 16:
[00195] [00195] In some embodiments, a variant IgG Fc polypeptide
[00196] [00196] In some embodiments, a variant IgG Fc polypeptide comprises 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, a variant IgG Fc polypeptide comprises an amino acid substitution at position 3 and / or at a position corresponding to position 20 of SEQ ID NO:
[00197] [00197] In some embodiments, a variant IgG Fc polypeptide comprises a proline in a 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, a variant IgG Fc polypeptide comprises a serine in a position corresponding to position 3 and / or a proline in a position corresponding to position 20 of SEQ ID NO: 129.
[00198] [00198] In some embodiments, a variant IgG Fc polypeptide comprises a 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, a variant IgG Fc polypeptide comprises a serine at position 3 and / or a proline at position 20 of SEQ ID NO: 129.
[00199] [00199] In some embodiments, the IgG Fc polypeptide variant 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: SEQ ID NO: 129,
[00200] [00200] In certain embodiments, a heterodimeric polypeptide provided herein is a bispecific antibody. A bispecific antibody has a binding specificity for two different epitopes or target molecules. In some embodiments, a bispecific antibody binds to two different epitopes on the same target molecule. Bispecific antibodies can be full-length antibodies or antibody fragments.
[00201] [00201] In some embodiments, the heterodimeric polypeptide comprises a first variant IgG Fc polypeptide comprising a "knob" mutation and a second variant IgG Fc polypeptide comprising a "hole" mutation. Non-limiting exemplary knob and hole mutations are described, for example, in Mer- chant, A. M. et al. An efficient route to human bispecific IgG. Nat Bio-technol, 16 (7): 677-81 (1998).
[00202] [00202] In some embodiments, a variant feline or canine variant IgG Fc polypeptide comprises a knob mutation. In some embodiments, a variant IgG Fc polypeptide comprising a tyrosine or a tryptophan in a position corresponding to position 138 of SEQ ID NO: 1. In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan in a position corresponding to position 137 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan in a position corresponding to position 137 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan in a position corresponding to position 138 of SEQ ID NO: 4. In some embodiments, a variant IgG Fc polypeptide comprises a tryptophan in a position corresponding to position 154 SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO:
[00203] [00203] In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan at position 138 of SEQ ID NO: 1. In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan at position 137 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan at position 137 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a tyrosine or a tryptophan in the position 138 of SEQ ID NO: 4. In some embodiments, a variant IgG Fc polypeptide comprises a 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:
[00204] [00204] In some embodiments, a variant IgG Fc polypeptide comprising a knob mutation comprises an 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:
[00205] [00205] In some embodiments, a variant canine or feline variant IgG Fc polypeptide comprises a hole mutation. In some embodiments, a variant IgG Fc polypeptide comprises a serine in a position corresponding to position 138, an aline in a position corresponding to position 140, and / or a threonine in a position corresponding to position 181 of the SEQ ID NO: 1. In some embodiments, a variant IgG Fc polypeptide comprises a serine in a position corresponding to position 137,
[00206] [00206] In some embodiments, a variant IgG Fc polypeptide comprises a serine at position 138, an alanine at position 140, and / or a threonine at position 181 of SEQ ID NO: 1. In some embodiments, an Fc polypeptide variant IgG comprises a serine at position 137, an alanine at position 139, and / or a threonine at position 181 of SEQ ID NO: 2. In some embodiments, a variant IgG Fc polypeptide comprises a serine at position 137, an alanine at position 139, and / or a threonine at position 181 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises a serine at position 138, an alanine at position 140, and / or a threonine at position 181 of SEQ ID NO: 4. In some embodiments, a variant IgG Fc polypeptide comprising a serine at position 154, an alanine at position 156, and / or a threonine at position 197 of SEQ ID NO: 16 , SEQ ID NO: 80, SEQ ID
[00207] [00207] In some embodiments, a variant IgG Fc polypeptide comprising a hole mutation comprises an 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:
[00208] [00208] In some embodiments, a contiguous polypeptide comprises a GLP1 polypeptide and a variant canine or feline IgG Fc polypeptide comprising a knob mutation. In some embodiments, a contiguous polypeptide comprises a glucagon polypeptide and a variant canine or feline IgG Fc polypeptide comprising a knob mutation. In some embodiments, a contiguous polypeptide comprises a GLP1 polypeptide and a variant canine or feline IgG Fc polypeptide comprising a hole mutation. In some embodiments, a contiguous polypeptide comprises a glucagon polypeptide and a variant canine or feline IgG Fc polypeptide comprising a hole mutation.
[00209] In some embodiments, the heterodimeric polypeptide comprises a first contiguous polypeptide comprising a GLP1 polypeptide and a canine or feline variant IgG Fc polypeptide comprising a knob mutation, and a second contiguous polypeptide comprising a glucagon polypeptide and a canine or feline variant IgG Fc polypeptide comprising a human mutation. In some embodiments, the heterodimeric polypeptide comprises a first contiguous polypeptide comprising a glucagon polypeptide and a variant canine or feline IgG Fc polypeptide comprising a knob mutation, and a second contiguous polypeptide comprising a GLP1and polypeptide an Fc polypeptide of Canine or feline IgG variant comprising a hole mutation.
[00210] [00210] "GLP1" or a "GLP1 polypeptide", as used herein, is a polypeptide comprising all or a fragment of glucagon-like peptide-1 that binds to a glucagon-like peptide-1 receptor. (GLP1R).
[00211] [00211] For example, "GLP1" refers to a GLP1 polypeptide from any vertebrate source, including mammals such as primates (for example, humans and cynomolgus monkeys), rodents (for example, mice and rats), and animals companionship (for example, dogs, cats and horses), unless otherwise indicated. In some embodiments, a GLP1 polypeptide is a wild type GLP1 polypeptide, such as GLP1 (7-37) (SEQ ID NO: 85). In some embodiments, a GLP1 polypeptide is a variant 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 comprises the amino acid sequence of SEQ ID NO: 20.
[00212] [00212] "GLP1R," as used herein, is a polypeptide comprising all or a fragment of a glucagon-like peptide-1 receptor that is capable of binding to a wild-type GLP1.
[00213] [00213] For example, "GLP1R" refers to a GLP1R polypeptide from any vertebrate source, including mammals such as primates (eg humans and cynomolgus monkeys), rodents (eg mice and rats), and companion animals (for example, dogs, cats and horses), unless otherwise indicated. In some embodiments, GLP1R is an extracellular domain fragment that binds a wild-type GLP1 polypeptide. In some embodiments, a feline GLP1R comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, a feline GLP1R comprises the amino acid sequence of SEQ ID NO: 48.
[00214] [00214] An "extracellular domain" ("ECD") is the portion of a polypeptide that extends beyond the transmembrane domain in the extracellular space. The term "extracellular domain", as used herein, can comprise a complete extracellular domain or can comprise a truncated extracellular domain lacking one or more amino acids, which binds to its ligand. The composition of the extracellular domain may depend on the algorithm used to determine which amino acids are in the membrane. Different algorithms can predict, and different systems can express, different extracellular domains for a given protein.
[00215] [00215] "Glucagon" or a "glucagon polypeptide", as used herein, is a polypeptide comprising all or a fragment of glucagon that binds to a glucagon receptor.
[00216] [00216] For example, "glucagon" refers to a glucagon polypeptide from any vertebrate source, including mammals such as primates (for example, humans and cynomolgus monkeys), rodents (for example, mice and rats), and animals company (for example, dogs, cats and horses), unless otherwise stated. In some embodiments, a glucagon polypeptide is a wild-type glucagon polypeptide, such as SEQ ID NO:
[00217] [00217] Polypeptides and other molecules can comprise a variant IgG Fc polypeptide. In some embodiments, a fusion molecule comprises a variant IgG Fc polypeptide, such as the variant IgG Fc polypeptides described herein. In some embodiments, an antibody or antibody fragment comprises a variant IgG Fc polypeptide, such as the variant IgG Fc polypeptides described in the present document.
[00218] [00218] A "fusion molecule", as used in this document, refers to a molecule comprising one or more "fusion partners". In some embodiments, the merging partners are covalently linked ("merged"). If two fusion partners are both polypeptides, the fusion partner polypeptides can be part of a contiguous amino acid sequence (for example, a contiguous polypeptide). A first fusion partner polypeptide can be attached to any N-terminal or C-terminal of a second fusion partner. In some embodiments, the fusion partners are translated as a single polypeptide from a coding sequence that encodes both fusion partners. The fusion partners can be covalently linked by other means, such as, for example, a chemical bond other than a peptide bond. Many known methods of covalently linking polypeptides to other molecules (for example, fusion partners) can be used. In other embodiments, the fusion partners are fused through a "linker", which is comprised of at least one amino acid or chemical moiety. In some embodiments, the merging partners are linked non-covalently. In some of these embodiments, they can be linked, for example, using link pairs. Exemplary binding pairs include, but are not limited to, biotin and avidin or streptavidin, an antibody and its antigen, etc.
[00219] [00219] In some embodiments, the fusion partners include an IgG Fc polypeptide and at least one GLP1 polypeptide.
[00220] [00220] The term "contiguous polypeptide" in this document is used to mean an uninterrupted sequence of amino acids. A contiguous polypeptide is typically translated from a single contiguous DNA sequence. It can be done through genetic engineering, for example, by removing the stop codon from the DNA sequence of the first protein, after attaching the DNA sequence of the second protein in structure, so that the DNA sequence be expressed as a single protein. Typically, this is achieved by cloning a cDNA into an expression vector in structure with an existing gene.
[00221] [00221] A "linker" refers to one or more amino acid residues than connecting a first polypeptide with a second polypeptide.
[00222] [00222] In some embodiments, the ligand is a flexible, non-structural ligand. In some embodiments, the ligand is a ligand rich in glycine, rich in serine, or rich in glycine and serine. In some embodiments, a linker comprises 100%, at least 95%, at least 90%, or at least 85% serine and / or glycine amino acid residues.
[00223] [00223] An "extension" as used herein refers to one or more amino acid residues that are connected to a polypeptide at its C-terminus or at its N-terminus.
[00224] [00224] In some modalities, an extension is flexible. And bad-
[00225] [00225] In some embodiments, the contiguous polypeptide comprises an IgG Fc polypeptide comprising an amino acid sequence of any of 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, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147 , 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172 , 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197 , 198 or 199 and a GLP1 polypeptide comprising an amino acid sequence of SEQ ID NO: 85. In some embodiments, the contiguous polypeptide comprises an IgG Fc polypeptide comprising a sequence amino acid of any of 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, 117, 118, 119, 120, 121, 122, 123,
[00226] [00226] In some embodiments, the contiguous polypeptide comprises an IgG Fc polypeptide comprising an amino acid sequence of any of 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, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147 , 148, 149, 150, 151, 152, 153, 154, 155, 156, 167, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172 , 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197 , 198 or 199 and a glucagon polypeptide comprising an amino acid sequence of SEQ ID NO: 21.
[00227] [00227] In some embodiments, a contiguous polypeptide comprises
[00228] [00228] In some embodiments, a contiguous polypeptide comprises a GLP1 polypeptide, a first ligand (L1), an IgG Fc polypeptide, a second ligand (L2), and a glucagon polypeptide (Gluc). In some embodiments, the contiguous polypeptide comprises: Formula (IV): GLP1 — L1 — Fc — L2 — Gluc; or Formula (V): Gluc — L1 — Fc — L2 — GLP1.
[00229] [00229] In some embodiments, the GLP1 fusion molecule has an increased serum half-life compared to a wild-type GLP1 polypeptide. The increased half-life of the GLP1 fusion molecules described herein may require lower doses and less frequent dosing regimens than wild-type GLP1 polypeptides.
[00230] [00230] In some modalities, GLP1B, if present, comprises the same amino acid sequence as GLP1A.
[00231] [00231] In some embodiments, GLP1, GLP1A or GLP1B, if present, comprise a wild-type GLP1 polypeptide. In some embodiments, GLP1, GLP1A or GLP1B, if present, comprise a variant GLP1 polypeptide. In some modes, GLP1, GLP1A or GLP1B, if present, comprise an 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 .
[00232] [00232] In some embodiments, the glucagon polypeptide comprises a wild-type glucagon polypeptide. In some embodiments, the glucagon polypeptide comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, the glucagon polypeptide comprises a variant glucagon polypeptide.
[00233] [00233] In some embodiments, the Fc polypeptide is a human IgG Fc. In some embodiments, the Fc polypeptide is a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc. In some embodiments, the Fc polypeptide is a human variant IgG Fc.
[00234] [00234] In some embodiments, the Fc polypeptide is an IgG Fc from a pet animal. In some embodiments, the Fc polypeptide is a canine IgG-A Fc, a canine IgG-B Fc, a canine IgG-C Fc, a canine IgG-D Fc. In some embodiments, the Fc is an equine IgG1 Fc, an equine IgG2 Fc, an equine IgG3 Fc, an equine IgG4 Fc, an equine IgG5 Fc, an equine IgG6 Fc or an Equine IgG7 Fc. In some embodiments, the Fc is a feline IgG1a Fc, a feline IgG1b Fc, or a feline IgG2 Fc.
[00235] [00235] In some embodiments, the Fc polypeptide is a variant IgG Fc. In some embodiments, the Fc polypeptide is a variant canine IgG-A Fc, a canine variant IgG-B Fc, a canine variant IgG-C Fc, a canine variant IgG-D Fc. In some embodiments, the Fc is a variant equine IgG1 Fc, a variant equine IgG2 Fc, a variant equine IgG3 Fc, a variant equine IgG4 Fc, a variant equine IgG5 Fc, a Variant equine IgG6 or a variant equine IgG7 Fc. In some embodiments, the Fc is a variant feline IgG1a Fc, a variant feline IgG1b Fc, or a variant feline IgG2 Fc
[00236] [00236] In some modalities, L1 and L2, if present, each independently is a flexible ligand. In some embodiments, the amino acid sequence of L1 and L2, if present, each independently comprises 100%, at least 95%, at least 90%, at least 85% serine amino acid residues and / or glycine amino acid residues .
[00237] [00237] In some embodiments, the contiguous polypeptide comprises an extension at its C-terminal. In some embodiments, the contiguous polypeptide comprises a glycine residue, two glycine residues, three glycine residues, four glycine residues, five glycine residues, six glycine residues, seven glycine residues, or eight glycine residues or more than eight glycine residues at its C-terminal. In some embodiments, the contiguous polypeptide comprises an 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 in your C-terminal.
[00238] [00238] In some embodiments, the contiguous polypeptide comprises 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.
[00239] [00239] A nucleotide sequence that encodes a polypeptide of interest, such as a variant IgG Fc polypeptide or other polypeptide described herein, can be inserted into an expression vector suitable for expression in a selected host cell. A variant IgG Fc polypeptide or other polypeptide described herein can be expressed by culturing a host cell transfected with an expression vector comprising the nucleotide sequence.
[00240] [00240] A "vector" is a plasmid that can be used to transfer DNA sequences from one organism to another or to express a gene of interest. A vector typically includes an origin of replication and regulatory sequences that regulate the expression of the gene of interest, and may or may not carry a selective marker gene, such as an antibiotic resistance gene. A vector is suitable for the host cell in which it is to be expressed. A vector can be called a "recombinant vector" when the gene of interest is present in the vector.
[00241] [00241] A "host cell" refers to a cell that can be or was a recipient of an isolated vector or polynucleotide. Host cells can be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells and CHO cells, and derivatives thereof, such as 293-6E, DG44, CHO- S and CHO-K. Host cells include progeny from a single host cell, and the progeny may not necessarily be completely identical (in morphology or in addition to genomic DNA) to the original parent cell due to natural, accidental or deliberate mutation. A host cell includes transfected cells
[00242] [00242] The term "isolated" as used in this document refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or it is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector within a host cell can be referred to as "isolated".
[00243] [00243] A "signal sequence" refers to a sequence encoding amino acid or polynucleotide residues, which facilitates the secretion of a polypeptide of interest and is typically cleaved by exporting the polypeptide outside the surface membrane cell phone.
[00244] [00244] In some embodiments, a variant IgG Fc polypeptide or a contiguous polypeptide comprising a variant IgG Fc polypeptide is isolated using chromatography, such as size exclusion chromatography, ion exchange chromatography, chromatography on protein A column, hydrophobic interaction chromatography and CHT chromatography.
[00245] A tag can be attached to variant IgG Fc polypeptides or a contiguous polypeptide comprising a variant IgG Fc polypeptide.
[00246] [00246] The variant IgG Fc polypeptides described in this document may have altered binding affinity to protein A and / or C1q and / or CD16. In some embodiments, a variant IgG Fc polypeptide has increased binding affinity to protein A relative to the wild-type IgG Fc polypeptide. Said variant IgG Fc polypeptides can be purified by protein A column chromatography. In some embodiments, a variant IgG Fc polypeptide has reduced C1q binding affinity relative to the IgG Fc polypeptide of the wild type. Said variant IgG Fc polypeptides may have reduced complement-mediated immune responses. In some embodiments, a variant IgG Fc polypeptide has reduced affinity for binding to CD16 relative to the wild-type IgG Fc polypeptide. Said variant IgG Fc polypeptides may have reduced ADCC immune responses. In some embodiments, a variant IgG Fc polypeptide has increased binding affinity to protein A relative to wild type IgG Fc polypeptide and / or has reduced C1q binding affinity relative to wild type IgG Fc polypeptide and / or has reduced CD16 binding affinity for the wild-type IgG Fc polypeptide.
[00247] [00247] "Protein A", as used herein, is a polypeptide comprising all or a portion of protein A that is capable of binding a wild-type canine IgG-B Fc, an IgG1 Fc of wild type equine, a wild type equine IgG3 Fc, a wild type equine IgG4 Fc, a wild type equine IgG7 Fc, a wild type feline IgG1a Fc or an IgG2 Fc of wild type feline.
[00248] [00248] "C1q" or "C1q complex" is used interchangeably to refer to a protein complex involved in the complement system or a portion of it, which can bind a canine IgG-B Fc of the wild type, a wild type canine IgG-C Fc, a wild type equine IgG1 Fc, a wild type equine IgG3 Fc, a wild type equine IgG4 Fc, a wild type Fc Wild-type equine IgG7, a wild-type feline IgG1a Fc, or a wild-type feline IgG1b Fc.
[00249] [00249] "CD16", as used herein, is a polypeptide comprising all or a portion of CD16 that is capable of binding a wild-type canine IgG-A Fc or a canine IgG-D Fc of the wild type. The term "binds" to a substance is a term that is well understood in the art, and methods for determining said bond are also well known in the art. A molecule is said to exhibit "bonding" if it reacts, associates with, or has an affinity for a specific cell or substance and the reaction, association or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA, KinEx A, bi-layer interferometry (BLI), surface plasmon resonance devices, etc.
[00250] [00250] "Protein A +," as used herein, means that the IgG Fc polypeptide has binding affinity for protein A. In some embodiments, a protein A + IgG Fc polypeptide comprises at least one modification in the amino acid that increases protein A binding affinity.
[00251] [00251] "Protein A -," as used herein,
[00252] [00252] "C1q +," as used herein, means that the IgG Fc polypeptide has binding affinity for C1q.
[00253] [00253] "C1q -," as used herein, means that the IgG Fc polypeptide has low or no C1q binding affinity. In some embodiments, a C1q– Fc polypeptide has at least one modification in the amino acid that reduces C1q binding affinity.
[00254] [00254] "CD16 +," as used herein, means that the IgG Fc polypeptide has binding affinity for CD16.
[00255] [00255] "CD16 -," as used herein, means that the IgG Fc polypeptide has low or no affinity for binding to CD16. In some embodiments, a CD16– Fc polypeptide has at least one modification in the amino acid that reduces the affinity for binding to CD16.
[00256] [00256] The term "affinity" means the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, a receptor) and its binding partner (for example, a ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, such as, for example, immunoblot, ELISA, KinEx A, bi-layer interferometry (BLI) or surface plasmon resonance devices.
[00257] [00257] "Surface plasmon resonance" denotes an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting changes in protein concentrations within a biosensor matrix, for example using the BIAcore ™ system (BIAcore International AB , GE Healthcare com-
[00258] [00258] "Bi-layer interferometry" refers to an optical analytical technique that analyzes the interference pattern of the reflected light from a layer of protein immobilized on a tip of the biosensor and an internal reference layer. Changes in the number of molecules attached to the tip of the biosensor cause deviations in the interference pattern that can be measured in real time. A non-limiting example device for bi-layer interferometry is an Octet® system (Pall ForteBio LLC). See, for example, Abdiche et al., 2008, Anal. Biochem. 377: 209-277.
[00259] [00259] The terms "KD," "Kd," "Kd" or "Kd value" as used interchangeably to refer to the equilibrium dissociation constant of a receptor-ligand interaction or antigen-body-antigen interaction.
[00260] [00260] In some embodiments, a variant IgG Fc polypeptide binds to protein A with a dissociation constant (KD) of less than 5 x 10-6 M, less than 1 x 10-6 M, less than 5 x 10-7 M, less than 1 x 10-7 M, less than 5 x 10-8 M, less than 1 x 10-8 M, less than 5 x 10-9 M, less than 1 x 10-9 M, less than 5 x 10-10 M, less than 1 x 10-10 M, less than 5 x 10-11 M, less than 1 x 10-11 M, less than 5 x 10-12 M or less than 1 x 10-12 M, as measured by bilayer interferometry.
[00261] [00261] In some embodiments, a variant IgG Fc polypeptide binds to C1q or CD16 with a dissociation constant (KD) greater than 5 x 10-6 M, greater than 1 x 10-5 M, greater greater than 5 x 10-5 M, greater than 1 x 10-4 M, greater than 5 x 10-4 M or greater than 1 x 10-3 M, as measured by bi-layer interferometry.
[00262] [00262] In some embodiments, the KD of an Fc polypeptide of
[00263] [00263] "Increase" or "stimulate" means to increase, improve or expand an activity, function or quantity as compared to a reference. In some modalities, it is understood to "increase" or "stimulate" the ability to cause a total increase of about 5% or more, of about 10% or more, of about 20% or more, of about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90 % or more, about 100% or more, about 125% or more, about 200% or more relative to a reference value. In some modalities, "increasing" or "stimulating" means the ability to cause a total increase of about 5% to about 50%, from about 10% to about 20%, from about 50 % to about 100%, from about 25% to about 70% relative to a reference value. In some modalities, "increasing" or "stimulating" means the ability to cause a total increase of 50% or more. In some modalities, "increasing" or "stimulating" means the ability to cause a total increase of about 75%, 85%, 90%, 95% or more. In some embodiments, the amount noted above is stimulated or increased over a period of time, relative to a control dose (such as a placebo) for the same period of time.
[00264] [00264] In some embodiments, a variant IgG Fc polypeptide is able to bind to protein A with an increased affinity of about 5% or more, about 10% or more, about 20% or more , about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 125% or more, about 150% or more, about 200% or more relative to a reference IgG Fc polypeptide. In some embodiments, a variant IgG Fc polypeptide is able to bind to protein A with an increased affinity of about 5% to about 50%, from about 10% to about 20%, from about 50 % to about 100%, from about 25% to about 70% relative to a reference IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a wild-type IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a different variant IgG Fc polypeptide.
[00265] [00265] "Reduce" or "inhibit" means to decrease, reduce or stop an activity, function or quantity as compared to a reference. In some modalities, "reducing" or "inhibiting" means the ability to cause a total reduction of about 5% or more, of about 10% or more, of about 20% or more, of about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more relative to a reference IgG Fc polypeptide. In some embodiments, "reducing" or "inhibiting" means the ability to cause a total reduction of about 5% to about 50%, from about 10% to about 20%, from about 50% to about 100%, from about 25% to about 70% relative to a reference value. In some modalities, it is understood to "reduce" or "inhibit" the ability to cause a total reduction of 50% or more. In some modalities, "reducing" or "inhibiting" means the ability to cause a total reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the amount noted above is inhibited or decreased for a period of time, relative to a control dose (such as a placebo) for the same period of time.
[00266] [00266] In some embodiments, a variant IgG Fc polypeptide is capable of binding to C1q or CD16 with a decreased affinity of about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more relative to a reference IgG Fc polypeptide. In some embodiments, a variant IgG Fc polypeptide is capable of binding to C1q or CD16 with a decreased affinity of about 5% to about 50%, about 10% to about 20%, about 50% up to about 100%, from about 25% to about 70% relative to a reference IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a wild-type IgG Fc polypeptide. In some embodiments, the reference IgG Fc polypeptide is a different variant IgG Fc polypeptide.
[00267] [00267] A "reference" as used in this document, refers to any sample, standard or level that is used for comparison purposes. A reference can be a wild type reference or a variant reference. A reference can be obtained from a healthy or disease-free sample. In some instances, a reference is obtained from a disease-free or untreated sample from a pet. In some examples, a reference is obtained from one or more healthy animals of a specific species, which are not the animal being tested or treated. Exemplary biological activity of variant GLP1 fusion molecules
[00268] [00268] In some embodiments, a GLP1 fusion molecule binds to GLP1R and activates cAMP production. In some embodiments, a GLP1 fusion polypeptide increases cAMP production in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, by 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 . Exemplary pharmaceutical compositions
[00269] [00269] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in that form in order to allow the biological activity of the active ingredient (s) to be effective, and does not contain any additional components that are unacceptably toxic to an individual to whom the formulation would be administered.
[00270] [00270] A "pharmaceutically acceptable vehicle" refers to a solid, non-toxic, semi-solid, or liquid charge, diluent, encapsulating material, auxiliary formulation or conventional vehicle in the art.
[00271] [00271] The pharmaceutical composition can be stored in lyophilized form. Thus, in some modalities, the preparation process includes a lyophilization step. The lyophilized composition can then be reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to the dog, cat or horse. In other embodiments, particularly where a variant IgG Fc polypeptide or other polypeptide described herein is highly stable to thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, that is, as an aqueous composition, which can be stored. be administered directly or with appropriate dilution to the dog, cat or mackerel. A lyophilized composition can be reconstituted with sterile water for injection (WFI). Bacteriostatic reagents, such as benzyl alcohol, can be included. Thus, the invention provides pharmaceutical compositions in solid or liquid form.
[00272] [00272] The pH of pharmaceutical compositions can be in the range of about pH 5 to about pH 8, when administered. The compositions of the invention are sterile if they need to be used for therapeutic purposes. Sterility can be achieved by any of the various means known in the art, including through filtration through sterile filtration membranes (for example, 0.2 micron membranes). Sterility can be maintained with or without antibacterial agents. Certain uses of Fc polypeptides and pharmaceutical compositions
[00273] [00273] A polypeptide comprising a variant IgG Fc polypeptide, such as a variant IgG Fc polypeptide, of the invention or pharmaceutical compositions comprising a variant IgG Fc polypeptide of the invention may be useful to extend the half-life of the invention. product in a pet, including, but not limited to, canine, feline or equine.
[00274] [00274] As used in this document, "treatment" is an approach to obtain beneficial or desired clinical results. The "treatment" as used herein, covers any administration or application of a therapeutic product for diseases in a mammal, including a pet animal. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: relief of one or more symptoms, decrease in the extent of the disease, prevention or delay of the spread of the disease , prevention or delay of disease recurrence, delay or delay in disease progression, improvement of disease state, inhibition of disease or disease progression, inhibition or halt of disease or progression, interrupting its development, and remission (either partial or total).
[00275] [00275] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist can vary according to factors such as the type of disease to be treated, the state of the disease, the severity and course of the disease, the type of therapeutic purpose, any previous therapy, clinical history, response to previous treatment, the criterion of the attending veterinarian, age, sex and weight of the animal, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the animal. A therapeutically effective amount is also one in which any toxic or harmful effects of the subunit / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be released in one or more administrations. A therapeutically effective amount refers to an effective amount, in dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
[00276] [00276] In some embodiments, a variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same is administered parenterally, through subcutaneous administration, intravenous infusion or intramuscular injection. In some embodiments, a variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, a variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising it is administered via an intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal or inhalation.
[00277] [00277] In some embodiments, a GLP1 fusion polypeptide or pharmaceutical compositions comprising the same can be used according to the methods in this document to treat conditions related to high blood glucose. In some embodiments, a GLP1 fusion polypeptide or pharmaceutical compositions are administered to a companion animal, such as a canine, feline or horse, to treat a condition related to high blood glucose.
[00278] [00278] In some embodiments, a variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same is administered in an amount ranging from 0.0001 mg / kg of body weight to 100 mg / kg body weight per dose. In some embodiments, the GLP1 analogue can be 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 analogue can be administered in an amount ranging from 1 mg / kg body weight to 10 mg / kg body weight per dose. In some embodiments, the GLP1 analogue can be administered in an amount ranging from 0.5 mg / kg body weight to 100 mg / kg body weight, in the range of 1 mg / kg body weight to 100 mg / kg of body weight, in the range of 5 mg / kg of body weight to 100 mg / kg of body weight, in the range of 10 mg / kg of body weight to 100 mg / kg of body weight, in the range of 20 mg / kg of body weight to 100 mg / kg body weight, in the range of 50 mg / kg body weight to 100 mg / kg body weight, in the range of 1 mg / kg body weight to 10 mg / kg body weight, in the 5 mg / kg body weight range to 10 mg / kg body weight, 0.5 mg / kg body weight range to 10 mg / kg body weight, or 5 mg / kg body weight range to 50 mg / kg of body weight.
[00279] [00279] In some embodiments, a variant IgG Fc polypeptide or other polypeptide described herein, or a pharmaceutical composition comprising the same is administered to a pet animal at one time or during a series of treatments. In some modalities, the dose is administered once a week for at least two or three consecutive weeks, and in some modalities, this treatment cycle is repeated two or more times, optionally interspersed with one or more weeks without treatment. In other modalities, the therapeutically effective dose is administered once a day for two to five consecutive days, and in some modalities, this treatment cycle is repeated two or more times, optionally interspersed with one or more days or weeks without treatment.
[00280] [00280] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concomitant) and consecutive or sequential administration in any order. The term "concurrently" is used in this document to refer to the administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of a therapeutic agent falls within a short period of time related to the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered with a time separation of no more than about a specified number of minutes. The term "sequentially" is used throughout this document to refer to the administration of two or more therapeutic agents where the administration of one or more agent (s) continues after discontinuing the administration of one or more other agent (s) ( (s), or where the administration of one or more agent (s) begins before the administration of one or more other agent (s). For example, the administration of two or more therapeutic agents is administered with a time separation of more than about a specified number of minutes. As used herein, "in conjunction with" refers to the administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to the administration of one treatment modality before, during or after the administration of the other treatment modality to the animal.
[00281] [00281] In some modalities, the dose is administered once a week for at least two or three consecutive weeks, and in some modalities, this treatment cycle is repeated two or more times, optionally interspersed with one or more weeks without treatment. In other modalities, the therapeutically effective dose is administered once a day for two to five consecutive days, and in some modalities, this treatment cycle is repeated two or more times, optionally interspersed with one or more days or weeks without treatment. .
[00282] [00282] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concomitant) and consecutive or sequential administration in any order. The term "concomitantly" is used in this document to refer to the administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of a therapeutic agent falls within a short period of time relative to the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered with a separation of time of no more than about a specified number of minutes. The term "sequentially" is used throughout this document to refer to the administration of two or more therapeutic agents where the administration of one or more agent (s) continues after discontinuing the administration of one or more other agent (s) , or where the administration of one or more agent (s) begins before the administration of one or more other agent (s). For example, the administration of two or more therapeutic agents is administered with a time separation of more than about a specified number of minutes. As used herein, "in conjunction with" refers to the administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to the administration of one treatment modality before, during or after the administration of the other treatment modality to the animal.
[00283] [00283] In some embodiments, the method comprises administering in combination with a GLP1 fusion polypeptide, insulin, a DPP4 inhibitor, a SGLT2 inhibitor, a sulfonylurea biguanides, a meglitinide derivative, an alpha inhibitor - glucosidase, a thiazolidinedione (TZD), an amylomimetic, a bile acid sequester, a dopamine agonist.
[00284] [00284] The following examples illustrate specific aspects of disclosure and are in no way intended to limit disclosure.
[00285] [00285] Purification of antibodies using protein A affinity is a well-developed process. However, among the four canine IgG subtypes, only IgG-B Fc (for example, SEQ ID NO: 2 or SEQ ID NO: 107) has binding affinity for protein A. IgG-A Fc (for example, SEQ ID NO: 1), IgG-C Fc (for example, SEQ ID NO: 3 or SEQ ID NO: 108), and canine IgG-D Fc (for example, SEQ ID NO: 4) have binding affinity to protein A weak or not measurable. IgG-A, IgG-C and IgG-D variant canine Fc polypeptides have been designed for binding to altered protein A.
[00286] [00286] Furthermore, IgG-B Fc and canine IgG-C Fc have complement activity and bind to C1q, while IgG-A Fc and canine IgG-D Fc have weak C1q binding affinity or not measurable. To potentially reduce the reduction to C1q and / or potentially reduce complement-mediated immune responses, the IgG-B Fc and canine IgG-C F polypeptides were designed.
[00287] [00287] Furthermore, IgG-B Fc and canine IgG-C Fc have CD16 binding activity. To potentially reduce the binding of CD16 to IgG-B Fc and IgG-C Fc, and / or potentially reduce ADCC, canine IgG-B Fc and IgG-C Fc polypeptides were designed.
[00288] [00288] Table 3, summarizes below the characteristics of binding to protein A and C1q, of canine IgG Fc subtypes. Notably, none of the subtypes of wild-type canine IgG Fc lacks binding to C1q and binds to protein A. Table 3. Wild-type canine IgG Fc binding to binding to protein A binding to C1q CD16 IgG-A Fc - - - IgG-B Fc + + + IgG-C Fc - + + IgG-D Fc - - - (-) denotes low or unmeasured binding activity.
[00289] [00289] Using protein sequence analysis and three-dimensional protein modeling, canine IgG-B Fc sequences that are likely to come into contact with protein A have been identified. Fig-
[00290] [00290] Two approaches have been used to designate IgG-A, IgG-C and IgG-D variant Fc polypeptides for binding to increased protein A. For the first approach, the variant canine IgG-A, IgG-C and IgG-D Fc polypeptides were designed to have the same protein A binding motif sequences as the canine IgG-B Fc (for example , SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively). For the second approach, canine IgG-A variant I (21) T / Q (207) H (SEQ ID NO: 60), canine IgG-C variant I (21) T (SEQ ID NO: 61), and canine IgG-D variant Fc (21) T / Q (207) H (SEQ ID NO: 62) were designated with one or two amino acid substitutions in the protein A binding region to correspond with the canine IgG-B Fc sequence.
[00291] [00291] In addition, IgG-A Fc, IgG-C Fc and canine IgG-D Fc polypeptides with increased protein A binding can be prepared having one or more of the amino acid substitutions listed in table 4 Table 4. Canine IgG Fc Amino Acid Substitutions * (Protein A +) Canine IgG-A Fc Canine IgG-C Fc Canine IgG-D Fc (SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 3) (SEQ ID NO: 4) Ile (21) Thr Ile (21) Thr Ile (23) Thr Arg (23) Leu Val (23) Leu Arg (23) Leu Thr (25) Thr Wing (24 ) Ile Thr (25) Ala Glu (80) Gly Glu (80) Gly Thr (205) Ala Gln (207) His Gln (207) His * The amino acid positions listed are relative to SEQ ID NO. indicated.
[00292] [00292] To potentially reduce the binding of C1q to IgG-B Fc and canine IgG-C Fc, and / or potentially reduce complement-mediated immune responses, IgG-B and IgG-C Fc polypeptides from canine variants can be prepared having a substitution
[00293] [00293] To potentially reduce the binding of CD16 to IgG-B Fc and IgG-C Fc, and / or potentially reduce ADCC, variant canine IgG-B and IgG-C Fc polypeptides can be prepared. - having one or more of the amino acid substitutions listed in table 5. The amino acid (s) substitution was identified after analyzing the protein sequence and modeling the 3-D structure of canine IgG-B and IgG-C compared to IgG-A and IgG-D, which do not show to exhibit ADCC activity. Table 5. Position of the original residue * Canine IgG-B Fc replacement (s) Canine IgG-C Fc (SEQ ID NO: 2) (SEQ ID NO: 3) Met (5) Leu (5) Any amino acid , except original residue, such as Pro Asp (38) Asp (38) Any amino acid, except original residue, such as Gly Pro (39) Pro (39) Any amino acid, except original residue, such as Arg Lys (97) Lys (97) Any amino acid, except original residue, such as Ile Ala (98) Ala (98) Any amino acid, except original residue, such as Gly * The amino acid positions listed are relative to SEQ ID NO. indicated.
[00294] [00294] Since canine IgG-C wild-type Fc lacks binding to protein A and is bound to C1q, a double canine IgG-C Fc that binds protein A and has reduced binding to C1q can be prepared by combining one or more of the amino acid substitutions listed in table 4 with a K (93) R substitution or K (93) X substitution, where X is any amino acid except Lys. A double variant canine IgG-B Fc or double variant canine IgG-C Fc with reduced binding to C1q and reduced binding to CD16 can be prepared by combining one or more of the amino acid substitutions listed in table 5 with a K (93) R substitution or K (93) X substitution, where X is any amino acid, except Lys. A triple variant canine IgG-C Fc that binds protein A and has reduced binding to C1q and CD16 can be prepared by combining one or more of the amino acid substitutions listed in table 4 and one or more of the amino acid substitutions listed in table 5 with a substitution K (93) R or substitution K (93) X, where X is any amino acid, except Lys.
[00295] [00295] The binding of any variant canine IgG Fc to protein A, CD16, and / or C1q can be determined and compared to a binding of another IgG Fc to protein A, CD16, and / or C1q (e.g. - example, the corresponding wild-type canine IgG Fc, another wild-type canine IgG Fc or variant, or a wild-type IgG Fc or variant of another companion animal, etc.).
[00296] [00296] The connection analysis can be performed using an Octet biosensor. In summary, the target molecule (for example, protein A, C1q, CD16, etc.) can be biotinylated and the unreacted free biotin removed (for example, through dialysis). The biotinylated target molecule is captured at the tips of the streptavidin sensor. The association of the target molecule with various concentrations (eg 10 μg / mL) of IgG Fc polypeptide is monitored for a specified time or until steady state is achieved. Dissociation is monitored for a specified time or until steady state is achieved. A blank curve of the buffer is subtracted to correct any deviations. The data is fitted to a 1: 1 link model using ForteBioTM data analysis software to determine the kon, koff and Kd. Example 2 Variant equine IgG Fc polypeptides for increased protein A binding and / or decreased complement binding
[00297] [00297] Of the seven IgG Fc subtypes, equine IgG1 (for example, SEQ ID NO: 63), IgG3 Fc (for example, SEQ ID NO: 65), IgG4 Fc (for example, SEQ ID NO: 66), IgG7 Fc (eg SEQ ID NO: 69) have protein A binding affinity. Equine IgG2 Fc (eg SEQ ID NO: 18, SEQ ID NO: 64), Fc IgG5 (for example, SEQ ID NO: 67), and IgG6 Fc (for example, SEQ ID NO: 68) have weak or unmeasurable protein A binding affinity. The IgG2, IgG5 and IgG6 variant equine Fc polypeptides have been designed for binding to altered protein A.
[00298] [00298] Furthermore, IgG2 Fc, IgG5 Fc and equine IgG6 Fc have weak or unmeasurable C1q binding affinity, while IgG1 Fc, IgG3 Fc, IgG4 Fc and equine IgG7 Fc bind to C1q. To potentially reduce binding to C1q and / or potentially reduce complement-mediated immune responses, IgG1 Fc, IgG3 Fc, IgG4 Fc and IgG7 Fc variant equine polypeptides were designed.
[00299] [00299] Table 6, summarizes below the protein A and C1q binding characteristics of equine IgG Fc subtypes. Notably, none of the Fc subtypes of wild-type equine IgG lack binding to C1q and bind to protein A.
[00300] [00300] Using protein sequence analysis and three-dimensional protein modeling, the IgG1 Fc, IgG3 Fc, IgG4 Fc and equine IgG7 Fc sequences that are likely to come into contact with protein A have been identified. The IgG2 Fc, IgG5 Fc and IgG6 Fc variant polypeptides with increased protein A binding can be prepared having one or more of the amino acid substitutions listed in Table 7. Table 7. Fc amino acid substitutions of Equine IgG variant * (Protein A +) Equine IgG2 Fc Equine IgG5 Fc Equine Ig6 Fc (SEQ ID NO: 64) (SEQ ID NO: 67) (SEQ ID NO: 68) Wing (15) Thr Val (199) Leu Ile (199) Leu Phe (203) Tyr Glu (200) Tyr Arg (200) His His (201) Asn Thr (202) His * The amino acid positions listed are relative to SEQ ID NO. indicated
[00301] [00301] For example, IgG2 Fc, IgG5 Fc, IgG6 Fc polypeptides have been designated with one or multiple amino acid substitutions in the protein A binding region to correspond with the wild-type equine IgG Fc sequence , which does not bind protein A. Equine IgG2 variant F (203) Y (SEQ ID NO: 71); A variant equine IgG2 Fc (15) T / F (203) Y (SEQ ID NO: 72); Equine variant V IgG5 (199) L / E (200) Y (SEQ ID NO: 75); and Fc of equine IgG6 variant I (199) L / R (200) H / H (201) N / T (202) H (SEQ ID NO: 76) with increased protein A binding can be prepared.
[00302] [00302] To potentially reduce the binding of C1q to IgG1 Fc, IgG3 Fc, IgG4 Fc and equine IgG7 Fc, and / or potentially reduce complement-mediated immune responses, IgG1 Fc polypeptides, IgG3 Fc, IgG4 Fc and variant canine IgG7 Fc can be prepared having an amino acid substitution of Lys with any amino acid except Lys in an amino acid position corresponding to position 87 of SEQ ID NO: 63, of SEQ ID NO: 65, from SEQ ID NO: 66, from SEQ ID NO: 69, respectively. These amino acid substitutions were identified after analyzing the protein sequence and modeling the 3-D structure of IgG1 Fc, IgG3 Fc, IgG4 Fc and equine IgG7 Fc compared to IgG2 Fc, IgG5 Fc and equine IgG6 Fc, which did not show complement activity. For example, equine variant K IgG1 Fc (87) S (SEQ ID NO: 70), equine variant IgG3 Fc K (87) S (SEQ ID NO: 73), equine variant K IgG4 Fc (87) ) S (SEQ ID NO: 74) and Fc of equine IgG7 variant K (87) S (SEQ ID NO: 77) can be prepared.
[00303] [00303] The binding of any variant equine IgG Fc to protein A and / or C1q can be determined and compared with the binding of another IgG Fc to protein A and / or C1q (e.g., equine IgG Fc wild type, another wild-type equine IgG Fc or variant or a wild type IgG Fc or variant of another pet animal, etc.). The binding assay described in Example 1 can be used. Example 3 The variant feline IgG Fc polypeptides for decreased complement binding
[00304] [00304] Each of the three subtypes of IgG Fc, feline IgG1a (SEQ ID NO: 80 or SEQ ID NO: 117), IgG1b Fc Fc (SEQ ID NO: 81 or SEQ ID NO: 118) and Fc of IgG2 (SEQ ID NO: 16) has protein A binding affinity. However, only feline IgG2 Fc has weak or not measurable C1q binding affinity, while IgG1a Fc, IgG1b Fc binds to C1q . To potentially reduce binding to C1q and / or potentially reduce complement-mediated immune responses, variant feline IgG1a and IgG1b Fc polypeptides were designed.
[00305] [00305] Table 8, summarizes below the protein A and C1q binding characteristics of the feline IgG Fc subtypes. Notably, none of the Fc subtypes of wild-type equine IgG lacks binding to C1q and binds to protein A. Table 8. Fc of wild-type feline IgG binding to protein A Binding to C1q IgG1a Fc + + IgG1b Fc + + IgG2 Fc + - (-) denotes low or unmeasured binding activity.
[00306] [00306] To potentially reduce the binding of C1q to feline IgG1a Fc and feline IgG1b Fc and / or potentially reduce complement-mediated immune responses, variant feline IgG1a Fc and IgG1b Fc polypeptides can be prepared having an amino acid substitution of Pro with any amino acid except Pro in an 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 modeling of 3-D structure of feline IgG1a Fc and IgG1b Fc compared to feline IgG2 Fc, which does not show to show complement activity. For example, feline IgG1a variant Fc P (198) A (SEQ ID NO: 82) and feline IgG1b variant Fc (198) A (SEQ ID NO: 83) can be prepared.
[00307] [00307] The binding of any variant feline IgG Fc to C1q can be determined and compared to the binding of another Fc of Ig1
[00308] [00308] To allow the preparation of a bispecific canine or feline antibody or a bifunctional canine or feline Fc fusion protein using a knob-in-hole heterodimerization approach, canine vari IgG Fc polypeptide pairing - before and variant feline IgG Fc polypeptides was investigated.
[00309] [00309] A threonine amino acid substitution in a position corresponding to canine IgG-A 138 (SEQ ID NO: 1), in a position corresponding to canine IgG-B Fc 137 (SEQ ID NO: 2 ), in a position corresponding to position 137 of IgG-C Fc (SEQ ID NO: 3), or in a position corresponding to position 138 of canine IgG-D Fc (SEQ ID NO: 4) to tyrosine (T138Y or T137Y) can be introduced to an Fc chain (heterodimeric chain1). Examples of the amino acid sequences of the heterodimeric chair 1 of IgG-A Fc, IgG-B Fc, IgG-C Fc and variant canine IgG-D Fc are SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, respectively.
[00310] [00310] A tyrosine amino acid substitution in a position corresponding to the position 181 of canine IgG-A (SEQ ID NO: 1), in a position corresponding to position 180 of canine IgG-B Fc (SEQ ID NO: 2) , in a position corresponding to canine IgG-C Fc position 180 (SEQ ID NO: 3) or in a position corresponding to canine IgG-D Fc position 181 (SEQ ID NO: 4) to threonine (Y181T or Y180T) can be introduced to a second Fc chain (heterodimeric chain 2). Examples of amino acid sequences of IgG-A Fc, IgG-B Fc, IgG-C Fc and heterodimeric chain variant canine IgG-D 2 are SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, respectively.
[00311] [00311] A second pairing of heterodimer 3 and 4 chains of canine IgG variant Fc was also investigated. A substitution of threonine amino acid in tryptophan in a position corresponding to position 138 of canine IgG-A (SEQ ID NO: 1) or canine IgG-D (SEQ ID NO: 4) (T138W), or in a position corresponding to the position137 of canine IgG-B Fc (SEQ ID NO: 2) or canine IgG-C (SEQ ID NO: 3) (T137W) can be introduced in a Fc chain ( heterodimeric chain 3). Examples of the amino acid sequences of the IgG-A Fc heterodimeric chain, IgG-B Fc, IgG-C Fc and variant canine IgG-D Fc are SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113 and SEQ ID NO: 115.
[00312] [00312] An amino acid substitution of threonine in serine in a position corresponding to position 138, of leucine in alanine in a position corresponding to position 140, and of tyrosine in threonine in a position corresponding to position 181 of canine IgG-A (SEQ ID NO: 1) or IgG-D (SEQ ID NO: 4) (T138S, L140A, Y181T), or serine threonine in a position corresponding to position 137, leucine in alanine in a position corresponding to position 139, and tyrosine to threonine in a position corresponding to the 180 Fc position of canine IgG-B (SEQ ID NO: 2) or IgG-C (SEQ ID NO: 3) (T137S, L139A, Y180T) can be introduced in a second Fc chain (heterodimeric chain 4). Examples of amino acid sequences of the heterodimeric chain 4 of IgG-A Fc, IgG-B Fc, IgG-C Fc and IgG-D Fc are SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114 and SEQ ID NO: 116.
[00313] [00313] A threonine amino acid substitution in tryptophan in a position corresponding to position 154 of feline IgG2 (SEQ ID NO: 16), of feline IgG1a Fc (SEQ ID NO: 80 or SEQ ID NO: 117), or feline IgG1b Fc (SEQ ID NO: 81 or SEQ ID NO: 118) (T154W) can be introduced into an Fc chain (heterodimeric chain 1). Examples of IgG2 Fc amino acid heterodimeric chain 1 sequences, IgG1b Fc and IgG1b Fc are SEQ ID NO: 119, SEQ ID NO: 121 and SEQ ID NO: 123, respectively.
[00314] [00314] An amino acid substitution of threonine in serine in a position corresponding to position 154, of leucine in alanine in a position corresponding to position 156, and of tyrosine in threonine in a position corresponding to position 197 of IgG2 Fc of feline (SEQ ID NO: 16), IgG-1a Fc (SEQ ID NO: 80 or SEQ ID NO: 117), or IgG-1b Fc (SEQ ID NO: 81 or SEQ ID NO: 118) ( T154S, L156A, Y197T) can be introduced in a second Fc chain (heterodimeric chain4). Examples of IgG2 Fc amino acid heterodimeric chain 1 sequences, IgG1a Fc and variant feline IgG1b Fc are SEQ ID NO: 120, SEQ ID NO: 122 and SEQ ID NO: 124.
[00315] [00315] Pairing of variant canine IgG Fc heterodimeric chains 1 and 2, pairing of variant canine IgG Fc heterodimeric chains 3 and 4, or pairing of heterodimeric chains 1 and 2 of IgG Fc of IgG variant feline may allow for Fc heterodimerization and prevent or reduce Fc homodimerization. The heterodimeric chain 1 of a canine IgG subtype can be combined with the heterodimeric chain 2 of the same or a different canine IgG subtype. The heterodimeric chain 3 of a canine IgG subtype can be combined with the heterodimeric chain 4 of the same or a different canine IgG subtype. The heterodimeric chain 1 of a feline IgG subtype can be combined with the heterodimeric chain 2 of the same or a different feline IgG subtype. The design may allow dimerization of bispecific canine or feline antibodies. In addition, two different peptides or proteins or a combination of different proteins can be fused to the heterodimeric Fc chains. For example, a double GLP1 and glucagon molecule can be created using canine IgG Fc heterodimer chains or variant feline IgG Fc heterodimer chains, such as a GLP1 polypeptide (for example, SEQ ID NO: 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) fused to a chain heterodimeric 1 variant canine IgG-D Fc (for example, SEQ ID NO: 14) and a glucagon polypeptide (for example, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO0: 94, or SEQ ID NO: 95) fused to a variant canine IgG-D Fc heterodimeric 2 chain (e.g., SEQ ID NO: 15). Example 5 GLP1 fusion proteins
[00316] [00316] The amino acid sequence of GLP1 is conserved among humans, felines, canines and horses, among other species. Proteolytic products of GLP1 (eg, GLP1 (amino acids 7-37) (SEQ ID NO: 85) are rapidly degraded by dipeptidyl peptidase-4 (DPP-4) and are understood to have a short half-life in serum for a few minutes.
[00317] [00317] The contiguous polypeptide design comprising at least one GLP1 polypeptide and feline, canine or equine IgG Fc polypeptides has been investigated to generate long-acting GLP1 fusion proteins. The constructs that follow were designated: Formula (I): GLP1A — L1 — Fc; Formula (II): Fc — L1 — GLP1A; or Formula (III): GLP1A — L1 — Fc — L2 — GLP1B. where GLP1A is a first GLP1 polypeptide, GLP1B is a
[00318] [00318] GLP1 has been modified to be resistant to DPP-4 by replacing alanine with glycine or serine in a position corresponding to position 8 of wild type GLP1 (7-37) (SEQ ID NO: 85). A minimal sequence of wild-type GLP1 (7-37) (SEQ ID NO: 87) to bind the N-terminal domain of mature feline GLP1R (SEQ ID NO: 49) was analyzed using three-dimensional protein modeling of the complex. Based on this modeling, the two C-terminal amino acids were removed from DPP-4 resistant GLP1 to generate GLP1-S8 (7-35) polypeptides (SEQ ID NO: 86) and GLP1-G8 (7-35) (SEQ ID NO: 87).
[00319] [00319] GLP1 polypeptides when positioned at the C-terminal of a construct, as in formulas II and III, are not susceptible to DPP-4 degradation. Therefore, substitution of alanine to glycine or serine is not necessary for GLP1 polypeptides positioned at the C-terminus. Consequently, wild type GLP1 (7-37) (SEQ ID NO: 85) can be used at the C-terminal.
[00320] [00320] The binder can be a flexible, non-structural binder, such as a binder rich in glycine and serine. A flexible extension can be added to the C-terminal of the contiguous polypeptide. The extension may comprise a 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. Example 6 Feline IgG GLP1 and Fc fusion proteins
[00321] [00321] Nucleotide sequences encoding (1) a polypeptide
[00322] [00322] The resulting vectors were separately transfected into CHO cells. The supernatant containing the contiguous polypeptides without the signal peptide (SEQ ID NOs: 24 and 23) was collected and filtered. Both proteins were affinity purified using a protein A column (CaptivA® protein A resin activity, Repligen). The proteins were determined to be monomeric as assessed by HPLC gel filtration. However, the SDS-PAGE analysis showed that a percentage of both contiguous polypeptides migrated to the same position on the gel in the absence and presence of a reducing agent (DTT) (Figure 2A). This result suggests that the wild-type feline IgG2 Fc hinge, which has only one pair of cysteine residues, may not be sufficient to form an effective disulfide bond.
[00323] [00323] Analysis of the three-dimensional protein modeling of various orthologous hinge structures was used to determine the approximate locations to modify the feline IgG2 hinge to increase disulfide formation. To increase disulfide formation in the feline IgG2 hinge, the hinge sequence can be modified by replacing an amino acid with cistein. For example, a variant feline IgG2 Fc (SEQ ID NO: 17) having a modified hinge was prepared by replacing Gly with Cys at an amino acid position corresponding to position 14 of SEQ ID NO: 16. Contiguous polypeptides run - ssGLP1-G8_I_VARfeIgG2 (SEQ ID NO: 39) and ssGLP1- G8 / GLP1-2G _III_WTfeIgG2 (SEQ ID NO: 38) components comprising feline IgG Fc variant of SEQ ID NO: 17 were designated, 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. The SDS-PAGE analysis of the feline variant IgG2 constructs showed a decrease in the amount of protein in the lower molecular weight range in the absence of the reducing agent compared to the wild-type feline IgG2 constructs (compare Figure 2 B with Figure 2A). These results suggest that covalent Fc pairing has been improved for both variant feline IgG2 constructs.
[00324] [00324] Furthermore, differential scanning fluorimetry was used to assess the stability of contiguous polypeptides at various pH, as reflected by the average melting point temperature (n = 3) (Table 9, below). The increased stability of the variant feline IgG2 hinge is most evident at pH 6. For example, constructs having variant feline IgG2 (SEQ ID NOs: 25 and 26) exhibited a higher Tm at pH 6 (56.9 and 59 , 7 ° C) than the corresponding constructs having wild-type feline IgG2 (SEQ ID NOs: 23 and 24), which had a Tm of 55.2 and 56.9 ° C, respectively.
[00325] [00325] Contiguous polypeptides of Formulas I, II and III comprising GLP1-S8 (7-35) (SEQ ID NO: 86) instead of GLP1-G8 (7- 35) (SEQ ID NO: 87) similarly designated 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-SII / GLP1- 3G_ NO: 27) can be prepared. Similar constructs having a wild type feline IgG variant instead of Fc, such as GLP1-S8_I_VARfeIgG2, GLP1-S8_I_ VAR- feIgG2, GLP1-S8 / GLP1-2G_III_VARfeIgG2 and GLP1-S8 / GLP1-G2 can be prepared.
[00326] [00326] While feline IgG2 Fc was used in this example, contiguous polypeptides of Formulas I, II and III comprising IgG1a Fc or feline IgG1b Fc instead of IgG2 can be designated and prepared. For example, similar contiguous polypeptides having wild-type feline IgG1a Fc, wild-type feline IgG1b Fc, variant feline IgG1a Fc or variant feline IgG1b Fc (SEQ ID NOs: 80-83 , respectively), can be designated and prepared. Example 7 Canine IgG GLP1 and Fc fusion proteins
[00327] [00327] Several contiguous polypeptides of formula I, II and III comprising a variant GLP1 and a canine IgG Fc can be designated and prepared. For example, a variant canine IgGD Fc (for example, SEQ ID NO: 7 or SEQ ID NO: 62) can be chosen based on its low or no binding to C1q for reduced complementary activity and protein A binding for ease of purification. In addition, a flexible, non-structural binder, such as a binder rich in glycine and / or serine, can be used.
[00328] [00328] GLP1-G8_I_VARcaIgGD (SEQ ID NO: 30) and GLP1- S8_I_VARcaIgGD (SEQ ID NO: 32) are examples of contiguous polypeptides of formula I comprising (1) whether GLP1-G8 (7-35) or GLP1-S8 ( 7-35), (2) a flexible linker, and (3) a variant canine IgGD Fc (for example, 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 contiguous polypeptides of formula III comprising (1) be GLP1-G8 (SEQ ID NO: 87) or GLP1-S8 (SEQ ID NO: 86), (2) GLP1 (7-35) (SEQ ID NO: 61), (3) two flexible binders, (4) a 3G C-terminal extension, and ( 5) a variant canine IgGD Fc.
[00329] [00329] Contiguous polypeptides can be designated with a signal sequence, a different GLP1 polypeptide (for example, SEQ ID NO: 85, 86, 87, 98, or 99), or different modifications to the IgG Fc of canine. Examples with such variations include ssGLP1-S8 / GLP1-2G_III_VARcaIgGD (SEQ ID NO: 41), ssGLP1-G8 / GLP1-2G_III_VARcaIgGD (SEQ ID NO: 42), ssGLP1- S8 / GLP1-3G_III_VARcaIg : 43), GLP1-S8 / GLP1- 2G_III_VARcaIgGD (SEQ ID NO: 105) and GLP1-G8 / GLP1- 2G_III_VARcaIgGD (SEQ ID NO: 106).
[00330] [00330] Furthermore, contiguous polypeptides of Formulas I, II and III can comprise a wild-type canine IgGD or a variant or wild-type canine IgGA, IgGB or IgGC, instead of a canine IgGD. For example, similar contiguous polypeptides can be designed and prepared having an IgGA Fc, IgGB Fc, IgGC Fc or wild type canine IgGD Fc (for example, SEQ ID NO: 1, 2, 3 or 4, respectively) ). Additional examples include contiguous polypeptides comprising a variant canine IgGA Fc (for example, SEQ ID NO: 5 or 60), a variant canine IgGB Fc (for example, SEQ ID NO: 78), or an Fc of variant canine IgGC (for example, SEQ ID NO: 6, 61, 79 or 84).
[00331] [00331] 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 supernatants containing the collected and filtered proteins. Both contiguous polypeptides were affinity purified by protein A chromatography. The SDS-PAGE profiles of the two contiguous polypeptides in the absence and presence of reducing agent (DTT) were compared and the results suggested that Fc disulfide binding was effectively formed for both polypeptides (data not shown). Example 8 Equine IgG Fc GLP1 and Fc Proteins
[00332] [00332] Several contiguous polypeptides of formula I, II and III comprising a variant GLP1 and an equine IgG Fc can be designated and prepared. For example, a variant canine IgG2 Fc (eg SEQ ID NO: 19, 71 or 72) can be chosen based on its low or no binding to C1q for reduced complementary activity and binding to protein A for ease of purification. In addition, a flexible, non-structural binder, such as a binder rich in glycine and / or serine, can be used.
[00333] [00333] GLP1-G8_I_VAReqIgG2 (SEQ ID NO: 34) and GLP1- S8_I_VAReqIgG2 (SEQ ID NO: 36) are examples of contiguous polypeptides of formula I comprising (1) whether GLP1-G8 or GLP1-S8,
[00334] [00334] Contiguous polypeptides can be designated with a signal sequence, a different GLP1 analog (for example, SEQ ID NO: 86, 87, 98 or 99), a glycine extension (for example, SEQ ID NO: 88 to 95) or additional modifications to the equine IgG Fc. Examples with such variations include ssGLP1- G8 / GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 44), ssGLP1- G8_I_VAReqIgG2 (SEQ ID NO: 45), ssGLP1-S8 / GLP1- 3G_III_VAReqIgG2 (SEQ ID NO: 46) S8_I_VAReqIgG2 (SEQ ID NO: 47).
[00335] [00335] Furthermore, contiguous polypeptides of Formulas I, II and III may comprise a wild type equine IgG2, or a wild type or variant IgG1, IgG3, IgG4, IgG5, IgG6, IgG7 Variant equine IgG2. For example, similar contiguous polypeptides can be designed and prepared having a wild-type equine IgG1 Fc (for example, SEQ ID NO: 63), IgG2 Fc (for example, SEQ ID NO: 18 or 64), Fc of IgG3 (for example, SEQ ID NO: 65), IgG4 Fc (for example, SEQ ID NO: 66), IgG5 Fc (for example, SEQ ID NO: 67), IgG6 Fc (for example, SEQ ID NO: 68), or IgG7 Fc (for example, SEQ ID NO: 69). Additional examples include contiguous polypeptides comprising a variant equine IgG1 Fc (for example, SEQ ID NO: 70), a variant equine IgG3 Fc (for example, SEQ ID NO: 73), a Fc of
[00336] [00336] 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 segregated in CHO cells. The supernatant containing the polypeptides contiguous after cleavage of the signal peptide (SEQ ID NOs: 103 and 104) was collected and filtered. Both proteins were affinity purified using a protein A column (CaptivA® Protein A activity resin, Repligen) were expressed separately in CHO cells and the supernatants containing the collected and filtered proteins. Both contiguous polypeptides were affinity purified by protein A chromatography. The SDS-PAGE profiles of the two contiguous polypeptides in the absence and presence of reducing agent (DTT) were compared and the results suggested that the binding of Fc disulfide was effectively formed for both polypeptides (data not shown). Example 9 Expression and purification of feline GLP1R N-terminal soluble domain
[00337] The mature feline GLP1R N-terminal domain (SEQ ID NO: 49) responsible for the binding of GLP1 to GLP1R was identified from the feline full-length GLP1R amino acid sequence (SEQ ID NO: 48). The nucleotide sequences encoding (1) a signal sequence, feline N-terminal GLP1R, human Fc and a poly-His marker (ssFeGLP1R-N-huFc_PolyHis; SEQ ID NO: 50) and
[00338] [00338] The binding affinity of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) to FeGLP1R-N-huFc_PolyHis was assessed using bi-layer interferometer (Octet). In summary, FeGLP1R-N-huFc_PolyHis was biotinylated, the unreacted free biotin was removed, and the bio-tinylated protein was captured at the tips of the streptavidin sensor. The association of different concentrations of GLP1-G8_I_VARfeIgG2 was monitored for ninety seconds. Dissociation was monitored for 600 seconds. A blank curve of the buffer is subtracted to correct any deviation. The data is adjusted for a 1: 1 connection model using ForteBio ™ data analysis software to determine kon, koff and Kd. The buffer for dilutions and all binding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd between feline GLP1R and GLP1-G8_I_VARfeIgG2 N-terminal domains was between 8.0x10-9 and 16x10-9 M. Example 11 Bioactivity of GLP1 fusion protein in a cell-based assay
[00339] [00339] The CHOK-1-GL1R cell line (Discoverx, cat # 95- 0062C2), an ovarian hamster cell line that overex-
[00340] [00340] By binding GLP1 to the GLP1R receptor coupled to GS, Gs stimulates adenylate cyclase to generate cAMP. At the end of the incubation, the cAMP antibody reagent and cAMP working detection solution, which contains lysis buffer, cAMPs attached to the small β-galactosidase fragment (β-gal) and substrates, were added the cells. The cells were incubated in the dark for 1 hour at room temperature to allow the immunocompetition reaction to occur between endogenously generated cAMPs and cAMPs conjugated to the small fragment of β-gal for binding to the cAMP antibody.
[00341] [00341] At the end of the 1-hour incubation, cAMP solution A containing large β-gal fragments, which can be complemented with small free β-gal fragment cAMPs (without binding to the antibody), to form functional enzymes , were added to the cell layer. The lysate was incubated for 3 to 6 hours in the dark at room temperature to allow β-gal to hydrolyze the substrate and generate luminescent signs.
[00342] [00342] The more small unbound free β-gal cAMPs remain, the more functional β-gal enzymes are formed. Therefore, the amount of signal produced is directly proportional to the amount of cAMP in the cell lysate. At the end of the incubation, the luminescence was read in a Synergy HT microplate reader (Biotek, Winooski, VT). EC50s were calculated with GraphPad Prism software (GraphPad Software, Inc. La Jolla, USA).
[00343] [00343] Figure 3 shows a graph of the relative light units (RLU) versus the concentration for a given tested sample. EC50s 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. Table 10. EC 50 (nM) sample GLP1 (7-37) 0.1449 Extendin-4 0.45596 GLP1-G8 / GLP1-2G_III_VARfeIgG2 0.01751 (SEQ ID NO: 25) GLP1-G8_I_VARfeIgG2 0.02806 (SEQ ID NO: 26) Example 12 Long-term stability of GLP1 fusion protein
[00344] [00344] GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored in PBS, pH7.2 at a concentration of 1.3 mg / ml, placed in a 1.5 ml Eppendorf tube and stored at 2-8 ° C for one year (Lot 2-29-2016). GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was also stored in PBS, pH7.2 at a concentration of 10 mg / mL, placed in a 1.5 ml Eppendorf tube, and stored at 2-8 ° C for one day (Lot 2-2-2017). To assess stability, the stored sample was analyzed by the cell-based assay using the same cell line CHOK-1-GL1R (Discoverx, cat # 95-0062C2) described in Example 11, but cell activity was assessed using an assay cAMP-Glo ™ Max (Promega, Cat # PAV1682).
[00345] [00345] CHOK-1-GL1R cells were plated on a 96-well plate (Corning, Cat # 3610) at a density of 20,000 cells per well in F-12K medium (ATCC, Cat # ATCC® 30-2004 ) supplemented with 10% fetal bovine serum, thermo-inactivated (Sigma, Cat # 2868) and incubated at 37 ° C, 5% CO2 for 24 hours. The cells were then stimulated with a human GLP1 control agonist (37 aa) (Prospec, Cat # HOR-236), Lot 2-20-2016 or Lot 2- 2-2017 in a series of 3-fold dilutions with free medium of serum followed by the addition of complete induction buffer which contains MgCl2 to a final concentration of 20 mM, isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich Cat. # I7018) to a final concentration of 500 μM and Ro 20- 1724 [4- (3-butoxy-4-methoxy-benzyl) imidazolidone] (Sigma Aldrich, Cat. # B8279) to a final concentration of 100 μM.
[00346] [00346] The cells were incubated at room temperature for 30 minutes. In this process, by binding GLP1 to the GLP1R receptor coupled to GS, Gs stimulates adenylate cyclase to generate cAMP. At the end of the incubation, the cAMP detection solution, which contains an inactive protein kinase A holoenzyme, protein kinase A substrate and lysis buffer, was added to the cells. The plates placed on an orbital shaker for 1–2 minutes and then incubated at room temperature (23 ° C) for 20 minutes. Cellular cAMP will activate protein kinase A by binding its regulatory inhibitor subunits and releasing the catalytic subunits. The free catalytic subunits catalyze the transfer of the terminal ATP phosphate to the protein kinase A substrate, consuming cellular ATP in the process. At the end of the incubation, a Kinase-Glo® reagent based on luciferase was added to the cell lysates and the plates were shaken on an orbital shaker for 2 min followed by incubation in the dark at room temperature for 10 min.
[00347] [00347] Luciferin mono-oxygenation was catalyzed by luciferase in the presence of Mg2 + and ATP that appeared in the cell lysate, resulting in a luminescent signal proportional to the amount of ATP in the cells. At the end of the 10 min incubation, the plate was read in a Synergy HT microplate reader (Biotek, Winooski, VT). The luminescence is proportional to the levels of ATP, but inversely proportional to the levels of cAMP. Thus, as the concentration of cAMP increases, luminescence decreases.
[00348] [00348] Figure 4 shows the results of the cell-based assay as a luminescence versus concentration graph for Lot 2-29-2016 compared to Lot 2-2-2017 and GLP1 (31 a.a.). The GLP1-G8_I_VARfeIgG2 sample maintained cellular activity after storage in PBS at 2-8 ° C for one year. Example 13 Stability of GLP1 fusion protein serum
[00349] [00349] GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored in PBS, pH7.2 with feline serum at 37 ° C for 24 hours to test the stability of the serum in vitro. The cell-based assay was performed as described in Example 12 and the results suggested that the activity was maintained (data not shown). In addition, no visible degradation was observed by Western blot analysis (Figure 5). Example 14 Pharmacokinetics of GLP1 fusion protein in vivo
[00350] [00350] GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was administered as a single dose (2 mg / kg) via subcutaneous injection to 5 cats. Serum samples were taken before dosing time (time 0) and in 4 hours, 8 hours, 12 hours, 24 hours, 48 hours,
[00351] [00351] The quantitative ELISA used an anti-GLP1 antibody (4F3, Novus Biologicals, Catalog No. NBP1-97413) and a conjugated IgG-Fc antibody, goat anti-cat HRP (Bethyl Laboratories, Inc., Catalog No. A20-117P) for quantification of GLP1-G8_I_VARfeIgG2 in feline serum samples from the in vivo pharmacokinetic study. A 96-well plate was coated with an anti-GLP1 antibody (5 μg / mL in coating buffer, 100 μl / well). The plate was sealed and incubated overnight at 4 ° C. The plate was washed in triplicate with 1X TBST (10X TBST, Teknova, Catalog No. T9511) and the blocking buffer was added. After removing the blocking buffer, serial dilutions of reference standard and samples in the blocking buffer were added (100 μl / well) and the plate was incubated for 2 hours at room temperature. The plate was washed in triplicate with 1X TBST and goat anti-cat IgG Fc antibody was added (0.1 μg / mL in blocking buffer, 100 μl / well). After incubation for 1 hour at room temperature, the plate was washed 5 times with 1X TBST. The TMB substrate (ScyTek, Catalog No. TM1999) was added (100 μl / well) and allowed to incubate at room temperature for 1 minute. The reaction was stopped by adding 2 M H2SO4 (50 μl / well). The absorbance at 450 nm was measured and the concentration of GLP1- G8_I_ VARfeIgG2 in the serum samples calculated.
[00352] [00352] Furthermore, the concentration of GLP1-G8_I_VARfeIgG2 in the same serum samples (without added DPP-4 inhibitor) was assessed using a cell-based activity assay. The same CHOK-1-GL1R cell line (Discoverx, cat # 95-0062C2) and the cAMP-Glo ™ Max assay (Promega, Cat # PAV1682) described in Example 12 were used. In this example, however, the cells were stimulated with a human GLP1 control agonist (37 aa) (Prospec, Cat # HOR-236) or cat serum samples taken before dosing (time 0) and at 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours and 168 hours diluted in serum-free medium (dilutions of 5%; 0.5% and 0.05%). The concentration of GLP1-G8_I_VARfeIgG2 in each sample was calculated using SoftMax pro 7 (Molecular Devices, Sunnyvale, CA). The average concentration of GLP1-G8_I_VARfeIgG2 for the 5 cats was plotted against time (Figure 7). The mean AUC was about 840 μg (h) / mL and the mean t1 / 2 was 36 hours. The concentrations calculated from the cell-based activity assay are consistent with the concentrations obtained from ELISA, which suggests that the GLP1 variant detected by ELISA is biologically active. Example 15 GLP1, glucagon and IgG Fc fusion proteins
[00353] [00353] To investigate a long-acting GLP1 receptor and glucagon receptor double agonist, contiguous polypeptides comprising a GLP1 polypeptide, a glucagon polypeptide, and an IgG Fc polypeptide having the following constructs have been designated : Formula (IV): GLP1 — L1 — Fc — L2 — Gluc; and Formula (V): Gluc — L1 — Fc — L2 — GLP1, where GLP1 is a GLP1 polypeptide, Gluc is a glucagon polypeptide, L1 and L2 are ligands, and Fc is an IgG Fc polypeptide.
[00354] [00354] As discussed above, GLP1 was modified to be resistant to DPP-4 by replacing alanine with either glycine or serine in a position corresponding to position 8 of GLP1 of the wild type (7-37) (SEQ ID NO: 85). In addition, DPP-4 resistant GLP1 was further modified by removing the two C-terminal amino acids to generate variant GLP1-S8 (7-35) polypeptides (SEQ ID NO: 86) and variant GLP1-G8 (7-35 ) (SEQ ID NO: 87).
[00355] [00355] GLP1 polypeptides when positioned at the C-terminal of a construct, as in Formula (V), are not susceptible to DPP-4 degradation. Therefore, substitution of alanine to glycine or serine is not necessary for GLP1 polypeptides positioned at the C-terminus. Consequently, wild type GLP1 (7-37) (SEQ ID NO: 85) can be used at the C-terminal.
[00356] [00356] The binder can be a flexible, non-structural binder, such as a binder rich in glycine and serine. A flexible extension can be added to the C-terminal of the contiguous polypeptide. The extension may comprise a 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.
[00357] The contiguous polypeptide can comprise a wild-type glucagon polypeptide (e.g., SEQ ID NO: 21) or a variant glucagon polypeptide.
[00358] The contiguous polypeptide may comprise a human IgG Fc or an IgG Fc from a species of companion animal, such as canine, feline or equine. The IgG Fc subtype used may be based on having low or no C1q binding activity and / or having protein A binding capacity. For example, a human, canine, equine or feline IgG Fc of wild type or variant having low or no C1q binding and / or endo protein A binding capacity can be used (for example, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 , 77, 78, 79, 80, 81, 82, 83 or 84).
[00359] Exemplary contiguous polypeptides comprising a GLP1 polypeptide, a glucagon polypeptide, and a feline IgG Fc polypeptide include GLP1-G8 / Gluc-3G_IV_WTfeIgG2 (SEQ ID NO: 52) and Gluc / GLP1-2GIG_V_W (TW SEQ ID NO: 53).
[00360] Exemplary contiguous polypeptides comprising a GLP1 polypeptide, a glucagon polypeptide, and a canine IgG Fc polypeptide include GLP1-G8 / Gluc-4G_IV_VARcaIgGD (SEQ ID NO: 54) and Gluc / GLP1-3GIG_VDAR (SEQ ID NO: 54) SEQ ID NO: 55).
[00361] Exemplary contiguous polypeptides comprising a GLP1 polypeptide, a glucagon polypeptide, and an equine IgG Fc polypeptide include GLP1-G8 / Gluc-4G_IV_VAReqIgGD (SEQ ID NO: 56) and Gluc / GLP1-3GIG_V_ SEQ ID NO: 57).
[00362] Exemplary contiguous polypeptides comprising a GLP1 polypeptide, a glucagon polypeptide, and a human IgG Fc polypeptide include GLP1-G8 / Gluc-4G_IV_huIgG4 (SEQ ID NO: 8) and Gluc / GLP1-3G_V_huIgG4 (SEQ ID NO: 8) ID NO: 59). Example 16 Variant IgG Fc polypeptides for formation of enlarged hinge disulfide
[00363] [00363] Analysis of the additional three-dimensional protein modeling of various orthologous hinge structures was used to modify feline and equine IgG hinges to increase disulfide formation. To increase the disulfide formation in the feline IgG hinge, the hinge sequence can be modified by replacing lysine with proline in a position corresponding to the position 16 of feline IgG2 (SEQ ID NO: 16), of feline IgG1a (SEQ ID NO: 80 or SEQ ID NO: 117), or feline IgG1b (SEQ ID NO: 81 or SEQ ID NO: 118) (e.g., K16P). Examples of amino acid sequences of variant feline IgG polypeptides having a modified hinge include SEQ ID NO: 125, SEQ ID NO: 126 and SEQ ID NO: 127.
[00364] [00364] To increase disulfide formation in the equine IgG hinge, the hinge sequence can be modified by replacing cysteine with serine in a position corresponding to position 3 of an equine IgG (for example, Fc de IgG2 (SEQ ID NO: 129)) and / or substitution of glutamine with proline in a position corresponding to position 20 of an equine IgG (for example, IgG2 Fc (SEQ ID NO: 129) (for example, C3S , Q20P). Examples of amino acid sequences of variant equine IgG polypeptides having a modified hinge include SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 134 and SEQ ID NO : 135. Example 17 Variant IgG Fc Polypeptides for increased recombinant production and / or increased hinge disulfide formation
[00365] [00365] Three-dimensional protein modeling was used to produce feline and equine variant IgG Fc polypeptides comprising sequences from the hinge region of a different IgG isotype for increased recombinant production and improved hinge disulfide formation. Variant feline IgG2 Fc polypeptides can be prepared which comprise the sequences from the hinge region of IgG1a or
[00366] [00366] Levels of recombinant production of variant IgG Fc polypeptides and / or levels of hinge disulfide formation can be determined and compared to that of another IgG Fc through SDS-PAGE analysis under reduction conditions and not reduction (for example, the corresponding wild-type IgG Fc of the same or different isotype, or a wild-type IgG Fc or variant of another companion animal, etc.).

权利要求:
Claims (127)
[1]
1. Polypeptide, characterized by the fact that it comprises a variant IgG Fc polypeptide comprising at least one amino acid modification relative to a wild-type IgG Fc polypeptide of a species of companion animal, wherein the variant IgG Fc polypeptide has increased binding affinity to protein A relative to the wild-type IgG Fc polypeptide.
[2]
2. Polypeptide, characterized by the fact that it comprises a variant IgG Fc polypeptide comprising at least one amino acid modification relative to a wild type IgG Fc polypeptide of a species of companion animal, wherein the variant IgG Fc polypeptide has reduced binding affinity to C1q and / or CD16 relative to the wild-type IgG Fc polypeptide.
[3]
3. Polypeptide according to claim 1 or r 2, characterized by the fact that the variant IgG Fc polypeptide binds to C1q and / or CD16 with a dissociation constant (Kd) greater than 5 x 10-6 M, greater than 1 x 10-5 M, greater than 5 x 10-5 M, greater than 1 x 10-4 M, greater than 5 x 10-4 M, or greater than 1 x 10- 3 M, as measured by bi-layer interferometry.
[4]
4. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide has increased binding affinity to protein A relative to the wild-type IgG Fc polypeptide.
[5]
5. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide binds to protein A with a dissociation constant (Kd) less than 5 x 10-6 M, less than 1 x 10-6 M, less than 5 x 10-7 M, less than 1 x 10-7 M, less than 5 x 10-8 M, less than 1 x 10-8 M, less than 5 x 10-9 M, less than 1 x 10-9 M, less than 5 x 10-10 M, less than 1 x 10-10 M, less than 5 x 10-11 M, less than 1 x 10-11 M, less than 5 x 10-12 M, or less than 1 x 10-12 M, as measured by bi-layer interferometry.
[6]
6. Polypeptide according to any of the preceding claims, characterized by the fact that the species of companion animal is canine, feline or equine.
[7]
7. Polypeptide according to any one of the preceding claims, characterized in that the wild-type IgG Fc polypeptide is a) an IgG-A Fc, IgG-B Fc, IgG-C Fc or Fc canine IgG-D; b) an IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc or equine IgG7 Fc c) an IgG1a Fc, IgG1b Fc, feline IgG2 Fc
[8]
8. Polypeptide, characterized by the fact that it comprises a variant IgG Fc polypeptide comprising at least one amino acid modification to a hinge region relative to a wild-type feline or equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide has increased recombinant production and / or formation of increased hinge dissulfer relative to the wild-type IgG Fc polypeptide, as determined by SDS-PAGE analysis under conditions of reduction and / or non-reduction.
[9]
9. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution in a 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 relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 3 of SEQ ID NO: 129; and / or c) at least one amino acid substitution relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 20 of SEQ ID NO :
129.
[10]
10. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises 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 relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 3 of SEQ ID NO: 129; and / or c) at least one amino acid substitution relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises an amino acid substitution at position 20 of SEQ ID NO: 129.
[11]
11. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide comprises:
a) at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a proline in a position corresponding to position 16 or in 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 relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a serine in a position corresponding to position 3 or position 3 of SEQ ID NO: 129; and / or c) at least one amino acid substitution relative to a wild-type equine IgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises a proline in a position corresponding to position 20 or position 20 of SEQ ID NO : 129.
[12]
12. Polypeptide according to any of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region from an IgG Fc polypeptide of a different isotype .
[13]
13. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises a hinge region or a portion of a hinge region from a feline IgG-1a Fc polypeptide wild-type, a wild-type feline IgG-1b Fc polypeptide or a wild-type equine IgG1 Fc polypeptide.
[14]
14. Polypeptide according to any one of the preceding claims, characterized in that the variant 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: 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]
15. Polypeptide, characterized by the fact that it comprises an 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 : 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]
16. Polypeptide, characterized by the fact that it comprises a variant IgG2 Fc polypeptide comprising at least one amino acid substitution relative to a wild-type feline IgG2 Fc polypeptide, where at least one amino acid substitution is a cysteine, and wherein the variant IgG2 Fc polypeptide is capable of forming at least one additional interchain disulfide bond relative to the wild-type feline IgG2 Fc polypeptide.
[17]
17. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises at least one amino acid substitution relative to a wild-type feline IgG Fc polypeptide, in that at least one amino acid substitution is a cysteine, and wherein the variant IgG Fc polypeptide is capable of forming at least one additional interchain disulfide bond relative to the wild-type feline IgG Fc polypeptide.
[18]
18. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises a cysteine in a position corresponding to position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, or position 16 of SEQ ID NO:
16.
[19]
19. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide comprises a cysteine in a position corresponding to position 14 of SEQ ID NO: 16.
[20]
20. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises a cysteine at position 14 of SEQ ID NO: 16.
[21]
21. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide is at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO : 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO : 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO : 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118 , SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, 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]
22. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises SEQ ID NO: 17.
[23]
23. Polypeptide, characterized by the fact that it comprises an amino acid sequence of SEQ ID NO: 17.
[24]
24. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide comprises: a) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 1, a amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 25 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 80 from SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 205 of SEQ ID NO: 1, and / or an amino acid substitution in a position corresponding to position 207 of SEQ ID NO: 1; b) an amino acid substitution in one position corresponding to position 21 of SEQ ID NO: 3, an amino acid substitution in one position corresponding to position 23 of SEQ ID NO: 3, and / or an amino acid substitution in one position corresponding to position 24 of SEQ ID NO: 3;
c) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to to position 25 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 80 of SEQ ID NO: 4, and / or an amino acid substitution in a position corresponding to position 207 of SEQ ID NO: : 4; d) an amino acid substitution in a position corresponding to position 15 of SEQ ID NO: 64, and / or an amino acid substitution in a position corresponding to position 203 of SEQ ID NO: 64; e) an amino acid substitution in a position corresponding to position 199 of SEQ ID NO: 67, and / or an amino acid substitution in a position corresponding to position 200 of SEQ ID NO: 67; and / or f) an amino acid substitution in one position corresponding to position 199 of SEQ ID NO: 68, an amino acid substitution in one position corresponding to position 200 of SEQ ID NO: 68, an amino acid substitution in one position corresponding to position 201 of SEQ ID NO: 68, and / or an amino acid substitution in a position corresponding to position 202 of SEQ ID NO: 68.
[25]
25. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acid substitution at position 23 of SEQ ID NO: 1, an amino acid substitution at position 25 of SEQ ID NO: 1, an amino acid substitution at position 80 of SEQ ID NO:
1, an amino acid substitution at position 205 of SEQ ID NO: 1, and / or an amino acid substitution at position 207 of SEQ ID NO: 1; b) an amino acid substitution at position 21 of SEQ ID NO: 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; c) an amino acid substitution at position 21 of SEQ ID NO: 4, an amino acid substitution at position 23 of SEQ ID NO: 4, an amino acid substitution at position 25 of SEQ ID NO: 4, an amino acid substitution at position 80 of SEQ ID NO: 4, and / or an amino acid substitution at position 207 of SEQ ID NO: 4; d) an amino acid substitution at position 15 of SEQ ID NO: 64, and / or an 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) an amino acid substitution at position 199 of SEQ ID NO: 68, an amino acid substitution at position 200 of SEQ ID NO: 68, an amino acid substitution at position 201 of SEQ ID NO: 68, and / or an amino acid substitution at position 202 of SEQ ID NO: 68.
[26]
26. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) a threonine in a position corresponding to position 21 of SEQ ID NO: 1, a leucine in a position corresponding to position 23 of SEQ ID NO: 1, an alanine in a position corresponding to position 25 of SEQ ID NO: 1, a glycine in a position corresponding to position 80 of SEQ ID NO: 1, an alanine in a position corresponding to position 205 of SEQ ID NO: 1, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 1; b) a threonine in a position corresponding to position 21 of SEQ ID NO: 3, a leucine in a position corresponding to position 23 of SEQ ID NO: 3, and / or an isoleucine in a position corresponding to position 24 of SEQ ID NO: 3; c) a threonine in a position corresponding to position 21 of SEQ ID NO: 4, a leucine in a position corresponding to position 23 of SEQ ID NO: 4, an alanine in a position corresponding to position 25 of SEQ ID NO: 4, a glycine in a position corresponding to position 80 of SEQ ID NO: 4, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 4; d) a threonine or a valine in a position corresponding to position 15 of SEQ ID NO: 64, and / or a tyrosine or a valine in a position corresponding to position 203 of SEQ ID NO: 64; e) a leucine in a position corresponding to position 199 of SEQ ID NO: 67, and / or a histidine in a position corresponding to position 200 of SEQ ID NO: 67; and / or f) a leucine in a position corresponding to position 199 of SEQ ID NO: 68, a histidine in a position corresponding to position 200 of SEQ ID NO: 68, an asparagine in a position corresponding to position 201 of SEQ ID NO: 68, and / or a histidine in a position corresponding to position 202 of SEQ ID NO: 68.
[27]
27. Polypeptide according to any one of the claims
preceding statements, characterized by the fact that the variant IgG Fc polypeptide comprises: a) a threonine at position 21 of SEQ ID NO: 1, a leucine at position 23 of SEQ ID NO: 1, an alanine at position 25 of SEQ ID NO: 1, a glycine at position 80 of SEQ ID NO: 1, an aline at position 205 of SEQ ID NO: 1, and / or a histidine at position 207 of SEQ ID NO: 1; b) a threonine at position 21 of SEQ ID NO: 3, a leucine at position 23 of SEQ ID NO: 3, and / or an isoleucine at position 24 of SEQ ID NO: 3; c) a threonine at position 21 of SEQ ID NO: 4, a leucine at position 23 of SEQ ID NO: 4, an alanine at position 25 of SEQ ID NO: 4, a glycine at position 80 of SEQ ID NO: 4 , and / or a histidine at position 207 of SEQ ID NO: 4; d) a threonine or valine at position 15 of SEQ ID NO: 64, and / or a tyrosine or valine at position 203 of SEQ ID NO: 64; e) a leucine at position 199 of SEQ ID NO: 67, and / or a histidine at position 200 of SEQ ID NO: 67; and / or f) a leucine at position 199 of SEQ ID NO: 68, a histidine at position 200 of SEQ ID NO: 68, an asparagine at position 201 of SEQ ID NO: 68, and / or a histidine at position 202 of SEQ ID NO: 68.
[28]
28. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises an amino acid sequence of: a) 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; or b) SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQ ID NO: 76.
[29]
29. Polypeptide, characterized by the fact that it comprises an 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]
30. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) an amino acid substitution in a position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 3; b) an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 63, an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 65, an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 66, or an amino acid substitution in a position corresponding to position 87 of SEQ ID NO: 69; or c) an amino acid substitution in a position corresponding to position 198 of SEQ ID NO: 80, or an amino acid substitution in a position corresponding to position 198 of SEQ ID NO: 81.
[31]
31. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) an amino acid substitution at position 93 of SEQ ID NO: 2, or an amino acid substitution at position 93 of SEQ ID NO: 3; b) an amino acid substitution at position 87 of the
SEQ ID NO: 63, an amino acid substitution at position 87 of SEQ ID NO: 65, an amino acid substitution at position 87 of SEQ ID NO: 66, or an amino acid substitution at position 87 of SEQ ID NO: 69; or c) an amino acid substitution at position 198 of SEQ ID NO: 80, or an amino acid substitution at position 198 of SEQ ID NO: 81.
[32]
32. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) an arginine in a position corresponding to position 93 of SEQ ID NO: 2, or an arginine in a position corresponding to position 93 of SEQ ID NO: 3; b) a serine in a position corresponding to position 87 of SEQ ID NO: 63, a serine substitution in a position corresponding to position 87 of SEQ ID NO: 65, a serine in a position corresponding to position 87 of SEQ ID NO: 66, or a serine in a position corresponding to position 87 of SEQ ID NO: 69; or c) an alanine in a position corresponding to position 198 of SEQ ID NO: 80, or an alanine in a position corresponding to position 198 of SEQ ID NO: 81.
[33]
33. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) an arginine at position 93 of SEQ ID NO: 2, or an arginine at position 93 of SEQ ID NO: 3; b) a serine at position 87 of SEQ ID NO: 63, a serine at position 87 of SEQ ID NO: 65, a serine at position 87 of SEQ ID NO: 66, or a serine at position 87 of SEQ ID NO: 69 ; or c) an alanine at position 198 of SEQ ID NO: 80, or alanine at position 198 of SEQ ID NO: 81.
[34]
34. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises the amino acid sequence of: a) SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO : 84; or b) SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 77; or c) SEQ ID NO: 82 or SEQ ID NO: 83.
[35]
35. Polypeptide, characterized by the fact that it comprises an 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]
36. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide comprises: a) an amino acid substitution in a position corresponding to position 5 of SEQ ID NO: 2, a amino acid substitution in one position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution in one position corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution in a position corresponding to position 98 of SEQ ID NO: 2; or b) an amino acid substitution in a position corresponding to position 5 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 38 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 39 of SEQ ID NO: 3, a substitution of
noacid in a position corresponding to position 97 of SEQ ID NO: 3, and / or an amino acid substitution in a position corresponding to position 98 of SEQ ID NO: 3.
[37]
37. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) an amino acid substitution at position 5 of SEQ ID NO: 2, an amino acid substitution at position 38 of SEQ ID NO: 2, an amino acid substitution at position 39 of SEQ ID NO: 2, an amino acid substitution at position 97 of SEQ ID NO: 2, and / or an amino acid substitution at position 98 of SEQ ID NO: 2; or b) an amino acid substitution at position 5 of SEQ ID NO: 3, an amino acid substitution at position 38 of SEQ ID NO: 3, an amino acid substitution at position 39 of SEQ ID NO: 3, an amino acid substitution at position 97 of SEQ ID NO: 3, and / or an amino acid substitution in position 98 of SEQ ID NO: 3.
[38]
38. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) a proline in a position corresponding to position 5 of SEQ ID NO: 2, a glycine in a position corresponding to position 38 of SEQ ID NO: 2, an arginine in a position corresponding to position 39 of SEQ ID NO: 2, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 2, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 2; or b) a proline in a position corresponding to position 5 of SEQ ID NO: 3, a glycine in a position corresponding to position 38 of SEQ ID NO: 3, an arginine in a position corresponding to position 39 of SEQ ID NO: : 3, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 3, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 3.
[39]
39. Polypeptide according to any one of the preceding claims, characterized by the fact that the variant IgG Fc polypeptide comprises: a) a proline at position 5 of SEQ ID NO: 2, a glycine at position 38 of SEQ ID NO: 2, an arginine at position 39 of SEQ ID NO: 2, an isoleucine at position 97 of SEQ ID NO: 2, and / or a glycine at position 98 of SEQ ID NO: 2; or b) a proline at position 5 of SEQ ID NO: 3, a glycine at position 38 of SEQ ID NO: 3, an arginine at position 39 of SEQ ID NO: 3, an isoleucine at position 97 of SEQ ID NO: : 3, and / or a glycine at position 98 of SEQ ID NO: 3.
[40]
40. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises an amino acid sequence of: a) 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, or SEQ ID NO: 147; or b) 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.
[41]
41. Polypeptide, characterized by the fact that it comprises an 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]
42. Polypeptide, characterized by the fact that it comprises a variant IgG Fc polypeptide comprising: a) a tyrosine or a tryptophan in a position corresponding to position 138 of SEQ ID NO: 1, a tyrosine or a tryptophan in the position corresponding to position 137 of SEQ ID NO: 2, a tyrosine or a tryptophan in a position corresponding to position 137 of SEQ ID NO: 3, or a tyrosine or a tryptophan in a position corresponding to position 138 of SEQ ID NO: 4; or b) a tyrosine or a tryptophan in a position corresponding to position 154 of SEQ ID NO: 16, a tyrosine or a tryptophan in a position corresponding to position 154 of SEQ ID NO: 80 or SEQ ID NO: 117 , or tyrosine or tryptophan in a position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO: 118.
[43]
43. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) a tyrosine or a tryptophan at position 138 of SEQ ID NO: 1, a tyrosine or a tryptophan in the position 137 of SEQ ID NO: 2, a tyrosine or a tryptophan in position 137 of SEQ ID NO: 3, or a tyrosine or a tryptophan in position 138 of SEQ ID NO: 4; or b) a tyrosine or a tryptophan at position 154 of SEQ ID NO: 16, a tyrosine or a tryptophan at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a tyrosine or a tryptophan in a position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO: 118.
[44]
44. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises an 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]
45. Polypeptide, characterized by the fact that it comprises an 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]
46. Contiguous polypeptide, characterized by the fact that it comprises the polypeptide according to any one of the preceding claims and a glucagon-like peptide-1 polypeptide (GLP1).
[47]
47. Contiguous polypeptide, characterized by the fact that it comprises the polypeptide according to any one of the preceding claims and a glucagon polypeptide.
[48]
48. Polypeptide, characterized by the fact that it comprises a variant IgG Fc polypeptide comprising: a) a serine in a position corresponding to position 138 of SEQ ID NO: 1, a serine in a position corresponding to position 137 of SEQ ID NO: 2, a serine in a position corresponding to position 137 of SEQ ID NO: 3, a serine in a position corresponding to position 138 of SEQ ID NO: 4, a serine in a position corresponding to position 154 of SEQ ID NO: 16, a serine in a position corresponding to position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a serine in a position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; b) an alanine in a position corresponding to position 140 of SEQ ID NO: 1, an alanine in a position corresponding to position 139 of SEQ ID NO: 2, an alanine in a position corresponding to position 139 of SEQ ID NO: 3, an alanine in a position corresponding to position 140 of SEQ ID NO: 4, an alanine in a position corresponding to position 156 of SEQ ID NO: 16, an alanine in a position corresponding to position 156 of SEQ ID NO: 80 or SEQ ID NO: 117, or an alanine in a position corresponding to position 156 of SEQ ID NO: 81 or SEQ ID NO: 118; and / or c) a threonine in a position corresponding to position 181 of SEQ ID NO: 1, a threonine in a position corresponding to position 180 of SEQ ID NO: 2, a threonine in a position corresponding to position 180 of SEQ ID NO: 3, a threonine in a position corresponding to position 181 of SEQ ID NO: 4, a threonine in a position corresponding to position 197 of SEQ ID NO: 16, a threonine in a position corresponding to position 197 of SEQ ID NO: 80 or SEQ ID NO: 117, or a threonine in a position corresponding to position 197 of SEQ ID NO: 81 or SEQ ID NO: 118.
[49]
49. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises: a) a serine at position 138 of SEQ ID NO: 1, a serine at position 137 of SEQ ID NO : 2, a serine at position 137 of SEQ ID NO: 3, a serine at position 138 of SEQ ID NO: 4, a serine at position 154 of SEQ ID NO: 16, a serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; b) an alanine at position 140 of SEQ ID NO: 1, an alanine at position 139 of SEQ ID NO: 2, an alanine at position 139 of SEQ ID NO: 3, an alanine at position 140 of SEQ ID NO: 4, an alanine at position 156 of SEQ ID NO: 16, an alanine at position
156 of SEQ ID NO: 80 or SEQ ID NO: 117, or an alanine at position 156 of SEQ ID NO: 81 or SEQ ID NO: 118; and / or; c) a threonine at position 181 of SEQ ID NO: 1, a threonine at position 181 of SEQ ID NO: 2, a threonine at position 181 of SEQ ID NO: 3, a threonine at position 181 of SEQ ID NO: 4, a threonine at position 197 of SEQ ID NO: 16, a threonine at position 197 of SEQ ID NO: 80 or SEQ ID NO: 117, or a threonine at position 197 of SEQ ID NO: 81 or SEQ ID NO: 118.
[50]
50. Polypeptide according to any one of the preceding claims, characterized in that the variant IgG Fc polypeptide comprises an 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]
51. Polypeptide, characterized by the fact that it comprises an 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]
52. Polypeptide according to any one of the preceding claims, characterized by the fact that the polypeptide is glycosylated.
[53]
53. Polypeptide according to any one of claims 1 to 51, characterized by the fact that the polypeptide is aglycosylated.
[54]
54. Contiguous polypeptide, characterized in that it comprises the polypeptide according to any one of claims 48 to 53 and a glucagon-like peptide-1 polypeptide (GLP1).
[55]
55. Contiguous polypeptide, characterized in that it comprises the polypeptide as defined in any of claims 48 to 53 and a glucagon polypeptide.
[56]
56. Heterodimeric protein, characterized by the fact that it comprises the contiguous polypeptide as defined in claim 46 and the contiguous polypeptide as defined in claim 54.
[57]
57. Heterodimeric protein, characterized by the fact that it comprises the contiguous polypeptide as defined in claim 47 and the contiguous polypeptide as defined in claim 55.
[58]
58. Contiguous polypeptide or heterodimeric protein according to any of claims 46, 47 or 54 to 57, characterized in that the GLP1 polypeptide is a wild-type GLP1 polypeptide, optionally comprising the amino acid sequence SEQ ID NO: 85.
[59]
59. Contiguous polypeptide or heterodimeric protein according to any one of claims 46, 47 or 54 to 58, characterized in that the GLP1 polypeptide is a variant GLP1 polypeptide.
[60]
60. Contiguous polypeptide or heterodimeric protein according to any one of claims 46, 47 or 54 to 59, characterized in that 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]
61. Contiguous polypeptide or heterodimeric protein according to any one of claims 46, 47 or 54 to 60, characterized in that the glucagon polypeptide is a wild-type glucagon polypeptide, optionally comprising the amino acid sequence SEQ ID NO: 21.
[62]
62. Contiguous polypeptide or heterodimeric protein according to any one of claims 46, 47 or 54 to 61, characterized in that the glucagon polypeptide is a polypeptide.
variant glucagon peptide.
[63]
63. Heterodimeric protein, characterized by the fact that it comprises: i) a first variant canine IgG Fc polypeptide comprising at least one amino acid modification relative to a first canine IgG wild type Fc polypeptide and a second variant canine IgG Fc polypeptide comprising at least one amino acid modification relative to a second canine wild-type IgG Fc polypeptide; or ii) a first variant feline IgG Fc polypeptide comprising at least one amino acid modification relative to a first feline IgG wild type Fc polypeptide and a second variant feline IgG Fc polypeptide comprising at least one amino acid modification relative to a second wild-type feline IgG Fc polypeptide, in which: a) the first variant canine IgG Fc polypeptide comprises a substitution of the amino acid in a 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 variant canine IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 138, position 140, and / or position 181 of SEQ ID NO: 1, position 137, position 139, and / or position 180 of SEQ ID NO: 2, position 137, position 139, and / or position 180 of SEQ ID NO: 3, or position 138, position 140, and / or position 181 of SEQ ID NO: 4; c) the first variant feline IgG Fc polypeptide comprises a amino acid substitution in a 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 variant feline IgG Fc polypeptide comprises an amino acid substitution in a position corresponding to position 154, position 156, and / or position 197 of SEQ ID NO: 6, of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.
[64]
64. Heterodimeric protein according to claim 63, characterized in that 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 polypeptide wild-type feline IgG Fc and the second wild-type feline IgG Fc polypeptide are of the same IgG subtype.
[65]
65. Heterodimeric protein according to claim 63, characterized in that the first wild-type canine IgG Fc polypeptide and the second wild-type canine IgG Fc polypeptide are of a different IgG subtype and / or the first polypeptide Wild type feline IgG Fc and the second wild type feline IgG Fc polypeptide are of the same IgG subtype.
[66]
66. Heterodimeric protein according to any one of claims 63 to 65, characterized in that: a) the first variant canine IgG Fc polypeptide comprises a tyrosine or tryptophan in a 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 variant feline IgG Fc polypeptide comprises a tryptophan in a 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.
[67]
67. Heterodimeric protein according to any one of claims 63 to 66, characterized by the fact that:
a) the second variant canine IgG Fc polypeptide comprises a serine in a position corresponding to position 138, an alanine in a position corresponding to position 140, and / or a threonine in a position corresponding to position 181 of SEQ ID NO: 1, a serine in a position corresponding to position 137, an alanine in a position corresponding to position 139, and / or a threonine in a position corresponding to position 180 of SEQ ID NO: 2, a serine in a position corresponding to position 137, a alanine in a position corresponding to position 139, and / or a threonine in a position corresponding to position 180 of SEQ ID NO: 3, and / or a serine in a position corresponding to position 138, an alanine in a position corresponding to position 140 , and / or a threonine in a position corresponding to position 181 of SEQ ID NO: 4; and / or b) the second variant feline IgG Fc polypeptide comprises a serine in a position corresponding to position 154, an alanine in a position corresponding to position 156, and / or a threonine in a position corresponding to position 197 of SEQ ID NO: 6, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.
[68]
68. Heterodimeric protein according to any one of claims 63 to 67, characterized in that: a) the first variant canine IgG Fc polypeptide comprises an 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 variant feline IgG Fc polypeptide comprises an amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123.
[69]
69. Heterodimeric protein according to any one of claims 63 to 68, characterized in that: a) the second variant canine IgG Fc polypeptide comprises an 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 variant feline IgG Fc polypeptide comprises an amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 123.
[70]
70. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 42 to 69, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises at least one additional amino acid modification relative to a wild-type IgG Fc polypeptide and has increased binding affinity to protein A relative to the wild-type IgG Fc polypeptide.
[71]
71. Polypeptide, contiguous polypeptide or heterodimeric protein according to any of claims 42 to 70, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises: a) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 25 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 80 of SEQ ID NO: 1, an amino acid substitution in a position corresponding to position 205 of SEQ ID NO: 1, and / or an amino acid substitution in a position corresponding to position 207 of SEQ
ID NO: 1; b) an amino acid substitution in one position corresponding to position 21 of SEQ ID NO: 3, an amino acid substitution in one position corresponding to position 23 of SEQ ID NO: 3, and / or an amino acid substitution in one position corresponding to position 24 of SEQ ID NO: 3; or c) an amino acid substitution in a position corresponding to position 21 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 23 of SEQ ID NO: 4, an amino acid substitution in a correct position corresponding to position 25 of SEQ ID NO: 4, an amino acid substitution in a position corresponding to position 80 of SEQ ID NO: 4, and / or an amino acid substitution in a position corresponding to position 207 of SEQ ID NO: 4.
[72]
72. Polypeptide, contiguous polypeptide or heterodimeric protein according to any of claims 42 to 71, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises: a) an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acid substitution at position 23 of SEQ ID NO: 1, an amino acid substitution at position 25 of SEQ ID NO: 1, an amino acid substitution at position 80 of SEQ ID NO: 1, an amino acid substitution at position 205 of SEQ ID NO: 1, and / or an amino acid substitution at position 207 of SEQ ID NO: 1; b) an amino acid substitution at position 21 of SEQ ID NO: 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) an amino acid substitution at position 21 of SEQ ID NO: 4, an amino acid substitution at position 23 of SEQ ID NO: 4, an amino acid substitution at position 25 of SEQ ID NO: 4, an amino acid substitution at position 80 of SEQ ID NO: 4, and / or an amino acid substitution at position 207 of SEQ ID NO: 4.
[73]
73. Polypeptide, contiguous polypeptide or hetero-dimeric protein according to any of claims 42 to 72, characterized in that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide of Variant IgG comprises: a) a threonine in a position corresponding to position 21 of SEQ ID NO: 1, a leucine in a position corresponding to position 23 of SEQ ID NO: 1, an alanine in a position corresponding to position 25 of SEQ ID NO: 1, a glycine in a position corresponding to position 80 of SEQ ID NO: 1, an alanine in a position corresponding to position 205 of SEQ ID NO: 1, and / or a histidine in a position corresponding to position 207 SEQ ID NO: 1; b) a threonine in a position corresponding to position 21 of SEQ ID NO: 3, a leucine in a position corresponding to position 23 of SEQ ID NO: 3, and / or an isoleucine in a position corresponding to position 24 of SEQ ID NO: 3; or c) a threonine in a position corresponding to position 21 of SEQ ID NO: 4, a leucine in a position corresponding to position 23 of SEQ ID NO: 4, an alanine in a position corresponding to position 25 of SEQ ID NO: 4 : 4, a glycine in a position corresponding to position 80 of SEQ ID NO: 4, and / or a histidine in a position corresponding to position 207 of SEQ ID NO: 4.
[74]
74. Polypeptide, contiguous polypeptide or heterodimeric protein according to any of claims 42 to 73, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises: a) a threonine at position 21 of SEQ ID NO: 1, a leucine at position 23 of SEQ ID NO: 1, an alanine at position 25 of SEQ ID NO: 1, a glycine at position 80 of SEQ ID NO: 1, an aline at position 205 of SEQ ID NO: 1, and / or a histidine at position 207 of SEQ ID NO: 1; b) a threonine at position 21 of SEQ ID NO: 3, a leucine at position 23 of SEQ ID NO: 3, and / or an isoleucine at position 24 of SEQ ID NO: 3; or c) a threonine at position 21 of SEQ ID NO: 4, a leucine at position 23 of SEQ ID NO: 4, an alanine at position 25 of SEQ ID NO: 4, a glycine at position 80 of SEQ ID NO: 4 , and / or a histidine at position 207 of SEQ ID NO: 4.
[75]
75. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 42 to 74, characterized in that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises at least one additional amino acid modification relative to a wild type IgG Fc polypeptide and has decreased CD16 binding affinity relative to the wild type IgG Fc polypeptide.
[76]
76. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 42 to 75, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises:
a) an amino acid substitution in one position corresponding to position 5 of SEQ ID NO: 2, an amino acid substitution in one position corresponding to position 38 of SEQ ID NO: 2, an amino acid substitution in a correct position - corresponding to position 39 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position 97 of SEQ ID NO: 2, and / or an amino acid substitution in a position corresponding to position 98 of SEQ ID NO: 2; or b) an amino acid substitution in a position corresponding to position 5 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 38 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 39 of SEQ ID NO: 3, an amino acid substitution in a position corresponding to position 97 of SEQ ID NO: 3, and / or an amino acid substitution in a position corresponding to position 98 of SEQ ID NO: 3.
[77]
77. Polypeptide, contiguous polypeptide or heterodimeric protein according to any of claims 42 to 76, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises: a) an amino acid substitution at position 5 of SEQ ID NO: 2, an amino acid substitution at position 38 of SEQ ID NO: 2, an amino acid substitution at position 39 of SEQ ID NO: 2, an amino acid substitution at position 97 of SEQ ID NO: 2, and / or an amino acid substitution at position 98 of SEQ ID NO: 2; or b) an amino acid substitution at position 5 of SEQ ID NO: 3, an amino acid substitution at position 38 of SEQ ID NO: 3, an amino acid substitution at position 39 of SEQ ID NO:
3, an amino acid substitution at position 97 of SEQ ID NO: 3, and / or an amino acid substitution at position 98 of SEQ ID NO: 3.
[78]
78. Polypeptide, contiguous polypeptide or hetero-dimeric protein according to any one of claims 42 to 77, characterized in that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises: a) a proline in a position corresponding to position 5 of SEQ ID NO: 2, a glycine in a position corresponding to position 38 of SEQ ID NO: 2, an arginine in a position corresponding to the position 39 of SEQ ID NO: 2, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 2, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 2; or b) a proline in a position corresponding to position 5 of SEQ ID NO: 3, a glycine in a position corresponding to position 38 of SEQ ID NO: 3, an arginine in a position corresponding to position 39 of SEQ ID NO: : 3, an isoleucine in a position corresponding to position 97 of SEQ ID NO: 3, and / or a glycine in a position corresponding to position 98 of SEQ ID NO: 3.
[79]
79. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 42 to 78, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises: a) a proline at position 5 of SEQ ID NO: 2, a glycine at position 38 of SEQ ID NO: 2, an arginine at position 39 of SEQ ID NO: 2, an isoleucine at position 97 of SEQ ID NO: 2, and / or a glycine at position 98 of SEQ ID NO: 2; or b) a proline at position 5 of SEQ ID NO: 3, a glycine at position 38 of SEQ ID NO: 3, an arginine at position 39 of SEQ ID NO: 3, an isoleucine at position 97 of SEQ ID NO: : 3, and / or a glycine at position 98 of SEQ ID NO: 3.
[80]
80. Polypeptide, contiguous polypeptide or heterodimeric protein according to any of claims 42 to 79, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises at least one additional amino acid modification relative to a wild type canine IgG Fc polypeptide and has decreased affinity for C1q binding relative to the wild type canine IgG Fc polypeptide.
[81]
81. Polypeptide, contiguous polypeptide or heterodimeric protein according to any of claims 42 to 80, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 2, or an amino acid substitution at a position corresponding to position 93 of SEQ ID NO: 3.
[82]
82. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 42 to 81, characterized in that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises an amino acid substitution at position 93 of SEQ ID NO: 2, or an amino acid substitution at position 93 of SEQ ID NO: 3.
[83]
83. Polypeptide, contiguous polypeptide or hetero-dimeric protein according to any one of claims 42 to 82, characterized in that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second Fc polypeptide from Variant IgG comprises an arginine in a position corresponding to position 93 of SEQ ID NO: 2, or an arginine in a position corresponding to position 93 of SEQ ID NO: 3.
[84]
84. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 42 to 83, characterized by the fact that the variant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and / or the second polypeptide Variant IgG Fc comprises an arginine at position 93 of SEQ ID NO: 2, or an arginine at position 93 of SEQ ID NO: 3.
[85]
85. Polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 1 to 84, characterized in that the polypeptide is an antibody, an antibody fusion or a fusion polypeptide.
[86]
86. Contiguous polypeptide, characterized by the fact that it comprises: a) a first glucagon-type peptide-1 (GLP1) (GLP1A) polypeptide; b) a first linker (L1); c) an IgG Fc (Fc) polypeptide from a species of companion animal; d) optionally, a second linker (L2); and e) optionally, a second GLP1 polypeptide (GLP1B).
[87]
87. Contiguous polypeptide according to claim 65, characterized in that it comprises: Formula (I): GLP1A — L1 — Fc; or Formula (II): Fc — L1 — GLP1A.
[88]
88. Contiguous polypeptide according to claim 65, characterized by the fact that it comprises:
Formula (III): GLP1A — L1 — Fc — L2 — GLP1B.
[89]
89. Contiguous polypeptide according to any one of claims 86 to 88, characterized in that GLP1B, if present, comprises the same amino acid sequence as GLP1A.
[90]
90. Contiguous polypeptide, characterized by the fact that it comprises: a) a glucagon-like peptide-1 polypeptide (GLP1); b) a first linker (L1); c) an IgG Fc (Fc) polypeptide; d) a second linker (L2); and e) a glucagon polypeptide (Gluc).
[91]
91. Contiguous polypeptide according to claim 90, characterized by the fact that it comprises: Formula (IV): GLP1 — L1 — Fc — L2 — Gluc; or Formula (V): Gluc — L1 — Fc — L2 — GLP1.
[92]
92. Contiguous polypeptide according to any one of claims 86 to 91, characterized in that GLP1A, GLP1, and / or GLP1B, if present, comprises a wild-type GLP1 polypeptide.
[93]
93. Contiguous polypeptide according to any one of claims 86 to 92, characterized in that GLP1A, GLP1, and / or GLP1B, if present, comprises a variant GLP1 polypeptide.
[94]
94. Contiguous polypeptide according to any one of claims 86 to 93, characterized in that GLP1A, GLP1, and / or GLP1B, if present, comprises an 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]
95. Contiguous polypeptide according to any one of claims 86 to 94, characterized in that the glucagon polypeptide comprises a wild-type glucagon polypeptide, optionally comprising the amino acid sequence of SEQ ID NO: 21.
[96]
96. Contiguous polypeptide according to any one of claims 86 to 95, characterized in that the glucagon polypeptide is a variant glucagon polypeptide.
[97]
97. Contiguous polypeptide according to any one of claims 86 to 96, characterized in that the IgG Fc polypeptide is a human IgG Fc.
[98]
98. Contiguous polypeptide according to any one of claims 86 to 97, characterized in that the IgG Fc polypeptide is an IgG1 Fc, IgG2 Fc, IgG3 Fc or human IgG4 Fc.
[99]
99. Contiguous polypeptide according to any one of claims 86 to 98, characterized in that the IgG Fc polypeptide is an Fc of a species of pet animal.
[100]
100. Contiguous polypeptide according to any one of claims 86 to 97 or 99, characterized in that the IgG Fc polypeptide comprises: a) an IgG-A Fc, IgG-B Fc, IgG-C Fc or Canine IgG-D Fc; b) an IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc or equine IgG7 Fc c) an IgG1a Fc, IgG1b Fc, feline IgG2 Fc
[101]
101. Contiguous polypeptide according to any one of claims 86 to 100, characterized in that the IgG Fc polypeptide is a wild-type IgG Fc polypeptide.
[102]
102. Contiguous polypeptide according to any one of claims 86 to 100, characterized in that the IgG Fc polypeptide is a variant IgG Fc polypeptide.
[103]
103. Contiguous polypeptide according to any one of claims 86 to 102, characterized in that the IgG Fc polypeptide comprises polypeptide, contiguous polypeptide or heterodimeric protein according to any one of claims 1 to 84.
[104]
104. Contiguous polypeptide according to any one of claims 85 to 102, characterized in that the contiguous polypeptide has a longer serum half-life than a wild-type GLP1 polypeptide.
[105]
105. Contiguous polypeptide according to any one of claims 86 to 104, characterized in that L1 and L2, if present, each independently is a flexible linker.
[106]
106. Contiguous polypeptide according to any one of claims 86 to 105, characterized by the fact that the amino acid sequence of L1 and L2, if present, each independently comprises 100%, at least 95%, at least 90 %, at least 85% amino acid residues of serine and / or glycine.
[107]
107. Contiguous polypeptide according to any one of claims 86 to 106, characterized in that the contiguous polypeptide comprises an extension at its C-terminus.
[108]
108. Contiguous polypeptide according to any one of claims 86 to 107, characterized in that the contiguous polypeptide comprises 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-terminal.
[109]
109. Contiguous polypeptide according to any one of claims 86 to 108, characterized in that the contiguous polypeptide comprises an 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 your C-terminal.
[110]
110. Contiguous polypeptide according to any one of claims 86 to 109, characterized in that the contiguous polypeptide comprises: 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) the amino acid sequence 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.
[111]
111. Polypeptide, characterized by the fact that it comprises an 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]
112. Polypeptide, the heterodimeric protein, or the contiguous polypeptide according to any one of the preceding claims, characterized by the fact that at least one amino acid modification or substitution comprises an amino acid substitution with an amino acid derivative.
[113]
113. Isolated nucleic acid encoding the polypeptide, heterodimeric protein, or contiguous polypeptide according to any of the preceding claims.
[114]
114. Host cell, characterized by the fact that it comprises the nucleic acid as defined in claim 113.
[115]
115. Method of producing a polypeptide, characterized in that it comprises culturing the host cell as defined in claim 114 and isolating the polypeptide.
[116]
116. Pharmaceutical composition, characterized in that it comprises the polypeptide, the heterodimeric protein, or the contiguous polypeptide as defined in any one of claims 1 to 112 and a pharmaceutically acceptable carrier.
[117]
117. Method of increasing cAMP production in a cell, characterized in that the method comprises exposing the cell to the polypeptide, the heterodimeric protein, the contiguous polypeptide, or the pharmaceutical composition as defined in any of claims 1 to 112 or 116 under permissive conditions for binding the polypeptide, heterodimeric protein, or contiguous polypeptide to GLP1R.
[118]
118. The method of claim 117, characterized in that the cell is exposed to the polypeptide, heterodimeric protein, contiguous polypeptide or the pharmaceutical composition ex vivo.
[119]
119. The method of claim 117, characterized in that the cell is exposed to the polypeptide, heterodimeric protein, contiguous polypeptide or the pharmaceutical composition in vivo.
[120]
120. Method according to any one of claims 118 and 119, characterized in that the cell is a human cell, a canine cell, a feline cell or an equine cell.
[121]
121. Method of releasing a polypeptide to an individual, characterized by the fact that it comprises administering the polypeptide, the heterodimeric protein, the contiguous polypeptide, or the pharmaceutical composition as defined in any of claims 1 to 112 or 116 parenterally.
[122]
122. Method of releasing a polypeptide to an individual, characterized by the fact that it comprises administering the polypeptide, the heterodimeric protein, the contiguous polypeptide, or the pharmaceutical composition as defined in any of claims 1 to 112 or 116 via an intramuscular route, an intraperitoneal route, an intracerebrospinal route, a subcutaneous route, an intra-arterial route, an intrasynovial route, an intrathecal route or through inhalation.
[123]
123. Method of treatment of an individual who has diabetes or obesity, characterized by the fact that the method comprises administering to the individual a therapeutically effective amount of the polypeptide, heterodimeric protein, contiguous polypeptide, or pharmaceutical composition as defined in any one of claims 1 to 112 or 116.
[124]
124. Method according to claim 123, characterized in that it comprises administering insulin, a DPP4 inhibitor, a SGLT2 inhibitor, a derivative of meguanitide sulfonylurea biguanides, an alpha-glucosidase inhibitor, a thiazolidinedion (TZD) ,
an amylinomimetic, a bile acid scavenger, a dopamine agonist.
[125]
125. Method according to any of claims 121 to 124, characterized by the fact that the individual is a human individual.
[126]
126. Method according to any of claims 121 to 124, characterized by the fact that the individual is a kind of pet animal.
[127]
127. Method according to claim 126, characterized by the fact that the species of pet animal is canine, equine or feline.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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