Compositions and methods of use for increasing the half-life of therapeutic agents in dogs

Abstract

Compositions and methods of use thereof for increasing the half-life of one or more polypeptides in dogs are provided. The compositions involve variant canine IgG Fc regions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 788,035, filed on January 3, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to polypeptides (e.g., fusion polypeptides such as polypeptide-Fc region fusions; or binding molecules such as ligand binding portions of antibodies or receptor-Fc fusions) having increased half-lives in dogs compared to their wild-type counterparts. Background Art

[0004] The Fc region of an antibody plays many functional roles, including, but not limited to, protecting the antibody from degradation via the lysosomal pathway and mediating antibody effector functions. With the increased use of canine antibodies as therapeutic agents, there is a growing focus not only on selecting the best Fab, but also on combining it with the appropriate Fc to achieve the desired half-life and effector functions.

[0005] There is little guidance in the art regarding increasing the half-life of polypeptide therapeutics (eg, antibodies) for canines. The present disclosure improves upon this by providing Fc region variants that improve the serum persistence of polypeptides (eg, antibodies) in dogs. Summary of the invention

[0006] Provided herein are platform technologies related to canine CH2, CH3 and Fc sequences useful for therapeutic polypeptides. The disclosure is characterized in that the polypeptide has an increased binding to canine FcRn than a control polypeptide (e.g., a wild-type counterpart IgG canine Fc region). In some cases, these polypeptides have an increased binding to canine FcRn than a control polypeptide at any pH (e.g., at any pH between about 5.0 and about 8.0). In some cases, these polypeptides have an increased binding to canine FcRn than a control polypeptide at pH 5.5 and / or pH 6.5. In some cases, these polypeptides can, for example, bind to canine FcRn at a higher level at an acidic pH (e.g., pH 5.5 or pH 6.5) than at a neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some cases, these polypeptides bind to canine FcRn at a higher level at pH 5.5 than at pH 7.4. The present disclosure relates in part to polypeptides having a half-life in dogs that is increased compared to its wild-type counterpart. For example, binding molecules (e.g., ligand binding portions of antibodies or receptors) with increased half-life are provided relative to forms in which these binding molecules are not connected to the Fc regions disclosed herein (e.g., CH2, CH3, or CH2+CH3 regions). Enzyme-Fc region fusions, ligand-Fc region fusions, and peptide-Fc region fusions are also provided, wherein the fusions have an increased half-life compared to their wild-type counterparts. In addition to having one or more substitutions that increase the half-life (relative to the wild-type canine Fc region), the Fc region may also include other substitutions such as increasing effector function, reducing effector function, and / or reducing the heterogeneity of the polypeptide (e.g., by removing one or more post-translational modifications in the Fc region). Canine CH2, CH3, and Fc region sequences may be from any canine antibody. In some cases, the canine CH2, CH3, and Fc region sequences are from canine IgG (eg, IgG.A, IgG.B, IgG.C, or IgG.D).

[0007] The present disclosure features a recombinant protein comprising (1) a binding domain, or a fragment thereof, which specifically binds to a ligand, or an epitope of a protein, wherein the binding domain is connected to (2) a domain containing a CH2 region, a CH3 region, or an Fc region (CH2+CH3 region) disclosed herein. In some cases, the binding domain comprises (i) six complementary determining regions (CDRs) of a canine or human / humanized antibody; (ii) VH and / or VL of a canine, caninized, humanized, or human antibody; (iii) a nanobody; (iv) a scFv; (v) a Fab; or (vi) a soluble receptor-binding domain that binds to a ligand, or a ligand-binding fragment thereof.

[0008] The present disclosure also provides a composition comprising: (1) a first polypeptide comprising a first Fc region (e.g., a CH2 region, a CH3 region, a CH2+CH3 region) comprising a canine IgG Fc region variant described herein; and (2) a second polypeptide comprising a second Fc region comprising a canine IgG Fc region variant described herein. The first and second polypeptides can be associated through the first and second Fc regions. In some cases, the amino acid sequences of the first and second Fc regions are identical. In other cases, the amino acid sequences of the first and second Fc regions are different (e.g., different from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). In some cases, the Fc region variant is a variant of the Fc region of a canine IgG.B antibody. In some cases, the Fc region variant is a variant of the Fc region of a canine IgG.A antibody. In some cases, the Fc region variant is a variant of the Fc region of a canine IgG.C antibody. In some cases, the Fc region variant is a variant of a canine IgG.D antibody Fc region.

[0009] Also disclosed are fusion molecules comprising canine IgG Fc region variants and polypeptides disclosed herein. In some embodiments, canine IgG Fc region variants are covalently linked to a polypeptide (e.g., via a hinge region or a linker). In some cases, the polypeptide is a ligand binding domain of a canine receptor protein, an extracellular domain of a canine receptor protein, or an antigen binding domain. In some cases, the polypeptide is selected from a ligand binding domain or an extracellular domain of canine IL-13Rα1, or IL-13Rα2, canine EPO, canine CTLA4, canine LFA3, canine VEGFR1 / VEGFR3, canine IL-1R, canine GLP-1 receptor agonist, and canine thrombopoietin binding peptide. In some cases, the polypeptide is a scFv, a nanobody, or a single domain antibody. In some cases, the IgG Fc region variant is a variant of a canine IgG.B antibody Fc region. In some cases, the IgG Fc region variant is a variant of a canine IgG.A antibody Fc region. In some cases, the IgG Fc region variant is a variant of a canine IgG.C antibody Fc region. In some cases, the IgG Fc region variant is a variant of a canine IgG.D antibody Fc region.

[0010] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc CH2 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 1 and comprising at least one of the following:

[0011] The amino acid other than Ile at amino acid position 250,

[0012] an amino acid other than Leu at amino acid position 251,

[0013] an amino acid other than Arg at amino acid position 252,

[0014] an amino acid other than Thr at amino acid position 254,

[0015] an amino acid other than Thr at amino acid position 256,

[0016] The amino acid other than His at amino acid position 285,

[0017] An amino acid other than Thr at amino acid position 286,

[0018] an amino acid other than Pro at amino acid position 307,

[0019] The amino acid other than Ile at amino acid position 308,

[0020] An amino acid other than Glu at amino acid position 309,

[0021] An amino acid other than Gln at amino acid position 311, or

[0022] An amino acid other than Thr at amino acid position 315.

[0023] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH2 region instead of a variant IgG Fc CH2 region; and / or (2) increased binding to canine FcRn compared to the one or more control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0024] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:1.

[0025] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc CH2 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 2 and comprising at least one of the following:

[0026] An amino acid other than Thr at amino acid position 250,

[0027] an amino acid other than Leu at amino acid position 251,

[0028] an amino acid other than Leu at amino acid position 252,

[0029] an amino acid other than Ala at amino acid position 254,

[0030] An amino acid other than Thr at amino acid position 256,

[0031] an amino acid other than Gln at amino acid position 285,

[0032] An amino acid other than Thr at amino acid position 286,

[0033] an amino acid other than Pro at amino acid position 307,

[0034] The amino acid other than Ile at amino acid position 308,

[0035] An amino acid other than Gly at amino acid position 309,

[0036] An amino acid other than Gln at amino acid position 311, or

[0037] An amino acid other than Lys at amino acid position 315.

[0038] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH2 region instead of a variant IgG Fc CH2 region; and / or (2) increased binding to canine FcRn compared to the one or more control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0039] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:2.

[0040] In one aspect, the present disclosure also provides one or more polypeptides comprising a canine IgG Fc CH2 region variant, the CH2 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 3 and comprising at least one of the following:

[0041] The amino acid other than Ile at amino acid position 250,

[0042] an amino acid other than Leu at amino acid position 251,

[0043] an amino acid other than Val at amino acid position 252,

[0044] an amino acid other than Ala at amino acid position 254,

[0045] An amino acid other than Thr at amino acid position 256,

[0046] an amino acid other than Gln at amino acid position 285,

[0047] An amino acid other than Thr at amino acid position 286,

[0048] an amino acid other than Pro at amino acid position 307,

[0049] The amino acid other than Ile at amino acid position 308,

[0050] An amino acid other than Gly at amino acid position 309,

[0051] An amino acid other than Gln at amino acid position 311, or

[0052] An amino acid other than Ser at amino acid position 315.

[0053] In some embodiments, the one or more polypeptides have: an increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH2 region instead of a variant IgG Fc CH2 region.

[0054] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:3.

[0055] In one aspect, the disclosure relates to one or more polypeptides comprising a canine IgG Fc CH2 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 4 and comprising at least one of:

[0056] The amino acid other than Ile at amino acid position 250,

[0057] an amino acid other than Leu at amino acid position 251,

[0058] an amino acid other than Arg at amino acid position 252,

[0059] an amino acid other than Thr at amino acid position 254,

[0060] An amino acid other than Thr at amino acid position 256,

[0061] The amino acid other than His at amino acid position 285,

[0062] An amino acid other than Thr at amino acid position 286,

[0063] an amino acid other than Pro at amino acid position 307,

[0064] The amino acid other than Ile at amino acid position 308,

[0065] An amino acid other than Glu at amino acid position 309,

[0066] An amino acid other than Gln at amino acid position 311, or

[0067] An amino acid other than Thr at amino acid position 315.

[0068] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH2 region instead of a variant IgG Fc CH2 region; and / or (2) increased binding to canine FcRn compared to the one or more control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0069] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:4.

[0070] In some embodiments, the one or more polypeptides comprise at least one of the following:

[0071] Glu or Gln at amino acid position 250,

[0072] Asp or Glu at amino acid position 251,

[0073] Tyr at amino acid position 252,

[0074] Thr at amino acid position 254,

[0075] Asp, Glu, or Phe at amino acid position 256,

[0076] Asn or Asp at amino acid position 285,

[0077] Asp at amino acid position 286,

[0078] Arg, Gln, or Ala at amino acid position 307,

[0079] Pro at amino acid position 308,

[0080] Pro at amino acid position 309,

[0081] Val at amino acid position 311, or

[0082] Asp at amino acid position 315.

[0083] In some embodiments, the one or more polypeptides comprise at least one of the following:

[0084] Tyr or Met at amino acid position 252,

[0085] Thr at amino acid position 254, or

[0086] Glu at amino acid position 256.

[0087] In some embodiments, the canine IgG Fc CH2 region variant comprises at least one of the following:

[0088] (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256;

[0089] (ii) Leu at amino acid position 428 and Ser at amino acid position 434;

[0090] (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311;

[0091] (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378;

[0092] (v) Asp at amino acid position 256, Asp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311;

[0093] (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315; or

[0094] (vii) Asp at amino acid position 251, and Pro at amino acid position 309.

[0095] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc CH3 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 5 and comprising at least one of the following:

[0096] An amino acid other than Asp at amino acid position 378,

[0097] The amino acid other than Glu at amino acid position 380,

[0098] an amino acid other than Met at amino acid position 428,

[0099] An amino acid other than Glu at amino acid position 430,

[0100] an amino acid other than Gln at amino acid position 433,

[0101] an amino acid other than Asn at amino acid position 434,

[0102] An amino acid other than His at amino acid position 435, or

[0103] An amino acid other than Tyr at amino acid position 436.

[0104] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH3 region instead of a variant IgG Fc CH3 region; and / or (2) increased binding to canine FcRn compared to the one or more control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0105] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:5.

[0106] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc CH3 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 6 and comprising at least one of the following:

[0107] An amino acid other than Asp at amino acid position 378,

[0108] The amino acid other than Glu at amino acid position 380,

[0109] an amino acid other than Met at amino acid position 428,

[0110] An amino acid other than Glu at amino acid position 430,

[0111] The amino acid other than His at amino acid position 433,

[0112] an amino acid other than Asn at amino acid position 434,

[0113] An amino acid other than His at amino acid position 435, or

[0114] An amino acid other than Tyr at amino acid position 436.

[0115] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH3 region instead of a variant IgG Fc CH3 region; and / or (2) increased binding to canine FcRn compared to the one or more control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0116] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:6.

[0117] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc CH3 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 7 and comprising at least one of the following:

[0118] An amino acid other than Asp at amino acid position 378,

[0119] The amino acid other than Glu at amino acid position 380,

[0120] an amino acid other than Met at amino acid position 428,

[0121] An amino acid other than Glu at amino acid position 430,

[0122] The amino acid other than His at amino acid position 433,

[0123] an amino acid other than Asn at amino acid position 434,

[0124] An amino acid other than His at amino acid position 435, or

[0125] An amino acid other than Tyr at amino acid position 436.

[0126] In some embodiments, the one or more polypeptides have: an increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH3 region instead of a variant IgG Fc CH3 region.

[0127] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:7.

[0128] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc CH3 region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 8 and comprising at least one of the following:

[0129] An amino acid other than Asp at amino acid position 378,

[0130] The amino acid other than Glu at amino acid position 380,

[0131] an amino acid other than Met at amino acid position 428,

[0132] An amino acid other than Glu at amino acid position 430,

[0133] an amino acid other than Gln at amino acid position 433,

[0134] an amino acid other than Asn at amino acid position 434,

[0135] An amino acid other than His at amino acid position 435, or

[0136] An amino acid other than Tyr at amino acid position 436.

[0137] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc CH3 region instead of a variant IgG Fc CH3 region; and / or (2) increased binding to canine FcRn compared to the one or more control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0138] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:8.

[0139] In some embodiments, the one or more polypeptides comprise at least one of the following:

[0140] Val at amino acid position 378,

[0141] Ala at amino acid position 380,

[0142] Leu at amino acid position 428,

[0143] Ala or Lys at amino acid position 430,

[0144] Lys at amino acid position 433,

[0145] Ser, Ala, or Phe at amino acid position 434,

[0146] Try at amino acid position 435,

[0147] His at amino acid position 436.

[0148] In some embodiments, the canine IgG Fc CH3 region variant comprises:

[0149] (i) Leu at amino acid position 428, and Ser at amino acid position 434;

[0150] (ii) Leu at amino acid position 428, and Ala at amino acid position 434;

[0151] (iii) Lys at amino acid position 430, and Lys at amino acid position 433; or

[0152] (iv) Tyr at amino acid position 435, and His at amino acid position 436.

[0153] In some embodiments, the one or more polypeptides comprise Tyr, Trp, Arg, or His at amino acid position 434.

[0154] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 9 and comprising at least one of:

[0155] The amino acid other than Ile at amino acid position 250,

[0156] an amino acid other than Leu at amino acid position 251,

[0157] an amino acid other than Arg at amino acid position 252,

[0158] an amino acid other than Thr at amino acid position 254,

[0159] an amino acid other than Thr at amino acid position 256,

[0160] The amino acid other than His at amino acid position 285,

[0161] An amino acid other than Thr at amino acid position 286,

[0162] an amino acid other than Pro at amino acid position 307,

[0163] The amino acid other than Ile at amino acid position 308,

[0164] An amino acid other than Glu at amino acid position 309,

[0165] An amino acid other than Gln at amino acid position 311,

[0166] an amino acid other than Thr at amino acid position 315,

[0167] An amino acid other than Asp at amino acid position 378,

[0168] The amino acid other than Glu at amino acid position 380,

[0169] an amino acid other than Met at amino acid position 428,

[0170] An amino acid other than Glu at amino acid position 430,

[0171] an amino acid other than Gln at amino acid position 433,

[0172] an amino acid other than Asn at amino acid position 434,

[0173] An amino acid other than His at amino acid position 435, or

[0174] An amino acid other than Tyr at amino acid position 436.

[0175] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc region instead of a variant IgG Fc region; and / or (2) increased binding to canine FcRn compared to the control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0176] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:9.

[0177] In one aspect, the present disclosure also provides one or more polypeptides comprising a canine IgG Fc region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 10 and comprising at least one of the following:

[0178] An amino acid other than Thr at amino acid position 250,

[0179] an amino acid other than Leu at amino acid position 251,

[0180] an amino acid other than Leu at amino acid position 252,

[0181] an amino acid other than Ala at amino acid position 254,

[0182] An amino acid other than Thr at amino acid position 256,

[0183] an amino acid other than Gln at amino acid position 285,

[0184] An amino acid other than Thr at amino acid position 286,

[0185] an amino acid other than Pro at amino acid position 307,

[0186] The amino acid other than Ile at amino acid position 308,

[0187] An amino acid other than Gly at amino acid position 309,

[0188] An amino acid other than Gln at amino acid position 311,

[0189] An amino acid other than Lys at amino acid position 315,

[0190] An amino acid other than Asp at amino acid position 378,

[0191] The amino acid other than Glu at amino acid position 380,

[0192] an amino acid other than Met at amino acid position 428,

[0193] An amino acid other than Glu at amino acid position 430,

[0194] The amino acid other than His at amino acid position 433,

[0195] an amino acid other than Asn at amino acid position 434,

[0196] An amino acid other than His at amino acid position 435, or

[0197] An amino acid other than Tyr at amino acid position 436.

[0198] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc region instead of a variant IgG Fc region; and / or (2) increased binding to canine FcRn compared to the control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0199] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:10.

[0200] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 11 and comprising at least one of:

[0201] The amino acid other than Ile at amino acid position 250,

[0202] an amino acid other than Leu at amino acid position 251,

[0203] an amino acid other than Val at amino acid position 252,

[0204] an amino acid other than Ala at amino acid position 254,

[0205] An amino acid other than Thr at amino acid position 256,

[0206] an amino acid other than Gln at amino acid position 285,

[0207] An amino acid other than Thr at amino acid position 286,

[0208] an amino acid other than Pro at amino acid position 307,

[0209] The amino acid other than Ile at amino acid position 308,

[0210] An amino acid other than Gly at amino acid position 309,

[0211] An amino acid other than Gln at amino acid position 311,

[0212] an amino acid other than Ser at amino acid position 315,

[0213] An amino acid other than Asp at amino acid position 378,

[0214] The amino acid other than Glu at amino acid position 380,

[0215] an amino acid other than Met at amino acid position 428,

[0216] An amino acid other than Glu at amino acid position 430,

[0217] The amino acid other than His at amino acid position 433,

[0218] an amino acid other than Asn at amino acid position 434,

[0219] An amino acid other than His at amino acid position 435, or

[0220] An amino acid other than Tyr at amino acid position 436.

[0221] In some embodiments, the one or more polypeptides have: an increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc region instead of a variant IgG Fc region.

[0222] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:11.

[0223] In one aspect, the present disclosure provides one or more polypeptides comprising a canine IgG Fc region variant comprising an amino acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO: 12 and comprising at least one of:

[0224] The amino acid other than Ile at amino acid position 250,

[0225] an amino acid other than Leu at amino acid position 251,

[0226] an amino acid other than Arg at amino acid position 252,

[0227] an amino acid other than Thr at amino acid position 254,

[0228] an amino acid other than Thr at amino acid position 256,

[0229] The amino acid other than His at amino acid position 285,

[0230] An amino acid other than Thr at amino acid position 286,

[0231] an amino acid other than Pro at amino acid position 307,

[0232] The amino acid other than Ile at amino acid position 308,

[0233] An amino acid other than Glu at amino acid position 309,

[0234] An amino acid other than Gln at amino acid position 311,

[0235] an amino acid other than Thr at amino acid position 315,

[0236] An amino acid other than Asp at amino acid position 378,

[0237] The amino acid other than Glu at amino acid position 380,

[0238] an amino acid other than Met at amino acid position 428,

[0239] An amino acid other than Glu at amino acid position 430,

[0240] an amino acid other than Gln at amino acid position 433,

[0241] an amino acid other than Asn at amino acid position 434,

[0242] An amino acid other than His at amino acid position 435, or

[0243] An amino acid other than Tyr at amino acid position 436.

[0244] In some embodiments, the one or more polypeptides have: (1) increased half-life in dogs compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc region instead of a variant IgG Fc region; and / or (2) increased binding to canine FcRn compared to the control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0245] In some embodiments, the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:12.

[0246] In some embodiments, the one or more polypeptides comprise at least one of the following:

[0247] Glu or Gln at amino acid position 250,

[0248] Asp or Glu at amino acid position 251,

[0249] Tyr at amino acid position 252,

[0250] Thr at amino acid position 254,

[0251] Asp, Glu, or Phe at amino acid position 256,

[0252] Asn or Asp at amino acid position 285,

[0253] Asp at amino acid position 286,

[0254] Arg, Gln, or Ala at amino acid position 307,

[0255] Pro at amino acid position 308,

[0256] Pro at amino acid position 309,

[0257] Val at amino acid position 311,

[0258] Asp at amino acid position 315,

[0259] Val at amino acid position 378,

[0260] Ala at amino acid position 380,

[0261] Leu at amino acid position 428,

[0262] Ala or Lys at amino acid position 430,

[0263] Lys at amino acid position 433,

[0264] Ser, Ala, or Phe at amino acid position 434,

[0265] Try at amino acid position 435, or

[0266] His at amino acid position 436.

[0267] In some embodiments, the one or more polypeptides comprise at least one of the following:

[0268] Tyr or Met at amino acid position 252,

[0269] Thr at amino acid position 254,

[0270] Glu at amino acid position 256, or

[0271] Tyr, Trp, Arg, or His at amino acid position 434.

[0272] In some embodiments, the canine IgG Fc region variant comprises at least one of the following:

[0273] (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256;

[0274] (ii) Leu at amino acid position 428 and Ser at amino acid position 434;

[0275] (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311;

[0276] (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378;

[0277] (v) Asp at amino acid position 256, Asp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311;

[0278] (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315;

[0279] (vii) Asp at amino acid position 256, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0280] (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378;

[0281] (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378;

[0282] (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378;

[0283] (xi) Asp at amino acid position 256, Asp at amino acid position 285, Asp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 378;

[0284] (xii) Asp at amino acid position 256, Asp at amino acid position 286, Arg at amino acid position 307, Val at amino acid position 311, and Val at position 378;

[0285] (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0286] (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378;

[0287] (xv) Asp at amino acid position 256, Asp at amino acid position 285, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0288] (xvi) Gln at amino acid position 307, Ala at amino acid position 380, Ser or Ala at amino acid position 434;

[0289] (xvii) Leu at amino acid position 428, and Ser or Ala at amino acid position 434; or

[0290] (xviii) Gln at amino acid position 250 and Leu at amino acid position 428.

[0291] In some embodiments, the one or more polypeptides are one or more antigen binding domains.

[0292] In some embodiments, the one or more antigen binding domains bind to an antigen selected from the group consisting of: NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, TNF-α, IgE, PD-1, PD-1 ligand, CD3, CD20, CD47, CD52, OX40, OX40 ligand, CTLA4, VEGF, EGFR, NAV 1.7, and a complement system complex (e.g., C1 complex, C2 complex, C3 complex, C4 complex, C5 complex, C6 complex, C7 complex, C8 complex, C9 complex).

[0293] In some embodiments, the one or more antigen binding domains are scFv, scFab, or nanobody.

[0294] In some embodiments, one or more polypeptides described herein further comprise a protein. In some embodiments, the protein is selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonist, and thrombopoietin binding peptide.

[0295] In one aspect, the present disclosure provides a pharmaceutical composition comprising (i) one or more polypeptides described herein, and (ii) a pharmaceutically acceptable excipient.

[0296] In one aspect, the disclosure provides one or more nucleic acids encoding one or more polypeptides described herein.

[0297] In one aspect, the disclosure provides one or more expression vectors comprising one or more nucleic acids described herein.

[0298] In one aspect, the disclosure provides a host cell comprising one or more nucleic acids described herein or one or more expression vectors described herein.

[0299] In one aspect, the present disclosure provides a method of preparing one or more polypeptides, the method comprising

[0300] (a) providing one or more nucleic acids described herein;

[0301] (b) expressing the one or more nucleic acids in host cell culture, thereby producing the one or more polypeptides; and

[0302] (c) collecting the one or more polypeptides produced in (b) from the host cell culture.

[0303] In some embodiments, the method further comprises formulating the one or more polypeptides into a pharmaceutical preparation.

[0304] In one aspect, the present disclosure provides a method of treating a canine disease or condition in a canine in need thereof, the method comprising administering to the canine an effective amount of a composition comprising a pharmaceutical composition described herein.

[0305] In one aspect, the present disclosure provides a method of preventing a canine disease or disorder in a canine in need thereof, the method comprising administering to the canine an effective amount of a composition comprising a pharmaceutical composition described herein.

[0306] In some embodiments, the disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, kidney disease, fertility-related disorder, infectious disease, or cancer.

[0307] In some embodiments, the disease or condition is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

[0308] Unless otherwise noted, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or test of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present application, including definitions, will dominate. Materials, methods, and examples are illustrative only and are not intended to be limiting.

[0309] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0310] Figure 1 is an amino acid sequence alignment of canine IgGγ chains. These chains contain V H , CH1, CH2, and CH3 domains and the hinge region between CH1 and CH2. N-glycosylation sites are shown in bold and marked as open blocks. These sequences are designated SEQ ID NOs.: 13, 14, 15, and 16, respectively.

[0311] Figure 2 is an alignment of the amino acid sequences of the CH2 region of canine IgG gamma chain. These sequences are designated SEQ ID NOs.: 1, 2, 3, and 4, respectively. Residues that are substituted to increase half-life are marked by underlining.

[0312] Figure 3 is an alignment of the amino acid sequences of the CH3 region of canine IgG gamma chain. These sequences are designated SEQ ID NOs.: 5, 6, 7, and 8, respectively. Residues that are substituted to increase half-life are marked by underlining.

[0313] Figure 4 is an alignment of the amino acid sequences of the Fc region of canine IgG gamma chain. These sequences are designated SEQ ID NOs.: 9, 10, 11, and 12, respectively. Residues that were substituted to increase half-life are marked by underlining.

[0314] Figure 5 is a table providing EU numbering for the CH2 region of canine IgG.

[0315] Figure 6 is a table providing EU numbering for the CH3 region of canine IgG.

[0316] Figure 7A-7U Depicts Biacore sensorgrams from an alanine scanning mutagenesis experiment. The light lines in each graph represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0317] Figures 8A-8C Depicted are Biacore sensorgrams of wild type and different variants from the NNK library at position 250. The light lines in each graph represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0318] Fig. 9A -9C depicts Biacore sensorgrams of wild type and different variants from the NNK library at position 252. The light lines in each graph represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0319] Fig. 10A and 10B Depicted are the Biacore sensorgrams of wild type and variant A254T. The light lines in each figure represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0320] Fig.11A and 11BBiacore sensorgrams of wild type and variant G309P are depicted. The light lines in each figure represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0321] Fig. 12A and 12B Depicted are the Biacore sensorgrams of wild type and variant Q311V. The light lines in each figure represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0322] Fig.13A and 13B Depicted are the Biacore sensorgrams of the wild type and variant D378V. The light lines in each figure represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0323] Fig.14A and 15B Depicted are the Biacore sensorgrams of wild type and variant E380A. The light lines in each figure represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0324] Figures 15A-15F Depicted are Biacore sensorgrams of wild type and different variants from the NNK library at position 434. The light lines in each graph represent the measured data and the dark lines represent the fitted curves using a 1:1 interaction model.

[0325] Figures 16A-16E Depicted are Biacore sensorgrams of different variants in a concentration series. The concentrations of canine FcRn used were 100 nM (white circles), 200 nM (black circles), 400 nM (black triangles), and 800 nM (white triangles). The light lines in each figure represent the measured data, and the dark lines represent the fitted curves using a 1:1 interaction model. DETAILED DESCRIPTION

[0326] With the increasing use of polypeptides (e.g., antibodies, ligand binding domains of receptors, enzymes, ligands, peptides) as therapeutic agents for the prevention and treatment of various canine diseases, the development of polypeptides with extended half-lives is important, particularly in the prevention or treatment of chronic diseases where repeated administration of the polypeptide is necessary.

[0327] Thus, the disclosure features canine immunoglobulin CH2, CH3, and Fc regions comprising mutations that enhance the half-life of one or more polypeptides containing these sequences. Also disclosed are polypeptides comprising these domains and methods of use thereof. These peptides can be used for a variety of therapeutic and diagnostic purposes.

[0328] Canine Antibodies

[0329] Dogs have four IgG heavy chains, referred to as A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, referred to as IgG.A, IgG.B, IgG.C, and IgG.D. The amino acid and DNA sequences of these heavy chains are available from Tang et al., Vet. Immunol. Immunopathol., 80:259-270 (2001) and the GENBANK database. For example, the amino acid sequence of the IgG.A heavy chain has GENBANK accession number AAL35301.1, IgG.B has GENBANK accession number AAL35302.1, IgG.C has GENBANK accession number AAL35303.1, and IgG.D has GENBANK accession number AAL35304.1. Canine antibodies also include two light chains: κ and λ. The DNA and amino acid sequences of these light chains are also available from the GENBANK database. For example, the canine kappa light chain amino acid sequence has accession number ABY 57289.1 and the canine lambda light chain has accession number ABY 55569.1.

[0330] CH2 region of canine Fc region:

[0331] The CH2 region of the canine antibody includes or consists of amino acids 237 to 340 (according to EU numbering) of the canine IgG antibody. It should be understood that the CH2 region may include 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at their N-terminal and / or C-terminal ends.

[0332] The amino acid sequence of the CH2 region of canine IgG.A is provided below:

[0333]

[0334] The amino acid sequence of the CH2 domain of canine IgG.B is provided below:

[0335]

[0336] The amino acid sequence of the CH2 domain of canine IgG.C is provided below:

[0337]

[0338] The amino acid sequence of the CH2 domain of canine IgG.D is provided below:

[0339]

[0340] CH3 region of canine Fc region:

[0341] The CH3 region of a canine antibody includes or consists of amino acids 345 to 447 (according to EU numbering) of a canine IgG antibody. It should be understood that the CH3 region may include 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at their N-terminal and / or C-terminal ends.

[0342] The amino acid sequence of the CH3 domain of canine IgG.A is provided below:

[0343]

[0344] The amino acid sequence of the CH3 domain of canine IgG.B is provided below:

[0345]

[0346] The amino acid sequence of the CH3 domain of canine IgG.C is provided below:

[0347]

[0348] The amino acid sequence of the CH3 domain of canine IgG.D is provided below:

[0349]

[0350] Fc region of canine Fc region:

[0351] The Fc region of a canine IgG antibody comprises or consists of amino acids 231 to 447 (according to EU numbering) of a canine IgG antibody.

[0352] The amino acid sequence of the Fc domain of canine IgG.A is provided below:

[0353]

[0354] The amino acid sequence of the Fc domain of canine IgG.B is provided below:

[0355]

[0356] The amino acid sequence of the Fc domain of canine IgG.C is provided below:

[0357]

[0358] The amino acid sequence of the Fc domain of canine IgG.D is provided below:

[0359]

[0360] Substitutions in canine IgG Fc that improve half-life

[0361] Increased serum persistence is a beneficial property of therapeutic polypeptides. The present disclosure features substitutions in wild-type canine IgG.A, IgG.B, IgG.C, and IgG.D Fc regions that enhance the half-life of one or more polypeptides comprising these Fc regions in dogs relative to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type canine IgG Fc region in place of an IgG Fc region variant. The half-life-increasing substitutions may occur in the canine CH2 region, the canine CH3 region, or in the case of the canine Fc (i.e., CH2+CH3) region.

[0362] In some cases, the present disclosure provides canine IgG CH2 region variants comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 4. Also provided are canine IgG CH2 region variants comprising an amino acid sequence that differs from any one of SEQ ID NOs.: 1 to 4 by 1 to 15 amino acids.

[0363] In other cases, the disclosure features a canine IgG CH3 region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs.: 5 to 8. Also provided is a canine IgG CH3 region variant comprising an amino acid sequence that differs from any one of SEQ ID NOs.: 5 to 8 by 1 to 15 amino acids.

[0364] In certain cases, the disclosure features a canine IgG Fc region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 12. Also provided is a canine IgG Fc region variant comprising an amino acid sequence that differs from any one of SEQ ID NOs.: 9 to 12 by 1 to 20 amino acids.

[0365] In some cases, at least one (e.g., 1, 2, or 3) of the following regions in the canine IgG Fc CH2 region variant is identical to the corresponding region in the wild-type canine IgG Fc CH2 region:

[0366] amino acid positions 250-256;

[0367] Amino acid positions 285-288; and

[0368] Amino acid positions 307-315, wherein the amino acid positions are based on EU numbering. In some cases, all of the above regions in the canine IgG Fc CH2 region variant are identical to the corresponding regions in the wild-type canine IgG Fc CH2 region.

[0369] In some cases, at least one (e.g., 1 or 2) of the following regions in the canine IgG Fc CH3 region variant is identical to the corresponding region in the wild-type canine IgG Fc CH3 region:

[0370] Amino acid positions 376-380; and

[0371] Amino acid positions 428-436, wherein the amino acid positions are based on EU numbering. In some cases, all of the above regions in the canine IgG Fc CH3 region variant are identical to the corresponding regions in the wild-type canine IgG Fc CH3 region.

[0372] In some cases, at least one (e.g., 1, 2, 3, 4, or 5) of the following regions in the canine IgG Fc variant is identical to the corresponding region in wild-type canine IgG Fc:

[0373] amino acid positions 250-256;

[0374] amino acid positions 285-288;

[0375] amino acid positions 307-315;

[0376] Amino acid positions 376-380; and

[0377] Amino acid positions 428-436, wherein the amino acid positions are based on EU numbering. In some cases, all of the above regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc.

[0378] In still other embodiments, one or more polypeptides comprising a canine IgG Fc CH2 region variant are provided, wherein the CH2 region variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 4.

[0379] In some embodiments, one or more polypeptides are characterized as comprising a canine IgG Fc CH3 region variant, wherein the CH3 region variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 5 to 8.

[0380] In some embodiments, one or more polypeptides are characterized as comprising a canine IgG Fc region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 12.

[0381] In some cases, the one or more polypeptides described above include a canine IgG CH2 region comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following:

[0382] an amino acid other than the wild-type amino acid present at amino acid position 250,

[0383] an amino acid other than the wild-type amino acid present at amino acid position 251,

[0384] an amino acid other than the wild-type amino acid present at amino acid position 252,

[0385] an amino acid other than the wild-type amino acid present at amino acid position 254,

[0386] an amino acid other than the wild-type amino acid present at amino acid position 256,

[0387] an amino acid other than the wild-type amino acid present at amino acid position 285,

[0388] an amino acid other than the wild-type amino acid present at amino acid position 286,

[0389] an amino acid other than the wild-type amino acid present at amino acid position 307,

[0390] an amino acid other than the wild-type amino acid present at amino acid position 308,

[0391] an amino acid other than the wild-type amino acid present at amino acid position 309,

[0392] An amino acid other than the wild-type amino acid present at amino acid position 311, or

[0393] an amino acid other than the wild-type amino acid present at amino acid position 315,

[0394] Wherein the amino acid positions are based on the EU numbering of canine IgG.A, IgG.B, IgG.C, and IgG.D antibodies.

[0395] In some embodiments, the one or more polypeptides described above include a canine IgG CH3 region comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following:

[0396] an amino acid other than the wild-type amino acid present at amino acid position 378,

[0397] an amino acid other than the wild-type amino acid present at amino acid position 380,

[0398] an amino acid other than the wild-type amino acid present at amino acid position 428,

[0399] an amino acid other than the wild-type amino acid present at amino acid position 430,

[0400] an amino acid other than the wild-type amino acid present at amino acid position 433,

[0401] an amino acid other than the wild-type amino acid present at amino acid position 434,

[0402] An amino acid other than the wild-type amino acid present at amino acid position 435, or

[0403] an amino acid other than the wild-type amino acid present at amino acid position 436,

[0404] Wherein the amino acid positions are based on the EU numbering of canine IgG.A, IgG.B, IgG.C, and IgG.D antibodies.

[0405] In certain embodiments, the one or more polypeptides described above include a canine IgG Fc region comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of the following:

[0406] an amino acid other than the wild-type amino acid present at amino acid position 250,

[0407] an amino acid other than the wild-type amino acid present at amino acid position 251,

[0408] an amino acid other than the wild-type amino acid present at amino acid position 252,

[0409] an amino acid other than the wild-type amino acid present at amino acid position 254,

[0410] an amino acid other than the wild-type amino acid present at amino acid position 256,

[0411] an amino acid other than the wild-type amino acid present at amino acid position 285,

[0412] an amino acid other than the wild-type amino acid present at amino acid position 286,

[0413] an amino acid other than the wild-type amino acid present at amino acid position 307,

[0414] an amino acid other than the wild-type amino acid present at amino acid position 308,

[0415] an amino acid other than the wild-type amino acid present at amino acid position 309,

[0416] an amino acid other than the wild-type amino acid present at amino acid position 311,

[0417] an amino acid other than the wild-type amino acid present at amino acid position 315,

[0418] an amino acid other than the wild-type amino acid present at amino acid position 378,

[0419] an amino acid other than the wild-type amino acid present at amino acid position 380,

[0420] an amino acid other than the wild-type amino acid present at amino acid position 428,

[0421] an amino acid other than the wild-type amino acid present at amino acid position 430,

[0422] an amino acid other than the wild-type amino acid present at amino acid position 433,

[0423] an amino acid other than the wild-type amino acid present at amino acid position 434,

[0424] an amino acid other than the wild-type amino acid present at amino acid position 435,

[0425] an amino acid other than the wild-type amino acid present at amino acid position 436,

[0426] Wherein the amino acid positions are based on the EU numbering of canine IgG.A, IgG.B, IgG.C, and IgG.D antibodies.

[0427] The present disclosure encompasses substitutions including one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of those disclosed in Table 1.

[0428] Table 1

[0429]

[0430] In some cases, the present disclosure encompasses substitutions including one or more (eg, 1, 2, 3, or 4) of those disclosed in Table 2.

[0431] Table 2

[0432]

[0433] The present disclosure includes all possible combinations and permutations of the substitutions disclosed above. In some cases, substitutions include one or more of the following substitutions:

[0434] (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256;

[0435] (ii) Leu at amino acid position 428 and Ser at amino acid position 434;

[0436] (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311;

[0437] (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378;

[0438] (v) Asp at amino acid position 256, Asp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311;

[0439] (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315;

[0440] (vii) Asp at amino acid position 256, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0441] (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378;

[0442] (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378;

[0443] (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378;

[0444] (xi) Asp at amino acid position 256, Asp at amino acid position 285, Asp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 378;

[0445] (xii) Asp at amino acid position 256, Asp at amino acid position 286, Arg at amino acid position 307, Val at amino acid position 311, and Val at position 378;

[0446] (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0447] (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378;

[0448] (xv) Asp at amino acid position 256, Asp at amino acid position 285, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0449] (xvi) Gln at amino acid position 307, Ala at amino acid position 380, Ser or Ala at amino acid position 434;

[0450] (xvii) Leu at amino acid position 428, and Ser or Ala at amino acid position 434;

[0451] (xviii) Gln at amino acid position 250 and Leu at amino acid position 428;

[0452] (xix) Glu at amino acid position 250 and Glu at amino acid position 251;

[0453] (xx) Phe at amino acid position 256 and Phe at amino acid position 309;

[0454] (xxi) Ala at amino acid position 430 and Lys at amino acid position 433;

[0455] (xxii) Phe at amino acid position 434 and His at amino acid position 436;

[0456] (xxiii) Tyr at amino acid position 435 and His at amino acid position 436;

[0457] In some cases, the substitution does not include a combination of Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256.

[0458] The substitution can be made on one or both chains of the CH2 domain, CH3 domain, or Fc domain. In some cases, the substitutions on both chains of the CH2 domain, CH3 domain, or Fc domain are the same. In some cases, the substitutions on both chains of the CH2 domain, CH3 domain, or Fc domain are different. In some cases, the Fc region includes one or more additional substitutions that increase or decrease effector function, improve product heterogeneity.

[0459] Other Substitutions That Can Be Combined with Half-Life Enhancing Substitutions

[0460] The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process involving the coordination of a complex series of activities to produce the desired polypeptide / protein. These include optimization of specificity, affinity, functional activity, expression levels in engineered cell lines, long-term stability, elimination or enhancement of effector function, and development of commercially viable manufacturing and purification methods. The present disclosure includes any additional substitutions that promote any one or more of the above-mentioned goals.

[0461] In some embodiments, substitutions are introduced to reduce the effector function of the canine Fc region. Such substitutions may be located at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions (numbered according to EU numbering) of canine IgG: 238, 265, 297, 298, 299, 327, and 329. The substitution (one or more) may be any of the other 19 amino acids. In some cases, the substitution is conservative. In certain non-limiting cases, the substituted amino acid at position 238 is Ala; the substituted amino acid at position 265 is Ala; the substituted amino acid at position 297 is Ala or Gln; the substituted amino acid at position 298 is Pro; the substituted amino acid at position 299 is Ala; the substituted amino acid at position 327 is Gly; the substituted amino acid at position 329 is Ala. In some cases, the variant Fc region is from a canine IgG.B or IgG.C antibody.

[0462] In some embodiments, substitutions are introduced into the wild-type canine IgG Fc region to enhance binding to Protein A, so as to facilitate purification by Protein A chromatography. Such substitutions may be at one or two (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions of canine IgG (numbered according to EU numbering): 252 and 254. The substitution (one or more) may be any of the other 19 amino acids. In some cases, the substitution is conservative. In certain non-limiting cases, the substituted amino acid at position 252 is Met; and the substituted amino acid at position 254 is Ser.

[0463] In some embodiments, substitutions are made to alter the binding affinity to FcRn (e.g., to increase or decrease the binding affinity to FcRn) compared to a parent polypeptide or a wild-type polypeptide. In some variations, the modification may be one, two, three, or four modifications selected from the group consisting of: 308F, 428L, 434M, and 434S, wherein the numbering is according to EU numbering. In some embodiments, the Fc variant includes one or more modifications selected from the group consisting of: 252Y / 428L, 428L / 434H, 428L / 434F, 428L / 434Y, 428L / 434A, 428L / 434M, and 428L / 434S, wherein the numbering is according to EU numbering. In some embodiments, the Fc variant includes one or more modifications selected from the group consisting of: 428L / 434S, 308F / 428L / 434S, wherein the numbering is according to EU numbering. In some embodiments, the Fc variant comprises one or more modifications selected from the group consisting of: 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S, and 308F / 319L / 434S, wherein the numbering is according to EU numbering. A detailed description of these modifications is described in, for example, US8883973B2, which is incorporated herein by reference in its entirety.

[0464] In some embodiments, the polypeptide includes the hinge region of a canine antibody. In some embodiments, the hinge region of a canine antibody may be modified to increase half-life. In some embodiments, according to EU numbering, the modification is 228P.

[0465] In some embodiments, binding to FcRn is pH dependent. H310 and H435 (EU numbering) may be important for pH dependent binding. Thus, in some embodiments, the amino acid at position 310 (EU numbering) is histidine. In some embodiments, the amino acid at position 435 (EU numbering) is histidine. In some embodiments, the amino acid at two positions is histidine.

[0466] In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering), or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering). In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residues at positions 234 and 235 (EU numbering) are Ala.

[0467] Polypeptides comprising canine IgG Fc variants

[0468] The present disclosure includes any polypeptide that may benefit from having increased half-life in dogs. To increase half-life, these polypeptides are designed to contain the Fc region variants disclosed above (eg, CH2 region, CH3 region, CH2+CH3 region).

[0469] Exemplary polypeptides include, but are not limited to, complete antibodies, scFv, nanobodies, ligand binding portions of receptors, cytokines, growth factors, enzymes, and peptides. For example, the CH3 domain variants disclosed above can be linked to scFv nanobodies, ligand binding portions of receptors (e.g., ligand binding portions of canine IL-13Rα1 or IL-13Rα2), cytokines, growth factors, enzymes, or peptides. Alternatively, the Fc region variants disclosed above can be linked to these polypeptides. In another embodiment, a canine or caninized antibody is modified to include an Fc region variant disclosed herein.

[0470] In certain embodiments, the polypeptides of the present disclosure include an antibody hinge region. The hinge region may be located between the ligand binding domain of an antigen or polypeptide and the Fc region variant. In some cases, the hinge region is connected to the C-terminus of a cytokine, growth factor, enzyme, or peptide, and the hinge region is connected to the N-terminus of the Fc region variant. Exemplary hinge region sequences are provided below.

[0471] IgG.A:FNECRCTDTPPCPVEPEP (SEQ ID NO:17);

[0472] IgG.B: PKRENGRVPRPDCPKCPAPEM (SEQ ID NO: 18);

[0473] IgG.C:AKECECKCNCNNCPCPGCGL (SEQ ID NO:19);

[0474] IgG.D: PKESTCKCISPCPVPES (SEQ ID NO: 20); and

[0475] IgG.Dmut:PKESTCKCIPPCPVPES (SEQ ID NO:21).

[0476] If used, the hinge region in the recombinant protein of the present disclosure may include 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions relative to the amino acid sequence recorded in any one of SEQ ID NOs.: 17-21. In some cases, the hinge region used in the recombinant protein of the present disclosure may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence recorded in any one of SEQ ID NOs.: 17-21.

[0477] In certain embodiments, a linker sequence may be used in place of an antibody hinge sequence to link a polypeptide (e.g., an antibody, a ligand binding domain of a receptor, an enzyme, a ligand, a peptide) to a canine Fc region variant disclosed herein. In certain embodiments, the linker may be composed of 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as understood by those skilled in the art. In other embodiments, 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is composed of a majority of amino acids that are not sterically hindered, such as glycine and alanine. Examples of peptide linkers include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 22); Ser Gly Gly Gly (SEQ ID NO: 23); Gly Gly Gly Gly Ser (SEQ ID NO: 24); Ser GlyGly Gly Gly (SEQ ID NO: 25); Gly Gly Gly Gly Gly Ser (SEQ ID NO:26); Ser Gly GlyGly Gly Gly (SEQ ID NO:27); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:28); Ser GlyGly Gly Gly Gly Gly (SEQ ID NO:29); (Gly Gly Gly Gly Gly Ser) n (SEQ ID NO: 24) n, wherein n is an integer greater than 1 (e.g., 1, 2, 3, 4, 5); and (Ser Gly Gly Gly Gly) n(SEQ ID NO: 25) n, wherein n is an integer greater than 1 (e.g., 1, 2, 3, 4, 5).

[0478] Non-peptide linkers can also be used to link one or more polypeptides of interest to the Fc region variants disclosed herein. For example, an alkyl linker, such as -NH(CH2) n C(O)-, wherein n = 2-20. These alkyl linkers may be further substituted by any non-sterically hindered groups such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0479] One or more polypeptides disclosed herein may include a binding domain. The binding domain may specifically bind to a protein, subunit, domain, motif, and / or epitope of a selected target described herein. In some embodiments, one or more polypeptides (e.g., fusion polypeptides) may include a protein, wherein the protein is a therapeutic protein described herein. In some embodiments, a target (e.g., a target for a binding domain) or a therapeutic protein (e.g., for a fusion protein) is selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIBALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, IgE, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA -1. BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenin, BMP -4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB(ALK-6 ), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A (CathepsinA), cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CC1, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL 21. CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR , CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6 、CD7、CD8、CD10、CD11a、CD11b、CD11c、CD13、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD27L、CD28、CD29、CD30、CD30L、CD32、CD33(p67 protein)、CD34、CD38、CD40、CD40L、CD44、CD45、CD46、CD47、 CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringenstoxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, C XCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin Leukoma-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, neprilysin, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6. GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, GLP1, GLP2, glucagon, Glut 4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, GnRH, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparinase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, cardiac myosin, cytomegalovirus (CMV), growth hormone (GH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptor (eg, IL- 1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), interferon (INF)-α, INF-β, INF-γ, inhibition Insulin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2 (Kallikrein2), Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y Antigen, Lewis-Y Related Antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, Lipoprotein, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung Surfactant, Luteinizing Hormone, Lymphotoxin β Receptor, Mac -1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Muellerian inhibitory substance, Mug, MuSK, NAIP, NAP, NAV 1.7, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, Neurturin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PD1, PDL1, PDGF, PD K-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, relaxin (Prorelaxin), protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan Specific, TGF-βR1(ALK-5), TGF-βR11, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, Thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAILR3DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA) TNFRSF18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF R1CD120a、p55-60)、TNFRSF1B(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DCTRAIL R2). TNFRH2, TNFRST23(DCTRAIL R1TNFRH1), TNFRSF25(DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 division, TL2, TNFSF11 (TRANCE / RANK division ODF, OPG division), TNFSF12 (TWEAK Apo-3 division, DR3 division), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF). BLYS、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT HVEM PLATE、LTg) TNFSF15(TL1A / VEGI) TNFSF18(GITR PLANT AITR PL6 TL6) TNFSF1A(TNF-a Connectin, DIF, TNFSF2, TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb).TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer protein (transferringreceptor), TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrates, TWEAK, TXB2, Ung, UPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1(fit-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrands factors, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors.

[0480] In some embodiments, the binding domain specifically binds to one or more therapeutic targets or antigens in dogs, such as, but not limited to: ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (Myostatin), GLP1, GLP2, GnRH, growth hormone releasing factor, IgE, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL -8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL -33. Interleukin receptors (e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15 R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-βPan Specific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, and VEGFR-3 (flt-4).

[0481] In some embodiments, the one or more polypeptides may comprise a protein, wherein the protein is a therapeutic protein, for example, EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, or a thrombopoietin binding peptide.In some embodiments, the therapeutic protein is ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (Myostatin), GLP1, GLP2, GnRH, growth hormone releasing factor, IgE, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL -8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL -33. Interleukin receptors (e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15 R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-βPan Specific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9(4-1BB CD137, ILA), VEFGR-1(fit-1), VEGF, VEGFR, or VEGFR-3(flt-4).

[0482] In some embodiments, the therapeutic protein is any protein described herein. In some embodiments, the one or more polypeptides further include a canine IgG CH2 domain, an IgG CH3 domain, or an IgG Fc region as described herein. The modified canine IgG CH2 domain, IgG CH3 domain, or IgG Fc region can enhance the half-life of the therapeutic protein in vivo.

[0483] Pharmaceutical composition

[0484] To prepare a pharmaceutical or sterile composition of one or more polypeptides described herein, one or more polypeptides may be mixed with a pharmaceutically acceptable carrier or excipient (see, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)).

[0485] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, one or more polypeptides of the invention are diluted to an appropriate concentration in sodium acetate solution pH 5-6, and NaCl or sucrose is added to adjust tonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0486] Toxicity and therapeutic effectiveness of a polypeptide composition, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., to determine the LD 50 (the dose lethal to 50% of the population) and ED 50 The ratio of doses that are toxic to those that are therapeutically effective is the therapeutic index (LD 50 / ED 50 ). In certain aspects, one or more polypeptides that exhibit a high therapeutic index are desired. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for dogs. The dosage of such compounds preferably falls within the range that includes ED with little or no toxicity.50 The dosage may vary within this range depending on the dosage form and route of administration employed.

[0487] Modes of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial. In some embodiments, one or more polypeptides may be administered by invasive routes such as injection. In further embodiments, one or more polypeptides are administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery.

[0488] The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of known implants and components for administering pharmaceutical compositions include: U.S. Pat. No. 4,487,603, which discloses a portable microinfusion pump for dispensing at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable-speed flow portable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment. Many other such implants, delivery systems, and components are known to those skilled in the art.

[0489] Alternatively, one or more polypeptides may be administered in a local rather than a systemic manner, for example, by injecting the antibody directly into a joint or a pathogen-induced lesion characterized by immunopathology, typically in a sustained or slow-release formulation. In addition, one or more polypeptides may be administered in a targeted drug delivery system, for example, in liposomes coated with tissue-specific antibodies that target, for example, a joint or a pathogen-induced lesion characterized by immunopathology, etc. The liposomes will target the afflicted tissue and be selectively taken up by the tissue.

[0490] The administration regimen depends on multiple factors, including, but not limited to, the age, weight, and physical condition of the dog to be treated, the serum or tissue conversion rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the one or more therapeutic polypeptides, and the accessibility of target cells in the biomatrix. Preferably, the administration regimen delivers enough one or more therapeutic polypeptides to effectively improve the target disease state while minimizing undesirable side effects. Therefore, the amount of the organism delivered depends in part on the severity of the specific one or more therapeutic polypeptides and the condition to be treated. Guidance for selecting appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Milgrom et al. New Engl. J. Med. 341: 1966-1973 (1999); Slamon et al. New Engl. J. Med. 344: 783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342: 613-619 (2000); Ghosh et al. New Engl. J. Med. 348: 24-32 (2003); Lipsky et al. New Engl. J. Med. 343: 1594-1602 (2000)).

[0491] Those skilled in the art, for example, use parameters or factors known in the art or suspected of affecting treatment to determine the appropriate dosage of one or more polypeptides. Typically, the dosage begins with an amount slightly less than the optimal dose and is increased in small increments thereafter until the desired or optimal effect is achieved relative to any negative effects. Important diagnostic measurements include those of symptoms of inflammation or the levels of inflammatory cytokines produced.

[0492] Nucleic acid, vector, host cell, and preparation method

[0493] The present disclosure also includes one or more nucleic acids encoding one or more polypeptides described herein, one or more vectors comprising the one or more nucleic acids, and host cells comprising the one or more nucleic acids or the one or more vectors.

[0494] One or more polypeptides described herein can be produced in bacterial cells or eukaryotic cells. Some polypeptides, such as Fab polypeptides, can be produced in bacterial cells such as Escherichia coli cells. Polypeptides can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS, 293T, Hela). In addition, polypeptides (e.g., scFv polypeptides) can be expressed in yeast cells such as Pichia, Hanseula, or Saccharomyces (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)). In order to produce antibodies of interest, one or more polynucleotides encoding one or more polypeptides are constructed, introduced into one or more expression vectors, and then expressed in suitable host cells. In order to improve expression, the nucleotide sequence of the gene can be recoded while the amino acid sequence is not changed (or minimally changed-e.g., removing the C-terminal residue of the heavy chain or light chain). The region for potential recoding includes those related to translation initiation, codon usage, and possible unintentional mRNA splicing. Those skilled in the art can easily envision polynucleotides encoding the Fc region variants described herein.

[0495] Standard molecular biology techniques can be used to prepare the recombinant expression vector(s), transfect the host cells, select for transformants, culture the host cells, and recover the polypeptide (eg, antibody).

[0496] If one or more polypeptides are to be expressed in bacterial cells (e.g., E. coli), the expression vector should have features that allow the vector to be amplified in the bacterial cell. In addition, when E. coli such as JM109, DH5α, HB101, or XL1-Blue are used as hosts, the vector should have a promoter, for example, a lacZ promoter (Ward et al., 341:544-546 (1989), an araB promoter (Better et al., Science, 240:1041-1043 (1988)), or a T7 promoter that allows efficient expression in E. coli. Examples of such vectors include, for example, M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIA Expression System" (QIAGEN), pEGFP, and pET (when using this expression vector, the host preferably expresses T7 The expression vector may contain a signal sequence for antibody secretion. For production in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) may be used as a signal sequence for antibody secretion. For bacterial expression, the calcium chloride method or electroporation method may be used to introduce the expression vector into bacterial cells.

[0497] If one or more polypeptides are to be expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, for example, the SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)) (e.g., early simian virus 40 promoter), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)), or CMV promoter (e.g., human cytomegalovirus immediate early promoter). In addition to the nucleic acid sequence encoding the Fc region variant, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., initiation of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, e.g., G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0498] In some embodiments, one or more polypeptides are produced in mammalian cells. Exemplary mammalian host cells for expressing one or more polypeptides include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selection marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159: 601 621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines such as NS0 myeloma cells and SP2 cells, and cells from transgenic animals such as transgenic mammals. For example, the cell is a mammary epithelial cell.

[0499] In an exemplary system for antibody expression, a recombinant expression vector encoding an antibody heavy chain and an antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, and adenovirus, such as a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high-level transcription of the gene. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select CHO cells transfected with the vector. The selected transformed host cells are cultured to allow expression of the antibody heavy and light chains, and the antibodies are recovered from the culture medium.

[0500] Treatment

[0501] One or more polypeptides disclosed herein can be used to treat or prevent any disease or condition in a dog in need thereof. The present invention is particularly useful for the treatment of chronic conditions requiring repeated dosing. Due to the increased half-life of protein drugs, less frequent dosing and / or reduced dosage levels are possible.

[0502] In some embodiments, the disease, disorder, condition or symptom to be treated or prevented is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, skeletal / musculoskeletal disease, cardiovascular disease, neurological disease, kidney disease, metabolic disease, immune disease, genetic / hereditary disease, fertility-related disorder, infectious disease or cancer. In certain embodiments, the disease or condition to be treated or prevented is atopic dermatitis, allergic dermatitis, food allergy, osteoarthritis pain, perioperative pain, dental pain, cancer pain, arthritis, anemia, obesity, or diabetes.

[0503] Antibodies can be used not only to treat or prevent disease, but also to regulate normal biological functions, such as managing fertility or behavior.

[0504] diagnosis

[0505] One or more polypeptides disclosed herein can also be used for various diagnostic purposes, for example, to determine whether a dog has any particular disease or condition. In some embodiments, one or more polypeptides may include a binding domain. The binding domain may specifically bind to a protein, subunit, domain, motif, and / or epitope (e.g., a marker of a cancer cell) as described herein. In some embodiments, one or more polypeptides also include a tag group. Typically, the tag group falls into various categories, depending on the test they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active parts; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, labeled as tags, etc.). In some embodiments, the tag group is coupled to the antibody by spacer arms of various lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be performed in the present invention.

[0506] In some embodiments, the tag group is a probe, a dye (e.g., a fluorescent dye), or a radioisotope (e.g., 3 H. 14 C. 22 Na, 36 Cl, 35 S. 33 P, or 125 I).

[0507] Specific labels may also include optical dyes, including but not limited to chromophores, phosphors and fluorophores, the latter being specific in many cases. Fluorophores may be "small molecule" sodium fluorescein (fluore), or protein fluorescein.

[0508] A fluorescent tag can be any molecule detectable by its intrinsic fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, and Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.) Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook by Richard P. Haugland, which is incorporated herein by reference in its entirety.

[0509] Suitable protein fluorescent tags also include, but are not limited to, green fluorescent protein, including GFP from Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U. Sci. SA85:2603-2607) and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019, U.S. Pat. Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, 5,925,558). All of the above-cited references in this paragraph are expressly incorporated herein by reference in their entirety.

[0510] test

[0511] Fc γ RI binding:

[0512] Binding to Fc γ RI is a measure of the ability of an antibody to mediate ADCC. To evaluate this property of an antibody, the antibody can be measured for Fc γ Test of RI binding.

[0513] C1q binding:

[0514] Binding to the first component of complement, CIq, is a measure of the ability of an antibody to mediate complement dependent cytotoxicity (CDC). To assess antibodies for this property, assays measuring antibody binding to CIq can be performed using methods known in the art.

[0515] half life:

[0516] Methods for measuring the half-life of antibodies are known in the art. See, for example, Booth et al., MAbs, 10(7): 1098-1110 (2018). As an example, the half-life of an antibody can be measured by injecting the antibody into an animal model and measuring the antibody level in serum over a certain period of time. Exemplary animal models include non-human primates and transgenic mouse models. Transgenic mouse models (e.g., Tg32 or Tg276 transgenic mice) can be null for mouse FcRnα chains and express human FcRnα transgenes (e.g., under the control of a constitutive promoter). Human FcRnα chains can be paired with mouse β2 microglobulin in vivo to form functional chimeric FcRn heterodimers.

[0517] Example

[0518] Example 1: Alanine scanning mutagenesis of the CH2 and CH3 domains of canine IgG.B

[0519] Alanine scanning mutagenesis was performed on residues 250, 251, 252, 254, 256, 285, 286, 307, 309, 311, 315 in the CH2 domain and residues 378, 380, 428, 430, 433, 434, 435 and 436 in the CH3 domain (Morrison and Weiss, Curr. Opin. Chem. Biol. 5: 302-307 (2001)). For this experiment, the wild-type (wt) sequence of the CH2 and CH3 domains of canine IgG.B was synthesized and used as a template for mutation. Each specific position except position 254 was individually changed to alanine by PCR mutagenesis using primers encoding the changes. Position 254 was alanine in the wild-type sequence and was modified to serine. The PCR product was subcloned into the GenScript FASEBA plasmid, transformed into E. coli and the sequence was verified for the presence of variants. Upstream of the CH2 domain is a SASA (single domain antibody to serum albumin) tag (see, e.g., US 2013 / 0129727A1), which has a pM affinity for albumin. A PelB (pectate lyase B) signal peptide is located at the N-terminus to promote secretion of Fc to the culture medium. The expression of the CH2-CH3 protein is regulated by the Lac promoter. The supernatant from the conditioned medium was analyzed for binding to canine FcRn (UniProtKB-E2R0L6 [FcRn] and UniProtKB-E2RN10 [canine beta-2-microglobulin]) at pH 5.5 using surface plasmon resonance (SPR).

[0520] For SPR analysis using Biacore 8K, bovine serum albumin (BSA) was fixed to a CM5 sensor chip. The sensor chip surfaces of flow cells 1 and 2 were activated by freshly mixing 50 mmol / L of N-hydroxysuccinimide and 200 mmol / L of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for 420 seconds (10 μL / min). Thereafter, BSA diluted in 10 mM sodium acetate (pH 4.5) was injected into flow cell 2 to activate conjugation, while flow cell 1 was set to blank. After the alanine coupling reaction, the remaining activated coupling sites on the chip surface were blocked by a 420 second injection of 1 mM ethanolamine hydrochloride. The running buffer used for the binding experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 5.5) and was run at 25 ° C. Supernatants from alanine variants were injected on the chip surface and captured on immobilized BSA via the SASA tag for 60 seconds. Canine FcRn at 400 nM was injected for 120 seconds and dissociation was completed by running buffer for 120 seconds. The flow rate of the stationary phase of BSA was 10 μl / min, and the flow rate of the binding and dissociation phases was 30 μl / min. All data were processed using Biacore8K Evaluation Software Version 1.1. Tabulated data are shown in Table 3, with the last column containing the average KD of the wild type divided by the variant KD. The sensor graph is shown in Figure 7A-7U .

[0521] Table 3

[0522]

[0523]

[0524]

[0525] Example 2: Generation of NNK saturation mutation libraries at selected positions and analysis of individual mutations

[0526] The NNK saturation mutagenesis method is an effective strategy for generating all 20 possible amino acids at the desired position (Hogrefe et al., Biotechniques. 33: 1158-1165 (2002)). A single NNK library at positions 250, 252, 254, 309, 311, 378, 380, and 434 (EU numbering) was generated. For this method, NNK (N = A / C / G / T, K = G / T) primers at specific positions were used together with the QuikChange Site-Directed Mutagenesis Kit (Agilent). The Biacore method described in Example 1 was used to test the binding of canine FcRn at pH 5.5 to the supernatant of 90 individual transformants from each library. The only difference was that the concentration of canine FcRn used for the test was 200nM to 400nM. The sensor diagrams for all NNK library variants are shown in Figures 8-15.

[0527] For the NNK pool at position 250, no variant showed increased binding to canine FcRn at pH 5.5. Data from variants T250E and T250Q and wild-type Fc are shown in Table 4. In competitive binding assays, variants T250E and T250Q in human IgG2 have been shown to bind more tightly to human FcRn at pH 6.0 compared to wild-type human IgG2 Fc (Hinton et al., J. Biol. Chem. 279: 6213-6216 (2004)).

[0528] Table 4

[0529] Variants ka(1 / Ms) kd(1 / s) KD(M) T250Q 9.96E+04 2.58E-01 2.59E-06 T250Q 9.43E+04 2.68E-01 2.84E-06 T250E 1.14E+05 2.84E-01 2.48E-06 T250E 1.72E+05 2.87E-01 1.66E-06 WT 3.87E+04 3.47E-01 8.99E-06 WT 1.14E+05 3.54E-01 3.11E-06

[0530] For the NNK pool at position 252, only variants L252Y and L252M had significantly higher affinity for canine FcRn at pH 5.5 (see Table 5 below). In 90 transformants, the L252F variant was not present, so no binding data were obtained for this variant.

[0531] Table 5

[0532] Variants ka(1 / Ms) kd(1 / s) KD(M) L252Y 4.02E+05 3.97E-02 9.87E-08 L252Y 3.58E+05 4.10E-02 1.14E-07 L252M 1.93E+05 1.68E-01 8.69E-07 L252M 2.18E+05 1.69E-01 7.74E-07 WT 1.68E+05 2.88E-01 1.71E-06 WT 1.23E+05 3.26E-01 2.66E-06

[0533] For the NNK library at position 254, the variant without test has significantly higher affinity to canine FcRn at pH 5.5. The data of A254T variant are shown in Table 6 and the corresponding variant in human IgG1 has been used for YTE variant (M252Y / S254T / T256E), which has increased affinity to human FcRn at pH 6.0 (Dall'Acqua et al., J.Immunol.169:5171-5180 (2002)) and has shown the half-life of increased human IgG in preclinical models and people (Borrok et al., J.Biol.Chem.290:4282-4290 (2015); Robbie et al., Antimicrob.Agents Ch.57:6147-6153 (2013)). In 90 transformants, there is no A254H variant, so the data of the variant are not obtained.

[0534] Table 6

[0535] Variants ka(1 / Ms) kd(1 / s) KD(M) A254T 1.63E+05 3.75E-01 2.29E-06 A254T 3.23E+05 4.33E-01 1.34E-06 WT 1.51E+05 3.10E-01 2.05E-06 WT 1.05E+05 3.15E-01 2.99E-06

[0536] For the NNK library at positions 309 and 311, the untested variants have significantly higher affinity for canine FcRn at pH 5.5. The data of variants G309P and Q311V are shown in Tables 7 and 8, and certain combinations of corresponding human variants (L309P and Q311V) and other variants in human IgG1 have been shown to have higher affinity for human FcRn at pH 6.0 (Dall'Acqua et al., J.Immunol.169:5171-5180 (2002); Booth et al., MAbs, 10 (7): 1098-1110 (2018)). Variants G309D, G309K and G311D were not identified in the NNK library, so FcRn binding was not tested for them.

[0537] Table 7

[0538] Variants ka(1 / Ms) kd(1 / s) KD(M) G309P 3.77E+05 2.39E-01 6.35E-07 G309P 5.58E+05 2.42E-01 4.34E-07 G309P 2.07E+05 1.32E-01 6.37E-07 G309P 2.02E+05 1.45E-01 7.18E-07 WT 2.37E+05 2.10E-01 8.84E-07 WT 2.53E+05 2.18E-01 8.62E-07

[0539] Table 8

[0540] Variants ka(1 / Ms) kd(1 / s) KD(M) Q311V 1.52E+06 5.66E-01 3.72E-07 Q311V 1.59E+06 6.96E-01 4.39E-07 WT 2.67E+05 1.70E-01 6.39E-07 WT 2.47E+05 1.71E-01 6.92E-07

[0541] For the NNK library at positions 378 and 380, the untested variants have significantly higher affinity for canine FcRn at pH 5.5. The data of variant D378V are shown in Table 9, and compared with wild-type Fc, the corresponding variants in human IgG1 have been used in combination with other IgG variants to show higher affinity for human FcRn at pH 6.0, and extend the half-life of human IgG in transgenic human FcRn mice (Monnet et al., MABS.6: 422-436 (2014); Booth et al., 2018). In addition, the data of variant E380A are shown in Table 10 and the corresponding variants in human IgG1 have been shown to have higher binding affinity for human FcRn at pH 6.0 (Shields et al., J.Biol.Chem.276: 6591-6604 (2001)). Variants D378E, D378I, D378K, and E380F were not present in the NNK library and were not screened for binding to canine FcRn.

[0542] Table 9

[0543] Variants ka(1 / Ms) kd(1 / s) KD(M) D378V 2.29E+05 1.59E-01 6.93E-07 D378V 1.84E+05 1.60E-01 8.73E-07 WT 3.36E+05 1.69E-01 5.02E-07 WT 2.64E+05 2.07E-01 7.84E-07

[0544] Table 10

[0545] Variants ka(1 / Ms) kd(1 / s) KD(M) E380A 1.68E+05 2.23E-01 1.32E-06 E380A 1.52E+05 2.39E-01 1.57E-06 WT 1.15E+05 1.79E-01 1.56E-06 WT 2.42E+05 1.82E-01 7.54E-07

[0546] For the NNK library at position 434, variants N434Y, N434W, and N434R have higher affinity for canine FcRn at pH 5.5, as shown in Table 11. Variants N434S and N434A do not have higher affinity for canine FcRn at low pH, unlike the corresponding human IgG1 variants (Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Yeung et al., J. Immunol. 182: 7663-7671 (2009); Zalevsky et al., Nat. Biotechnol. 28: 157-159 (2010); Deng et al., Drug Metab. Dispos. 38: 600-605 (2010)). The NNK library screened at position 434 did not include the N434F variant, so this variant was not tested for binding to canine FcRn.

[0547] Table 11

[0548] Variants ka(1 / Ms) kd(1 / s) KD(M) N434Y 6.07E+05 9.68E-03 1.59E-08 N434W 6.93E+05 2.80E-02 4.04E-08 N434W 4.20E+06 3.18E-01 7.57E-08 N434R 4.88E+05 5.24E-02 1.07E-07 N434R 3.99E+05 6.33E-02 1.59E-07 N434S 2.25E+05 2.07E-01 9.24E-07 N434S 1.99E+05 2.09E-01 1.05E-06 N434A 2.72E+05 1.56E-01 5.73E-07 N434A 2.61E+05 1.64E-01 6.29E-07 WT 1.90E+05 1.81E-01 9.55E-07 WT 1.59E+05 2.09E-01 1.31E-06

[0549] Example 3: Binding kinetics to L252Y, N434Y, N434W, N434R, N434H and YET (L252Y / A254T / T256E) variants and wild-type Fc

[0550] Several canine IgG.Bs that showed higher affinity for canine FcRn at pH 5.5 were further evaluated for binding kinetics to canine FcRn. In this study, variants (L252Y, N434Y, N434W, N434R, N434H), YTE variants (L252Y / A254T / T256E) and wild-type canine Fc were evaluated for binding to canine FcRn at pH 5.5 and pH 7.4. The Biacore method for pH 5.5 conditions was the same as described in Example 1, except that 4 concentrations of FcRn (100 nM, 200 nM, 400 nM, 800 nM) were tested to produce more accurate binding kinetics. For Biacore conditions at pH 7.4, the running buffer used was 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4, and the concentration of canine FcRn tested was 200 nM. All variants including YTE and wild type did not bind to canine FcRn at pH 7.4. The binding kinetics at pH 5.5 are shown in Table 12 and the sensorgrams are shown in Figures 16A-16E The tested variants showed increased affinity for canine FcRn at pH 5.5 compared to wild-type Fc.

[0551] Table 12

[0552] Variants ka(1 / Ms) kd(1 / s) KD(M) L252Y 2.75E+05 4.76E-02 1.73E-07 N434Y 5.20E+05 1.51E-02 2.91E-08 N434W 4.46E+05 4.50E-02 1.01E-07 N434R 4.40E+05 8.01E-02 1.82E-07 N434H 4.11E+05 1.02E-01 2.47E-07 wild type 1.68E+05 5.27E-01 3.15E-06 YTE 2.50E+05 4.60E-02 1.84E-07

[0553] Other Implementations

[0554] While the invention has been described in conjunction with the detailed description, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A polypeptide comprising a canine IgG.B Fc region variant, wherein the canine IgG.B Fc region variant is mutated to His at amino acid position 434 of the wild-type canine IgG.B Fc region, wherein the amino acid positions are based on EU numbering, wherein the polypeptide has increased binding to canine FcRn relative to a control polypeptide, and wherein the control polypeptide is identical to the polypeptide except for having a wild-type canine IgG.B Fc region instead of a variant IgG.B Fc region.

2. A polypeptide comprising a canine IgG.B Fc region variant, wherein the canine IgG.B Fc region variant is mutated to Trp at amino acid position 434 of the wild-type canine IgG.B Fc region, wherein the amino acid positions are based on EU numbering, wherein the polypeptide has increased binding to canine FcRn relative to a control polypeptide, and wherein the control polypeptide is identical to the polypeptide except for having a wild-type canine IgG.B Fc region instead of a variant IgG.B Fc region.

3. A polypeptide comprising a canine IgG.B Fc region variant, wherein the canine IgG.B Fc region variant is mutated to Arg at amino acid position 434 of the wild-type canine IgG.B Fc region, wherein the amino acid positions are based on EU numbering, wherein the polypeptide has increased binding to canine FcRn relative to a control polypeptide, and wherein the control polypeptide is identical to the polypeptide except for having a wild-type canine IgG.B Fc region instead of a variant IgG.B Fc region.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide further comprises a binding domain.

5. A polypeptide according to claim 4, wherein the binding domain comprises (i) six complementarity determining regions (CDRs) of an immunoglobulin molecule; (ii) a ligand binding domain of a canine receptor protein; (iii) a nanobody; or (iv) an extracellular domain of a canine receptor protein.

6. A polypeptide according to claim 4, wherein the binding domain specifically binds to an antigen selected from the group consisting of: NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and complement system complexes.

7. The polypeptide according to any one of claims 1 to 3, further comprising a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonist, and thrombopoietin binding peptide.

8. The polypeptide of any one of claims 1 to 3, wherein the polypeptide binds to canine FcRn at a higher level at acidic pH than at neutral pH.

9. A pharmaceutical composition comprising (i) a polypeptide according to any one of claims 1 to 8, and (ii) a pharmaceutically acceptable excipient.

10. One or more nucleic acids encoding a polypeptide according to any one of claims 1 to 8.

11. One or more expression vectors comprising one or more nucleic acids according to claim 10.

12. A host cell comprising one or more nucleic acids according to claim 10.

13. A host cell comprising one or more expression vectors according to claim 11.

14. A method for preparing a polypeptide, the method comprising: include: (a) providing one or more nucleic acids according to claim 10; (b) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; and (c) collecting the polypeptide produced in (b) from the host cell culture.

15. The method of claim 14, further comprising formulating the polypeptide into a pharmaceutical preparation.

16. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for treating a canine disease or condition in a dog in need thereof by the following method, the method comprising administering to the dog an effective amount of a composition comprising the pharmaceutical composition of claim 9, wherein the canine disease or condition is gastrointestinal disease, cardiovascular disease, kidney disease, infectious disease, cancer, atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

17. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing a canine disease or condition in a dog in need thereof by the following method, the method comprising administering to the dog an effective amount of a composition comprising the pharmaceutical composition of claim 9, wherein the canine disease or condition is gastrointestinal disease, cardiovascular disease, kidney disease, infectious disease, cancer, atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

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