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

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 023,083 filed on May 11, 2020 and U.S. Provisional Application No. 63 / 122,417 filed on December 7, 2020, the contents of each of which are incorporated 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.

[0004] Sequence Listing Submitted via EFS-WEB

[0005] The entire contents of the following sequence listing electronically submitted via the USPTO EFS-WEB server, authorized and set forth in MPEP §1730 II.B.2(a), are incorporated herein by reference in their entirety for all purposes. The sequence listing is in the electronically archived text file identified below:

[0006] File name :47406-0015WO1_Sequence_Listing.txt

[0007] Creation Date : May 6, 2021

[0008] Size (bytes) : 34,000 bytes Background Art

[0009] The Fc region of an antibody plays many functional roles, including but not limited to protecting the antibody from degradation by the lysosomal pathway and mediating antibody effector functions. With the increasing use of canine antibodies as therapeutic agents, attention has increased not only in selecting the best Fab, but also in combining it with the appropriate Fc to obtain the desired half-life and effector functions.

[0010] There is little guidance in the art regarding increasing the half-life of polypeptide therapeutics (eg, antibodies) for dogs. The present disclosure remedies this deficiency by providing Fc region variants that improve the serum persistence of polypeptides (eg, antibodies) in dogs. Summary of the invention

[0011] Provided herein are canine Fc (e.g., canine IgG Fc region variants) or canine FcRn binding fragments thereof that can be used for therapeutic polypeptides. The disclosure is characterized in that the polypeptide has an increased binding to canine FcRn compared to a control polypeptide (e.g., a wild-type counterpart IgG canine Fc region). In some cases, at pH 5.5, pH 6.0, and / or pH 6.5, these polypeptides have an increased binding to canine FcRn compared to a control polypeptide. In some cases, compared to at neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5), these polypeptides can, for example, bind to canine FcRn at a higher level at acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5). In some cases, compared to at pH 7.4, these polypeptides bind to canine FcRn at a higher level at pH 5.5 and / or 6.0. The present disclosure relates in part to polypeptides with increased half-life in dogs compared with their wild-type counterparts.For example, a binding molecule (e.g., a ligand binding portion of an antibody or receptor) is provided, which has an increased half-life relative to versions of these binding molecules not connected to Fc regions or their dog FcRn binding regions disclosed herein.Enzyme-Fc region fusions, ligand-Fc region fusions, nanobody-Fc fusions and peptide-Fc region fusions are also provided, wherein the fusions have an increased half-life compared with their wild-type counterparts.In addition to one or more substitutions (relative to wild-type dog Fc regions) with increased half-life, the Fc region may also include other substitutions, such as increasing effector functions, reducing effector functions, increasing the combination with protein A and / or reducing the heterogeneity of polypeptides (e.g., by removing one or more post-translational modifications in the Fc region).Canine Fc region sequences may be from any canine antibody.In some cases, canine Fc region sequences are from canine IgG (e.g., IgGA, IgGB, IgGC or IgGD).

[0012] The present disclosure features a recombinant protein comprising (1) a binding domain or fragment thereof that specifically binds to a ligand or protein epitope, wherein the binding domain is linked to (2) a domain comprising an Fc region (CH2+CH3 region) or a canine FcRn binding region thereof as disclosed herein. In some cases, the binding domain comprises (i) six complementarity determining regions (CDRs) of a canine or human / humanized antibody; (ii) a nanobody; (iii) a soluble receptor binding domain or a ligand binding fragment thereof that binds to a ligand, and (iv) an extracellular domain of a canine receptor protein.

[0013] 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), the first Fc region comprising a canine IgG Fc region variant described herein; and (2) a second polypeptide comprising a second Fc region, the second Fc region comprising a canine IgG Fc region variant described herein. The first polypeptide and the second polypeptide can be associated through the first Fc region and the second Fc region. In some cases, the amino acid sequences of the first Fc region and the second Fc region are identical. In other cases, the amino acid sequences of the first Fc region and the second Fc region are different (e.g., there are 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 that are different). In some cases, the Fc region variant is a variant of the canine IgGB antibody Fc region. In some cases, the Fc region variant is a variant of a canine IgGA antibody Fc region. In some cases, the Fc region variant is a variant of a canine IgGC antibody Fc region. In some cases, the Fc region variant is a variant of a canine IgGD antibody Fc region.

[0014] Also disclosed are fusion molecules comprising canine IgG Fc region variants and polypeptides disclosed herein. In some cases, canine IgG Fc region variants are covalently linked to polypeptides (e.g., through hinge regions or joints). 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 agonists, and canine thrombopoietin binding peptides. In some cases, the polypeptide is a scFv, a nanobody, or a single domain antibody. In some cases, IgG Fc region variants are variants of canine IgGB antibody Fc regions. In some cases, IgG Fc region variants are variants of canine IgGA antibody Fc regions. In some cases, IgG Fc region variants are variants of canine IgGC antibody Fc regions. In some cases, the IgG Fc region variant is a variant of a canine IgGD antibody Fc region.

[0015] In some aspects, the disclosure provides a polypeptide comprising a canine IgG Fc region variant or a canine FcRn binding region thereof, wherein the polypeptide comprises at least one amino acid substitution at a position selected from the group consisting of:

[0016] (i) the position corresponding to amino acid position 286 of wild-type canine IgG;

[0017] (ii) the position corresponding to amino acid position 312 of wild-type canine IgG;

[0018] (iii) a position corresponding to amino acid position 426 of wild-type canine IgG; and

[0019] (iv) the position corresponding to amino acid position 436 of wild-type canine IgG,

[0020] wherein the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of Tyr, Phe, Leu and Trp, wherein the amino acid position is based on EU numbering, and wherein the binding affinity of the polypeptide to canine FcRn is increased when compared to the Fc domain of wild-type canine IgG.

[0021] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 312 of wild-type canine IgG.

[0022] In some embodiments, the polypeptide comprises a Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG.

[0023] In some embodiments, at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises Tyr, His, or Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0024] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 436 of wild-type canine IgG. In some embodiments, the polypeptide comprises His at an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0025] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

[0026] In some embodiments, the polypeptide comprises at least one additional amino acid substitution at a position selected from the group consisting of:

[0027] (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0028] (ii) the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0029] (iii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0030] (iv) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0031] (v) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0032] (vi) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0033] (vii) the amino acid position corresponding to amino acid position 286 of wild-type canine IgG,

[0034] (viii) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0035] (ix) the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0036] (x) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0037] (xi) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0038] (xii) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0039] (xiii) the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0040] (xiv) the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0041] (xv) the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0042] (xvi) the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0043] (xvii) the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0044] (xviii) the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and

[0045] (xix) Amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0046] In some embodiments, the polypeptide comprises:

[0047] (i) Glu or GIn at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0048] (ii) Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0049] (iii) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0050] (iv) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0051] (v) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0052] (vi) Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0053] (vii) Asp, Tyr, Phe, Leu or Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG,

[0054] (viii) Arg, GIn or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0055] (ix) Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0056] (x) Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0057] (xi) Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0058] (xii) Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0059] (xiii) Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0060] (xiv) Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0061] (xv) Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0062] (xvi) Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0063] (xvii) Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0064] (xviii) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and / or

[0065] (xix) Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0066] In some embodiments, at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0067] In some embodiments, the polypeptide comprises at least one additional amino acid substitution at a position selected from the group consisting of:

[0068] (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0069] (ii) the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0070] (iii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0071] (iv) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0072] (v) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0073] (vi) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0074] (vii) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0075] (viii) the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0076] (ix) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0077] (x) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0078] (xi) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0079] (xii) the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0080] (xiii) the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0081] (xiv) the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0082] (xv) the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0083] (xvi) the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0084] (xvii) the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and

[0085] (xviii) The amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0086] In some embodiments, the polypeptide comprises:

[0087] (i) Glu or GIn at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0088] (ii) Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0089] (iii) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0090] (iv) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0091] (v) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0092] (vi) Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0093] (vii) Arg, GIn or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0094] (viii) Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0095] (ix) Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0096] (x) Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0097] (xi) Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0098] (xii) Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0099] (xiii) Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0100] (xiv) Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0101] (xv) Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0102] (xvi) Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0103] (xvii) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and / or

[0104] (xviii) Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0105] In some embodiments, at least one additional amino acid substitution is at a position selected from the group consisting of:

[0106] (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0107] (ii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0108] (iii) the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0109] (iv) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0110] (v) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0111] (vi) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0112] (vii) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0113] (viii) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0114] (ix) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0115] (x) the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, and

[0116] (xi) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0117] In some embodiments, the polypeptide comprises:

[0118] (i) Glu or GIn at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0119] (ii) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0120] (iii) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0121] (iv) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0122] (v) Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0123] (vi) Arg, GIn or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0124] (vii) Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0125] (viii) Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0126] (ix) Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0127] (x) Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, and

[0128] (xi) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0129] In some embodiments, at least one additional amino acid substitution is at a position selected from the group consisting of:

[0130] (i) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0131] (ii) the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0132] (iii) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and

[0133] (iv) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0134] In some embodiments, the polypeptide comprises:

[0135] (i) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0136] (ii) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0137] (iii) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or

[0138] (iv) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0139] In some embodiments, the polypeptide comprises:

[0140] (i) Tyr at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0141] (ii) Thr at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0142] (iii) Glu at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or

[0143] (iv) Trp, Tyr, Arg or His at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0144] In some embodiments, the polypeptide further comprises at least one of the following:

[0145] (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256;

[0146] (ii) Leu at amino acid position 428 and Ser at amino acid position 434;

[0147] (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311;

[0148] (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378;

[0149] (v) Asp at amino acid position 256, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311;

[0150] (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315;

[0151] (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;

[0152] (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378;

[0153] (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378;

[0154] (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378;

[0155] (xi) Asp at amino acid position 256, Asp at amino acid position 285, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 378;

[0156] (xii) Asp at amino acid position 256, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, Val at amino acid position 311, and Val at position 378;

[0157] (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0158] (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378;

[0159] (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;

[0160] (xvi) Gln at amino acid position 307, Ala at amino acid position 380, Ser or Ala at amino acid position 434;

[0161] (xvii) Leu at amino acid position 428 and Ser or Ala at amino acid position 434; or

[0162] (xviii) Gln at amino acid position 250 and Leu at amino acid position 428.

[0163] In some aspects, the disclosure provides a polypeptide comprising a canine IgG Fc region variant or a canine FcRn binding region thereof, wherein the polypeptide comprises amino acid substitutions at two or more positions selected from the group consisting of:

[0164] (i) the position corresponding to amino acid position 286 of wild-type canine IgG;

[0165] (ii) the position corresponding to amino acid position 312 of wild-type canine IgG;

[0166] (iii) the position corresponding to amino acid position 426 of wild-type canine IgG;

[0167] (iv) a position corresponding to amino acid position 434 of wild-type canine IgG; and

[0168] (v) the position corresponding to amino acid position 436 of wild-type canine IgG,

[0169] wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity for canine FcRn when compared to the Fc domain of wild-type canine IgG.

[0170] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from T286L, T286Y, and any of the foregoing conservative amino acid substitutions.

[0171] In some embodiments, the polypeptide of claim 1, wherein the amino acid substitution at the position corresponding to amino acid position 312 of wild-type canine IgG is D312P or a conservative amino acid substitution thereof.

[0172] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG is selected from A426Y, A426H, and any of the foregoing conservative amino acid substitutions.

[0173] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 434 of wild-type canine IgG is N434R or a conservative amino acid substitution thereof.

[0174] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG is Y436H or a conservative amino acid substitution thereof.

[0175] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG.

[0176] In some embodiments, the polypeptide comprises amino acid substitutions at two or more positions selected from the group consisting of:

[0177] (i) positions corresponding to amino acid positions 426 and 286 of wild-type canine IgG;

[0178] (ii) positions corresponding to amino acid positions 426 and 312 of wild-type canine IgG;

[0179] (iii) positions corresponding to amino acid positions 426 and 434 of wild-type canine IgG;

[0180] (iv) positions corresponding to amino acid positions 426 and 436 of wild-type canine IgG; and

[0181] (v) Positions corresponding to amino acid positions 286, 426 and 436 of wild-type canine IgG.

[0182] In some embodiments, the polypeptide comprises an amino acid substitution selected from the group consisting of:

[0183] (i) A426Y and T286L;

[0184] (ii) A426Y and D312P;

[0185] (iii) A426Y and Y436H;

[0186] (iv) A426H and T286L;

[0187] (v) A426H and T286Y;

[0188] (vi) A426H and D312P; and

[0189] (vii) T286L, A426Y and Y436H.

[0190] In some embodiments, the two or more amino acid substitutions are selected from the group consisting of:

[0191] (i) A426Y in combination with one or more of T286L, D312P, N434R and Y436H;

[0192] (ii) A426H in combination with one or more of T286L, T286Y, D312P, N434R and Y436H; and

[0193] (iii) a combination of N434R and one or more of T286L, T286Y, D312P and Y436H.

[0194] In some embodiments, the wild-type canine IgG is a canine IgGA comprising an Fc domain having an amino acid sequence at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO:9, a canine IgGB comprising an Fc domain having an amino acid sequence at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO:10, a canine IgGC comprising an Fc domain having an amino acid sequence at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO:11, or a canine IgGD comprising an Fc domain having an amino acid sequence at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO:12.

[0195] In some embodiments, the wild-type canine IgG is canine IgGA, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 9. In some embodiments, the wild-type canine IgG is canine IgGB, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 10. In some embodiments, the wild-type canine IgG is canine IgGC, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 11. In some embodiments, the wild-type canine IgG is canine IgGD, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 12.

[0196] In some embodiments, the wild-type canine IgG is canine IgGA, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:9.

[0197] In some embodiments, the wild-type canine IgG is canine IgGB, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:10.

[0198] In some embodiments, the wild-type canine IgG is a canine IgGC, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:11.

[0199] In some embodiments, the wild-type canine IgG is canine IgGD, and the canine IgG Fc region variant or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:12.

[0200] In some embodiments, the polypeptide further comprises a binding domain.

[0201] In some embodiments, 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.

[0202] In some embodiments, 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.

[0203] In some embodiments, the polypeptide further comprises a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, and a thrombopoietin binding peptide.

[0204] In some embodiments, the polypeptide binds to canine FcRn at a higher level at acidic pH than at neutral pH in a binding assay. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 5.5 than at pH 7.4 in a binding assay. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4 in a binding assay.

[0205] In some embodiments, the polypeptide has: (1) increased half-life in dog 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 the IgG Fc region variant; and / or (2) increased binding to canine FcRn compared to the control polypeptide; and wherein the amino acid positions are based on EU numbering.

[0206] In some embodiments, the polypeptide comprises an amino acid sequence that 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 SEQ ID NO:9.

[0207] In some embodiments, the polypeptide comprises an amino acid sequence that 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 SEQ ID NO:10.

[0208] In some embodiments, the polypeptide comprises an amino acid sequence that 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 SEQ ID NO:11.

[0209] In some embodiments, the polypeptide comprises an amino acid sequence that 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 SEQ ID NO:12.

[0210] In some aspects, the present disclosure provides pharmaceutical compositions comprising (i) a polypeptide described herein and (ii) a pharmaceutically acceptable excipient.

[0211] In some aspects, the disclosure provides one or more nucleic acids encoding the polypeptides described herein.

[0212] In some aspects, the disclosure provides one or more expression vectors comprising one or more nucleic acids described herein.

[0213] In some aspects, the disclosure provides a host cell comprising one or more nucleic acids described herein or one or more expression vectors described herein.

[0214] In some aspects, the present disclosure provides methods of preparing one or more polypeptides, the methods comprising:

[0215] (a) providing one or more nucleic acids described herein;

[0216] (b) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; and

[0217] (c) collecting the polypeptide produced in (b) from the host cell culture.

[0218] In some embodiments, the method further comprises formulating the polypeptide into a pharmaceutical preparation.

[0219] In some aspects, the present disclosure provides methods of treating a canine disease or condition in a dog in need thereof, the method comprising administering to the dog an effective amount of a composition comprising a pharmaceutical composition described herein.

[0220] In some aspects, the present disclosure provides methods of preventing a canine disease or condition in a dog in need thereof, the method comprising administering to the dog an effective amount of a composition comprising a pharmaceutical composition described herein.

[0221] In some aspects, the present disclosure provides a pharmaceutical composition described herein for use in a method of treating a canine disease or disorder in a dog in need thereof.

[0222] In some aspects, the present disclosure provides a pharmaceutical composition described herein for use in a method of preventing a canine disease or disorder in a dog in need thereof.

[0223] In some aspects, the disclosure provides use of a polypeptide described herein in the manufacture of a medicament for treating a canine disease or disorder in a dog in need thereof.

[0224] In some aspects, the disclosure provides use of a polypeptide described herein in the manufacture of a medicament for preventing a canine disease or disorder in a dog in need thereof.

[0225] In some embodiments, the disease or disorder is an allergic disease, chronic pain, acute pain, an inflammatory disease, an autoimmune disease, an endocrine disease, a gastrointestinal disease, a cardiovascular disease, a renal disease, a fertility-related disorder, an infectious disease, or cancer.

[0226] In some embodiments, the disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0227] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing 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) shall prevail. The materials, methods and examples are illustrative only and are not intended to be limiting.

[0228] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0229] 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 with blocks. These sequences are designated as SEQ ID NOs: 13, 14, 15 and 16, respectively.

[0230] Figure 2 is an alignment of the amino acid sequences of the CH2 region of canine IgG gamma chain. These sequences are designated as SEQ ID NOs: 1, 2, 3 and 4, respectively. Residues that are substituted to increase half-life are identified by underlining.

[0231] Figure 3 is an alignment of the amino acid sequences of the CH3 region of canine IgG gamma chain. These sequences are designated as SEQ ID NOs: 5, 6, 7 and 8, respectively. Residues that are substituted to increase half-life are identified by underlining.

[0232] Figure 4 is an alignment of the amino acid sequences of the Fc region of canine IgG gamma chain. These sequences are designated as SEQ ID NOs: 9, 10, 11 and 12, respectively. Residues that were substituted to increase half-life are identified by underlining.

[0233] Figure 5 This is a table providing EU numbering for the CH2 region of canine IgG.

[0234] Figure 6 This is a table providing EU numbering for the CH3 region of canine IgG.

[0235] Figure 7A-7U Biacore sensorgrams from alanine scanning mutagenesis experiments are depicted. The lighter line in each graph represents the measured data and the darker line represents the fitted curve using a 1:1 interaction model.

[0236] Figures 8A-8C Depicted are Biacore sensorgrams of wild type and different variants from the NNK library at position 250. The lighter line in each graph represents the measured data and the darker line is the fitted curve using a 1:1 interaction model.

[0237] Figures 9A-9C Depicted are Biacore sensorgrams of wild type and different variants from the NNK library at position 252. The lighter line in each graph represents the measured data and the darker line is the fitted curve using a 1:1 interaction model.

[0238] Fig. 10A and 10B Depicted are the Biacore sensorgrams of wild type and variant A254T. The lighter lines represent the measured data and the darker lines represent the fitted curves using a 1:1 interaction model.

[0239] Fig.11A and 11B Biacore sensorgrams of wild type and variant G309P are depicted. The lighter lines represent measured data and the darker lines represent fitted curves using a 1:1 interaction model.

[0240] Fig. 12A and 12B Depicted are the Biacore sensorgrams of wild type and variant Q311V. The lighter lines represent the measured data and the darker lines represent the fitted curves using a 1:1 interaction model.

[0241] Fig.13A and 13B Depicted are the Biacore sensorgrams of wild type and variant D378V. The lighter lines represent the measured data and the darker lines represent the fitted curves using a 1:1 interaction model.

[0242] Fig.14A and 14B Biacore sensorgrams of wild type and variant E380A are depicted. The lighter lines represent measured data and the darker lines represent fitted curves using a 1:1 interaction model.

[0243] Figures 15A-15F Depicted are Biacore sensorgrams of wild type and different variants from the NNK library at position 434. The lighter lines represent measured data and the darker lines represent fitted curves using a 1:1 interaction model.

[0244] Figures 16A-16E Depicted are the Biacore sensorgrams of the 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 lighter line in each figure is the measured data and the darker line is the fitted curve using a 1:1 interaction model.

[0245] Figures 17A-17B Depicted are the Biacore NNK library sensorgrams of different variants. The concentration of canine FcRn used was 200 nM. The brighter line in each graph is the measured data and the darker line is the fitted curve using a 1:1 interaction model.

[0246] Figures 18A-18B Depicted are the Biacore sensorgrams of the different variants in a concentration series. The concentrations of canine FcRn used were (lines from bottom to top): 100 nM, 200 nM, 400 nM and 800 nM. The lines are fitted curves using a 1:1 interaction model.

[0247] Figures 19A-19CBiacore sensorgrams of IgGA wild type, IgGA A426Y variant and IgGA A426H variant binding to canine FcRn at pH 6.0 are shown. The concentrations of canine FcRn used were 50 nM (white squares), 100 nM (black circles), 200 nM (white circles), 400 nM (black triangles) and 800 nM (white triangles). The brighter lines in each sensorgram represent the measured data, and the darker lines represent the fitted curves using a 1:1 interaction model.

[0248] Figures 20A-20F Biacore sensorgrams of wild-type canine IgGB Fc, single variants (A426Y) of canine IgGB Fc, and combination variants combined with canine FcRn at pH 6.0 are shown. The concentration of canine FcRn for wild-type IgG is 200nM (white circles), 400nM (black triangles), 800nM (white triangles), 1600nM (black diamonds), and 3200nM (white diamonds). The concentration of canine FcRn for the remaining variants is 50nM (white squares), 100nM (black circles), 200nM (white circles), 400nM (black triangles), and 800nM (white triangles). The brighter lines in each sensorgram represent measured data, and the darker lines represent the fitting curves using the 1:1 interaction model.

[0249] Figures 21A-21F Shown are Biacore sensorgrams of single variants (A426H) and combination variants of canine IgGB Fc binding to canine FcRn at pH 6.0. The concentration of canine FcRn used for A426H-N434R IgG variants is 200nM (white circles), 400nM (black triangles), 800nM (white triangles), 1600nM (black diamonds) and 3200nM (white diamonds). The concentration of canine FcRn used for the remaining variants is 50nM (white squares), 100nM (black circles), 200nM (white circles), 400nM (black triangles) and 800nM (white triangles). The brighter lines in each sensorgram represent measured data, and the darker lines represent the fitting curves using the 1:1 interaction model.

[0250] Figures 22A-22EBiacore sensorgrams of single variants (N434R) and combined variants of canine IgGB Fc binding to canine FcRn at pH 6.0 are shown. The concentrations of canine FcRn used for the variants were 50 nM (white squares), 100 nM (black circles), 200 nM (white circles), 400 nM (black triangles), and 800 nM (white triangles). The brighter lines in each sensorgram represent the measured data, and the darker lines represent the fitted curves using a 1:1 interaction model.

[0251] Fig.23 Biacore sensorgrams of the N434Y variant of canine IgGB Fc binding to canine FcRn at pH 6.0 are shown. The concentrations of canine FcRn used for the variants were 50 nM (white squares), 100 nM (black circles), 200 nM (white circles), 400 nM (black triangles), and 800 nM (white triangles). The brighter lines in each sensorgram represent the measured data, and the darker lines represent the fitted curves using a 1:1 interaction model.

[0252] Figures 24A-24F Biacore sensorgrams of wild-type canine IgGB Fc, single variants (A426Y) and combined variants of IgGB Fc binding to canine FcRn at pH 7.4 are shown. The brighter lines in each sensorgram represent measured data and the darker lines represent fitted curves using a 1:1 interaction model.

[0253] Figures 25A-25F Biacore sensorgrams of single variant (A426H) and combination variants of IgGB Fc binding to canine FcRn at pH 7.4 are shown. The brighter lines in each sensorgram represent the measured data and the darker lines represent the fitted curves using a 1:1 interaction model.

[0254] Figures 26A-26E Biacore sensorgrams of single variant (N434R) and combined variants of IgGB Fc binding to canine FcRn at pH 7.4 are shown. The brighter lines in each sensorgram represent the measured data and the darker lines represent the fitted curves using a 1:1 interaction model.

[0255] Fig. 27 Biacore sensorgrams of the N434Y variant of IgGB Fc binding to canine FcRn at pH 7.4 are shown. The lighter line represents the measured data and the darker line represents the fitted curve using a 1:1 interaction model.

[0256] Fig.28The amino acid sequences of canine IgGB Fc and human IgG1 Fc are aligned. The positions of amino acid substitutions made to generate canine Fc variants (according to EU numbering) are underlined.

[0257] Fig.29 The terminal half-life (days; Y axis) of canine anti-nerve growth factor (NGF) IgGB Fc variants carrying single amino acid substitutions or combinations of amino acid substitutions after intravenous administration to male (M) and female (F) beagles is shown. The animals were randomly divided into eight groups, with one male and one female in each group. A single intravenous dose of 2 mg / kg of the antibody was administered to each animal, and approximately 1.5 ml of whole blood was collected at the following time points: 0 hours (pre-dose), 4 hours after injection, and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days. Serum was separated from the whole blood and the presence of anti-NGF antibodies was determined by ELISA. Non-compartmental PK analysis (NCA) was performed on each individual serum antibody measurement using PKSolver (Yong Zhang et al., Comput. Methods Prog rams Biomed.; 2010 Sep;99(3):306-14. doi:10.1016 / j.cmpb.2010.01.007).

[0258] Figures 30A-30K Biacore sensorgrams of different variants of IgGB binding to canine FcRn at pH 5.9 are shown.

[0259] Fig.31 is a schematic illustration of a two-compartment pharmacokinetic (PK) model with linear clearance using nonlinear mixed effects modeling.

[0260] Figures 32A-32B Serum concentrations of different variants of IgGB over time are shown.

[0261] Fig.33 Predicted serum concentration curves for antibodies carrying wild-type IgGB Fc or IgGB variants A426Y, A426Y+Y436H, A426Y+Y436H+T286L, N434R, N434Y and YTE are shown.

[0262] Fig.34 Structural models of canine Fc positions 286, 426, and 436 are shown.

[0263] Fig.35The structural model of the canine Fc position of A426H is shown. The left structure is the FcRn large subunit p51, where the WT structure is shown in dark gray and the mutant structure is shown in light gray. The right structure is the IgGB Fc, where the WT structure is shown in dark gray and the mutant structure is shown in light gray.

[0264] Fig.36 A structural model of the canine Fc position of A426Y is shown. The left structure is the FcRn large subunit p51, where the WT structure is shown in dark gray and the mutant structure is shown in light gray. The right structure is the IgGB Fc, where the WT structure is shown in dark gray and the mutant structure is shown in light gray.

[0265] Fig.37 Structural models of the canine Fc position of Y436H are shown. The left structure is the FcRn large subunit p51, where the WT structure is shown in dark grey and the mutant structure is shown in light grey. The right structure is the IgGB Fc, where the WT structure is shown in dark grey and the mutant structure is shown in light grey.

[0266] Fig.38 The structural model of the canine Fc position of T286L is shown. The left structure (upper part) is β2 microglobulin, wherein the WT structure is shown in dark gray and the mutant structure is shown in light gray; and FcRn large subunit p51 (lower part), wherein the WT structure is shown in dark gray and the mutant structure is shown in light gray. The right structure is IgGB Fc, wherein the WT structure is shown in dark gray, and the mutant structure is shown in light gray.

[0267] Fig.39 Structural model of canine Fc positions showing T286L, A426Y and Y436H. The left structure is β2 microglobulin and FcRn large subunit p51. The right structure is IgGB Fc. DETAILED DESCRIPTION

[0268] 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 a variety of canine diseases, it is important to develop polypeptides with extended half-lives, particularly for the prevention or treatment of chronic diseases in which repeated administration of the polypeptide is necessary.

[0269] Thus, the disclosure features canine immunoglobulin Fc regions or canine FcRn binding regions thereof, comprising mutations that enhance the half-life of one or more polypeptides containing these sequences. Also disclosed are polypeptides containing these domains and methods of using them. These peptides can be used for a variety of therapeutic and diagnostic purposes.

[0270] Where values ​​are described in the form of ranges, it is to be understood that the description includes disclosure of all possible subranges within such ranges, as well as specific numerical values ​​falling within such ranges, regardless of whether specific numerical values ​​or specific subranges are explicitly stated. All numerical designations (e.g., pH, temperature, time, concentration, and molecular weight, including ranges) are approximate values, varying by (+) or (-) increments of 1.0 or 0.1, as the case may be, or alternatively by + / -15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%. It is to be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about". It is also to be understood that, although not always explicitly stated, the agents described herein are exemplary only and equivalents of such agents are known in the art.

[0271] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0272] Canine Antibodies

[0273] Dogs have four IgG heavy chains, called A, B, C and D. These heavy chains represent four different subclasses of dog IgG, which are called IgGA, IgGB, IgGC and IgGD. The amino acid and DNA sequences of these heavy chains can be obtained from Tang et al., Vet. Immunol. Immunopathol., 80:259-270 (2001) and the GENBANK database. For example, the amino acid sequence of the IgGA heavy chain has GENBANK accession number AAL35301.1, IgGB has GENBANK accession number AAL35302.1, IgGC has GENBANK accession number AAL35303.1, and IgGD has GENBANK accession number AAL35304.1. Canine antibodies also include two types of light chains: κ and λ. The DNA and amino acid sequences of these light chains can also be obtained from the GENBANK database. For example, the dog κ light chain amino acid sequence has accession number ABY57289.1, and the dog λ light chain has accession number ABY55569.1.

[0274] CH2 region of canine Fc region:

[0275] The CH2 region of a canine antibody comprises or consists of amino acids 237 to 340 (according to EU numbering) of a canine IgG antibody. It should be understood that the CH2 region may include one to six (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N and / or C terminus.

[0276] The amino acid sequence of the CH2 region of canine IgGA is provided below:

[0277] GPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISW FVDGK EVHTAKTQSREQQFNGTYRV VSVLPIEHQD WLTGKE FKCR VNHIDLPSPI ERTISKAR(SEQ ID NO:1)

[0278] The amino acid sequence of the CH2 domain of canine IgGB is provided below:

[0279] GPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQFNGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKAR(SEQ ID NO:2)

[0280] The amino acid sequence of the CH2 domain of canine IgGC is provided below:

[0281] GPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQSNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTP(SEQ ID NO:3)

[0282] The amino acid sequence of the CH2 domain of canine IgGD is provided below:

[0283] GPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWF VD GKEVHTAKTQPREQQFNSTY RVVSVLPIEH QDWLTGKEF K CRVNHIGLPS PIERTISKAR(SEQ ID NO:4)

[0284] CH3 region of canine Fc region:

[0285] The CH3 region of a canine antibody comprises 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 one to six (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N and / or C terminus.

[0286] The amino acid sequence of the CH3 domain of canine IgGA is provided below:

[0287] KPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLDEDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK(SEQ ID NO:5)

[0288] The amino acid sequence of the CH3 domain of canine IgGB is provided below:

[0289] QP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSYFLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK(SEQ ID NO:6)

[0290] The amino acid sequence of the CH3 domain of canine IgGC is provided below:

[0291] Q PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQS NG QQEPESKYRM TPPQLDEDGSYFLYSKLSVD KSRWQRGDT F ICAVMHEALH NHYTQISLSH SPGK(SEQ ID NO:7)

[0292] The amino acid sequence of the CH3 domain of canine IgGD is provided below:

[0293] QPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSN GQPEP ESKYHTTAPQLDEDGSYFLY SKLSVDKSRW QQGDTF TCAV MHEALQNHYT DLSLSHSPGK(SEQ ID NO:8)

[0294] Fc region of canine Fc region:

[0295] 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.

[0296] The amino acid sequence of the Fc domain of canine IgGA is provided below:

[0297] VPEPLGGPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPEVQISWFVDGK EVHTAKTQSREQQFNGTYRV VSVLPIEHQDWLTGKEFKCR VNHIDLPSPI ERTISKARGR AHKPSVYVLP PSPKELSSSDTVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMHETLQNHYTDL SLSHSPGK(SEQ ID NO:9)

[0298] The amino acid sequence of the Fc domain of canine IgGB is provided below:

[0299] APEMLGGPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQTAKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKARGQAHQP SVYVLPPSREELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTTPPQLDEDGSY FLYSKLSVDK SRWQRGDTFICAVMHEALHN HYTQESLSHS PGK(SEQ ID NO:10)

[0300] The amino acid sequence of the Fc domain of canine IgGC is provided below:

[0301] GCGLLGGPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQTANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTPGQAHQPNVYVLPPSRDEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVDKSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK(SEQ ID NO:11)

[0302] The amino acid sequence of the Fc domain of canine IgGD is provided below:

[0303] VPESLGGPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTAKTQPREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR GQAHQPSVYV LPPSPKELSSSDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQLDEDGSYFLY SKLSVDKSRW QQGDTFTCAVMHEALQNHYTDLSLSHSPGK(SEQ ID NO:12)

[0304] Substitutions in canine IgG Fc that increase half-life

[0305] Increased serum persistence is a beneficial property of therapeutic polypeptides. The disclosure is characterized by substitutions in wild-type canine IgGA, IgGB, IgGC, and IgGD Fc regions that increase 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 having a corresponding wild-type canine IgG Fc region instead of an IgG Fc region variant. Substitutions that increase half-life can be performed in one or more of a canine CH2 region, a canine CH3 region, or in the context of a canine Fc (e.g., CH2+CH3) region.

[0306] The present disclosure provides a polypeptide comprising an Fc domain of a canine IgG or a canine FcRn binding region thereof, wherein the polypeptide comprises at least one amino acid substitution at a position selected from the group consisting of:

[0307] (i) the position corresponding to amino acid position 286 of wild-type canine IgG;

[0308] (ii) the position corresponding to amino acid position 312 of wild-type canine IgG;

[0309] (iii) a position corresponding to amino acid position 426 of wild-type canine IgG; and

[0310] (iv) the position corresponding to amino acid position 436 of wild-type canine IgG;

[0311] Wherein the amino acid substitution at the position corresponding to the amino acid position 286 of wild-type canine IgG is selected from the group consisting of Tyr, Phe, Leu and Trp, wherein the amino acid position is based on EU numbering, and wherein when compared with the Fc domain of wild-type canine IgG, the binding affinity of the polypeptide to canine FcRn increases. In some embodiments, when compared with the Fc domain of wild-type canine IgG in a binding assay, the binding affinity of the polypeptide to canine FcRn increases at a pH of about 5.0 to about 6.5 (e.g., about 5.5 or about 6.0). In some embodiments, the binding assay refers to a comparison, for example, of the binding affinity of the polypeptide variants described herein to canine FcRn at a pH of about 5.0 to about 6.5 and the binding affinity of the wild-type canine IgG to canine FcRn at the same pH (e.g., about 5.0 to about 6.5). In some embodiments, the binding assay is performed using comparable conditions. In some embodiments, the binding assay is a surface plasmon resonance (SPR) assay.

[0312] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 312 of wild-type canine IgG.

[0313] In some embodiments, the polypeptide comprises a Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG.

[0314] In some embodiments, at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises Tyr, His, or Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0315] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 436 of wild-type canine IgG. In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0316] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

[0317] In some cases, the present disclosure provides a canine IgG CH2 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%, or at least 99% identical 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids.

[0318] 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%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8. It also features 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids.

[0319] In some 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%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 12. Also disclosed are canine IgG Fc region variants comprising an amino acid sequence that differs from any one of SEQ ID NOs: 9 to 12 by 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids.

[0320] 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:

[0321] amino acid positions 250-256;

[0322] Amino acid positions 285-288; and

[0323] Amino acid positions 307-315,

[0324] 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.

[0325] 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:

[0326] Amino acid positions 376-380; and

[0327] Amino acid positions 428-436,

[0328] 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.

[0329] 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:

[0330] amino acid positions 250-256;

[0331] amino acid positions 285-288;

[0332] amino acid positions 307-315;

[0333] Amino acid positions 376-380; and

[0334] Amino acid positions 428-436,

[0335] Wherein the amino acid positions are based on EU numbering. In some cases, all of the following regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc.

[0336] 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:

[0337] amino acid positions 250-256;

[0338] amino acid positions 285, 287, and 288;

[0339] amino acid positions 307-315;

[0340] Amino acid positions 376-380; and

[0341] Amino acid positions 428-436,

[0342] Wherein the amino acid positions are based on EU numbering. In some cases, all of the following regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc.

[0343] In some 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 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% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 4.

[0344] In some embodiments, it is characterized by comprising one or more polypeptides of a canine IgG Fc CH3 region variant, wherein the CH3 region variant comprises 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% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 5 to 8.

[0345] In some embodiments, it is characterized by comprising one or more polypeptides 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%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 9 to 12.

[0346] In some embodiments, the polypeptide comprises:

[0347] (i) Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG; and / or

[0348] (ii) Tyr, His or Phe at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG; and / or

[0349] (iii) Tyr, Phe, Leu and Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0350] In some embodiments, the polypeptide comprises:

[0351] (i) Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG; and / or

[0352] Tyr at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG; and / or

[0353] Tyr, Phe, Leu and Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0354] In some embodiments, the polypeptide comprises:

[0355] (i) Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG; and / or

[0356] (ii) His at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG; and / or

[0357] (iii) Tyr, Phe, Leu and Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0358] As noted elsewhere, in some embodiments, the polypeptide further comprises at least one additional amino acid substitution in a region corresponding to amino acid positions 250-256, amino acid positions 285-288, amino acid positions 307-315, amino acid positions 376-380, or amino acid positions 428-436 of wild-type canine IgG, wherein the amino acid positions are numbered based on EU, and wherein the polypeptide has increased binding to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0359] In some embodiments, the polypeptide further comprises at least one additional amino acid substitution in a region corresponding to amino acid positions 250-256, amino acid positions 285, 287, and 288, amino acid positions 307-315, amino acid positions 376-380, or amino acid positions 428-436 of a wild-type canine IgG, wherein the amino acid positions are numbered based on EU, and wherein the polypeptide has increased binding to canine FcRn compared to the Fc domain of a wild-type canine IgG. The at least one additional amino acid substitution encompassed by the present disclosure includes 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.

[0360] Table 1

[0361]

[0362]

[0363] In some cases, at least one additional amino acid substitution encompassed by the present disclosure includes one or more (e.g., 1, 2, 3, or 4) of those substitutions disclosed in Table 2.

[0364] Table 2

[0365]

[0366] All possible combinations and permutations of the substitutions disclosed above are encompassed by the present disclosure. In some cases, the polypeptide comprises at least one (e.g., two or more, three or more, four or more, five or more) additional amino acid substitutions selected from the group consisting of:

[0367] (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256;

[0368] (ii) Leu at amino acid position 428 and Ser at amino acid position 434;

[0369] (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311;

[0370] (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378;

[0371] (v) Asp at amino acid position 256, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311;

[0372] (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315;

[0373] (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;

[0374] (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378;

[0375] (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378;

[0376] (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378;

[0377] (xi) Asp at amino acid position 256, Asp at amino acid position 285, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 378;

[0378] (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;

[0379] (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378;

[0380] (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378;

[0381] (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;

[0382] (xvi) Gln at amino acid position 307, Ala at amino acid position 380, Ser or Ala at amino acid position 434;

[0383] (xvii) Leu at amino acid position 428 and Ser or Ala at amino acid position 434;

[0384] (xviii) Gln at amino acid position 250 and Leu at amino acid position 428;

[0385] (xix) Glu at amino acid position 250 and Glu at amino acid position 251;

[0386] (xx) Phe at amino acid position 256 and Phe at amino acid position 309;

[0387] (xxi) Ala at amino acid position 430 and Lys at amino acid position 433;

[0388] (xxii) Phe at amino acid position 434 and His at amino acid position 436; and

[0389] (xxiii) Tyr at amino acid position 435 and His at amino acid position 436;

[0390] 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.

[0391] In some embodiments, at least one additional amino acid substitution is at a position selected from the group consisting of:

[0392] (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0393] (ii) the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0394] (iii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0395] (iv) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0396] (v) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0397] (vi) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0398] (vii) the amino acid position corresponding to amino acid position 286 of wild-type canine IgG,

[0399] (viii) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0400] (ix) the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0401] (x) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0402] (xi) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0403] (xii) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0404] (xiii) the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0405] (xiv) the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0406] (xv) the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0407] (xvi) the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0408] (xvii) the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0409] (xviii) the amino acid position corresponding to amino acid position 434 of wild-type canine IgG,

[0410] (xix) the amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and

[0411] (xx) Amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0412] In some embodiments, the polypeptide comprises:

[0413] (i) Glu or GIn at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0414] (ii) Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0415] (iii) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0416] (iv) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0417] (v) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0418] (vi) Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0419] (vii) Asp, Tyr, Phe, Leu or Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG,

[0420] (viii) Arg, GIn or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0421] (ix) Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0422] (x) Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0423] (xi) Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0424] (xii) Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0425] (xiii) Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0426] (xiv) Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0427] (xv) Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0428] (xvi) Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0429] (xvii) Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0430] (xviii) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG,

[0431] (xix) Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and / or

[0432] (xx) His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0433] In some embodiments, at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide comprises at least one additional amino acid substitution at a position selected from the group consisting of:

[0434] (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0435] (ii) the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0436] (iii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0437] (iv) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0438] (v) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0439] (vi) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0440] (vii) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0441] (viii) the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0442] (ix) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0443] (x) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0444] (xi) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0445] (xii) the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0446] (xiii) the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0447] (xiv) the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0448] (xv) the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0449] (xvi) the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0450] (xvii) the amino acid position corresponding to amino acid position 434 of wild-type canine IgG,

[0451] (xviii) the amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and

[0452] (xix) Amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0453] In some embodiments, the polypeptide comprises:

[0454] (i) Glu or GIn at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0455] (ii) Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG,

[0456] (iii) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0457] (iv) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0458] (v) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0459] (vi) Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0460] (vii) Arg, GIn or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0461] (viii) Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG,

[0462] (ix) Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0463] (x) Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0464] (xi) Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0465] (xii) Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG,

[0466] (xiii) Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG,

[0467] (xiv) Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG,

[0468] (xv) Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG,

[0469] (xvi) Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0470] (xvii) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG,

[0471] (xviii) Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and / or

[0472] (xix) His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0473] In some embodiments, at least one additional amino acid substitution is at a position selected from the group consisting of:

[0474] (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0475] (ii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0476] (iii) the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0477] (iv) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0478] (v) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0479] (vi) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0480] (vii) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0481] (viii) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0482] (ix) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0483] (x) the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0484] (xi) the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and

[0485] (xii) The amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0486] In some embodiments, the polypeptide comprises:

[0487] (i) Glu or GIn at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG,

[0488] (ii) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0489] (iii) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0490] (iv) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG,

[0491] (v) Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG,

[0492] (vi) Arg, GIn or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG,

[0493] (vii) Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG,

[0494] (viii) Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG,

[0495] (ix) Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG,

[0496] (x) Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG,

[0497] (xi) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and

[0498] (xii) His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0499] In some embodiments, at least one additional amino acid substitution is at a position selected from the group consisting of:

[0500] (i) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0501] (ii) the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0502] (iii) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and

[0503] (iv) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0504] In some embodiments, the polypeptide comprises:

[0505] (i) Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0506] (ii) Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0507] (iii) Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or

[0508] (iv) Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0509] In some embodiments, the polypeptide comprises:

[0510] (i) Tyr at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG,

[0511] (ii) Thr at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG,

[0512] (iii) Glu at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or

[0513] (iv) Trp, Tyr, Arg or His at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0514] Substitutions may be made on one or both chains of a CH2 domain, a CH3 domain, or an Fc domain. In some cases, the substitutions on both chains of a CH2 domain, a CH3 domain, or an Fc domain are identical. In some cases, the substitutions on both chains of a CH2 domain, a CH3 domain, or an Fc domain are not identical. In some cases, the Fc region comprises one or more additional substitutions that increase or decrease effector function and / or improve product heterogeneity.

[0515] The present disclosure also provides a polypeptide comprising a canine IgG Fc region variant or a canine FcRn binding region thereof, wherein the polypeptide comprises amino acid substitutions at two or more (e.g., two, three, four, or five) positions selected from the group consisting of:

[0516] (i) the position corresponding to amino acid position 286 of wild-type canine IgG;

[0517] (ii) the position corresponding to amino acid position 312 of wild-type canine IgG;

[0518] (iii) the position corresponding to amino acid position 426 of wild-type canine IgG;

[0519] (iv) a position corresponding to amino acid position 434 of wild-type canine IgG; and

[0520] (v) the position corresponding to amino acid position 436 of wild-type canine IgG,

[0521] wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity for canine FcRn when compared to the Fc domain of wild-type canine IgG.

[0522] All possible combinations and permutations of the substitutions disclosed herein are encompassed by the present disclosure. In some embodiments, two or more amino acid substitutions provide a synergistic effect in that the binding affinity of the polypeptide to canine FcRn is increased when compared to a polypeptide comprising only one of the aforementioned two or more amino acid substitutions.

[0523] In some embodiments, the polypeptides described herein comprise at least one additional amino acid substitution at a position other than those corresponding to positions 286, 312, 426, 434, and 436 of wild-type canine IgG. For example, the polypeptides described herein may include from about 1, 2, 3, 4, or 5 to about 30 or fewer additional amino acid substitutions of canine IgG.

[0524] In some embodiments, the polypeptide has an increased binding affinity for canine FcRn at a pH of about 5.0 to about 6.5 (e.g., about 5.5 or about 6.0) when compared to the Fc domain of wild-type canine IgG. Methods for determining FcRn binding affinity will be familiar to those of skill in the art, illustrative examples of which are described elsewhere herein.

[0525] Comparable or similar assays for each variable (e.g., pH value, number of amino acid substitutions, position of amino acid substitutions, type of amino acid substitutions, etc.) may be used appropriately to determine differences in FcRn binding activity. Comparable assays in this context refer to assays that operate in essentially the same or similar manner to minimize or otherwise avoid unnecessary variables that may have a significant impact on the way the assay is performed and the results that are independent of the variables being evaluated. However, it should be understood that the conditions necessary for performing an assay for determining FcRn binding at, for example, pH 6.0 may be different from the conditions necessary for performing a similar assay at pH 7.4, taking into account, for example, the effect of pH on the way the assay is performed. In some embodiments, the polypeptide has an increased binding affinity for canine FcRn at a pH of about 5.0 to about 6.5 (e.g., about 5.5 or about 6.0) when compared to the Fc domain of wild-type canine IgG using a comparable assay.

[0526] In some embodiments, the polypeptide binds to canine FcRn at a higher level at acidic pH than at neutral pH in comparable assays. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 5.5 than at pH 7.4 in comparable assays. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4 in comparable assays.

[0527] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from T286L, T286Y, and any of the foregoing conservative amino acid substitutions.

[0528] In some embodiments, the polypeptide of claim 1, wherein the amino acid substitution at the position corresponding to amino acid position 312 of wild-type canine IgG is D312P or a conservative amino acid substitution thereof.

[0529] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG is selected from A426Y, A426H, and any of the foregoing conservative amino acid substitutions.

[0530] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 434 of wild-type canine IgG is N434R or a conservative amino acid substitution thereof.

[0531] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG is Y436H or a conservative amino acid substitution thereof.

[0532] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG.

[0533] As used herein, the term "conservative amino acid substitution" refers to an amino acid residue being substituted with another amino acid residue having similar properties such as charge, hydrophobicity, and size. In some embodiments, a conservative amino acid substitution refers to a substitution that results in a property or function similar to another amino acid substitution. For example, a conservative amino acid substitution for A426Y can be A426F or A426T.

[0534] In some embodiments, the polypeptide comprises amino acid substitutions at two or more positions selected from the group consisting of:

[0535] (i) positions corresponding to amino acid positions 426 and 286 of wild-type canine IgG;

[0536] (ii) positions corresponding to amino acid positions 426 and 312 of wild-type canine IgG;

[0537] (iii) positions corresponding to amino acid positions 426 and 434 of wild-type canine IgG; and

[0538] (iv) Positions corresponding to amino acid positions 426 and 436 of wild-type canine IgG.

[0539] In some embodiments, the two or more amino acid substitutions are selected from the group consisting of:

[0540] (i) A426Y in combination with one or more of T286L, T286Y, D312P, N434R and Y436H;

[0541] (ii) A426H in combination with one or more of T286L, T286Y, D312P, N434R and Y436H; and

[0542] (iii) a combination of N434R and one or more of T286L, T286Y, D312P and Y436H.

[0543] In some embodiments, the polypeptide comprises an amino acid substitution selected from the group consisting of:

[0544] (i) A426Y and T286L;

[0545] (ii) A426Y and D312P;

[0546] (iii) A426Y and Y436H;

[0547] (iv) A426H and T286L;

[0548] (v) A426H and T286Y; and

[0549] (vi) A426H and D312P.

[0550] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

[0551] In some cases, the present disclosure provides a canine IgG CH2 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%, or at least 99% identical 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids.

[0552] 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%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8. It also features 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids.

[0553] In some 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%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 12. Also disclosed are canine IgG Fc region variants comprising an amino acid sequence that differs from any one of SEQ ID NOs: 9 to 12 by 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids.

[0554] 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:

[0555] Amino acid positions 250-256; and

[0556] Amino acid positions 307-311,

[0557] 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.

[0558] 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:

[0559] Amino acid positions 376-380; and

[0560] Amino acid positions 428-433,

[0561] 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.

[0562] 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:

[0563] amino acid positions 250-256;

[0564] amino acid positions 307-311;

[0565] Amino acid positions 376-380; and

[0566] Amino acid positions 428-433,

[0567] Wherein the amino acid positions are based on EU numbering. In some cases, all of the following regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc.

[0568] 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:

[0569] amino acid positions 250-256;

[0570] amino acid positions 285, 287, and 288;

[0571] amino acid positions 307-311;

[0572] Amino acid positions 376-380; and

[0573] Amino acid positions 428-433,

[0574] Wherein the amino acid positions are based on EU numbering. In some cases, all of the following regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc.

[0575] In some 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 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% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 4.

[0576] In some embodiments, it is characterized by comprising one or more polypeptides of a canine IgG Fc CH3 region variant, wherein the CH3 region variant comprises 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% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 5 to 8.

[0577] In some embodiments, it is characterized by comprising one or more polypeptides 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%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 9 to 12.

[0578] Other Substitutions That Can Be Combined with Half-Life Enhancing Substitutions

[0579] The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process that requires coordination of a complex set 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 functions, and development of commercially viable manufacturing and purification methods. The present disclosure encompasses substitutions at one or more additional amino acid positions of Fc region variants that promote any one or more of the above-mentioned goals.

[0580] In some embodiments, the Fc region variant comprises amino acid substitutions at one or more additional amino acid positions that increase or decrease effector function and / or improve product heterogeneity.

[0581] In some embodiments, substitutions are introduced to reduce the effector function of the canine Fc region. Such substitutions may be at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions of canine IgG (numbered according to EU numbering): 238, 265, 297, 298, 299, 327, and 329. The substitution may be any of the other 19 amino acids. In some cases, the substitution is conservative. In certain non-limiting examples, 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; and the substituted amino acid at position 329 is Ala. In some cases, the variant Fc region is from a canine IgGB or IgGC antibody.

[0582] In some embodiments, substitutions are introduced into the wild-type canine IgG Fc region to enhance binding to protein A, thereby facilitating 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 may be any of the other 19 amino acids. In some cases, the substitution is conservative. In certain non-limiting examples, the substituted amino acid at position 252 is Met; and the substituted amino acid at position 254 is Ser.

[0583] In some embodiments, substitutions are made to change the binding affinity to FcRn compared to a parent polypeptide or a wild-type polypeptide (e.g., to increase or decrease the binding affinity to FcRn). In some variations, the modification can 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 variants include 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 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).

[0584] In some embodiments, the polypeptide comprises the hinge region of a canine antibody. In some embodiments, the hinge region of a canine antibody can be modified to increase half-life. In some embodiments, the modification is 228P according to EU numbering.

[0585] In some embodiments, the binding to FcRn is pH dependent. H310 and H435 (EU numbering) may be critical for pH dependent binding. Therefore, 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 both positions is histidine.

[0586] 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 LALA mutation is P234A, M234A or S234A. 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.

[0587] Polypeptides comprising canine IgG Fc variants

[0588] The present disclosure encompasses any polypeptide that may benefit from increased half-life in dogs. To increase half-life, these polypeptides are designed to include the Fc region variants disclosed above (eg, CH2 region, CH3 region, CH2+CH3 region).

[0589] Exemplary polypeptides include, but are not limited to, whole 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. As used herein, the terms "nanoantibodies," "VHH," "VHH antibody fragments," and "single domain antibodies" are used interchangeably herein to represent the variable domains of single heavy chains of those types of antibodies found in the Camelidae (Camelidae), which are generally naturally found to lack light chains. Those skilled in the art will be familiar with suitable nanobodies, exemplary examples of which include nanobodies of camels, dromedaries, llamas, and alpacas. Alternatively, the Fc region variants disclosed above can be linked to these polypeptides. In another embodiment, a dog or caninized antibody is modified to include an Fc region variant disclosed herein.

[0590] In some embodiments, the polypeptides of the present disclosure include an antibody hinge region. The hinge region can be located between the antigen or ligand binding domain of the polypeptide and the Fc region variant. In some cases, the hinge region is connected to the C-terminus of a cytokine, a growth factor, an enzyme or a peptide, and the hinge region is connected to the N-terminus of the Fc region variant. Exemplary hinge region sequences are provided below.

[0591] IgGA: FNECRCTDTPPCPVEPEP (SEQ ID NO: 17);

[0592] IgGB: PKRENGRVPRPDCPKCPAPEM (SEQ ID NO: 18);

[0593] IgGC: AKECECKCNCNNCPCPGCGL (SEQ ID NO: 19);

[0594] IgGD: PKESTCKCISPCPVPES (SEQ ID NO: 20); and

[0595] IgGDmut:PKESTCKCIPPCPVPES (SEQ ID NO:21).

[0596] The hinge region in the recombinant protein of the present disclosure, if used, includes zero to six (i.e., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NOs: 17-21. In some cases, the hinge region used in the recombinant protein of the present disclosure 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 amino acid sequence set forth in any one of SEQ ID NOs: 17-21.

[0597] In some embodiments, a linker sequence can be used instead of an antibody hinge sequence to connect 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 consists of 1 to 20 amino acids connected by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. As is well known to those skilled in the art, some of these amino acids may be glycosylated. In other embodiments, 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker consists of most sterically unhindered amino acids (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 Gly GlyGly Gly (SEQ ID NO:25); Gly Gly Gly Gly Gly Ser (SEQ ID NO:26); Ser Gly Gly GlyGly Gly (SEQ ID NO:27); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:28); Ser Gly GlyGly Gly Gly Gly (SEQ ID NO:29); (Gly Gly Gly Gly Gly Ser) n (SEQ ID NO: 24) n, wherein n is an integer of one or more (e.g., 1, 2, 3, 4, 5); and (Ser Gly Gly Gly Gly) n (SEQ ID NO:25)n, wherein n is an integer of one or greater (e.g., 1, 2, 3, 4, 5).

[0598] 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(CH 2 ) n C(O)-, wherein n=2-20. These alkyl linkers may be further substituted with any non-sterically hindered groups, such as lower alkyl (e.g., C 1 -C 6 ) lower acyl, halogen (e.g., Cl, Br), CN, NH 2 , phenyl, etc.

[0599] One or more polypeptides disclosed herein may comprise a binding domain. The binding domain may specifically bind to a protein, subunit, domain, motif and / or epitope of a selected target as described herein. In some embodiments, one or more polypeptides (e.g., a fusion polypeptide) may comprise a protein, wherein the protein is a therapeutic protein as described herein. In some embodiments, the target (e.g., for a target of a binding domain) or a therapeutic protein (e.g., for a fusion polypeptide) 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 RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APR IL, AR, IgE, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, ARC, ART, Artemin, anti-Id, aspartate (ASPARTIC), atrial natriuretic peptide, 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, B CA-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(A LK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, 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, 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, CCL21, CCL22, CCL23, CCL24, CC L25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CC R2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11 a. CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD2 9. CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD49a, CD52, CD 54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNA enzyme, Dpp, DPPIV / CD26, Dtk, ECAD, EDA,EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMPRIN, ENA, endothelin receptor, neprilysin, eNOS, Eot, eotaxin 1, 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 (GP IIb / 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, heparanase, 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 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, 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, I L-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptor (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), interferon (INF)-α, INF-β, INF-γ, inhibin, 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, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 1 2. 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), Potential TGF-1, Potential 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, Pulmonary surfactant, Luteinizing hormone, Lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinases, 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,Mullerian inhibitory substance, Mug, MuSK, NAIP, NAP, NAV 1.7, NCAD, N-cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4 or -6, Neurturin, Neuron 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 , PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, 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, testis P LAP-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(TRAIL R3DcR1、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(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY CROWN), TNFRSF19L(RELT), TNFRSF1A(TNF R1CD120a, p55-60), TNFRSF1B(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1R), 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). R2TNFRH2、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). LIGHT、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT Whether LTg, TNFSF15 (TL1A / VEGI), TNFSF18 (GITR, AITR, TL6), TNFSF1A (TNF-binding factor (Conectin), 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 receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expression Lewis Y-related carbohydrate, 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 antigen, VLA, VLA-1, VLA-4, VNR integrin, vascular vascular disease factor, 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. 、

[0600] 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 peptide, 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-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP (TGF-1), potential TGF-1, potential TGF-1bp1, LFA-1, neuronal growth factor (NGF) F), NGFR, NGF-β, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TG F-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).

[0601] 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, GLP-1 receptor agonist, and 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 peptide, 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-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP (TGF-1), potential TGF-1, potential TGF-1bp1, LFA-1, neuronal growth factor (NGF) F), NGFR, NGF-β, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TG F-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).

[0602] In some embodiments, the therapeutic protein is any protein described herein. In some embodiments, one or more polypeptides further comprise 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 in vivo half-life of the therapeutic protein.

[0603] Pharmaceutical composition

[0604] To prepare a pharmaceutical or sterile composition of one or more polypeptides described herein, the 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)).

[0605] Formulations of therapeutic and diagnostic agents can be prepared, for example, in the form of a lyophilized powder, slurry, aqueous solution or suspension by mixing with an acceptable carrier, excipient or stabilizer (see, e.g., Hardman et al. (2001) Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaró (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). Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dek ker, Inc., New York, NY). In one embodiment, one or more polypeptides of the invention are diluted to an appropriate concentration in a sodium acetate solution at 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.

[0606] Toxicity and therapeutic efficacy 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, for example, to determine the LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 ). In certain aspects, it is desirable for one or more polypeptides to exhibit a high therapeutic index. 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 is preferably within the range that includes the ED 50The dosage may vary within this range depending on the dosage form employed and the route of administration employed.

[0607] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, directly intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial. In some embodiments, one or more polypeptides can be administered by an invasive route such as by injection. In other embodiments, administration is by intravenous, subcutaneous, intramuscular, intraarterial, intratumoral or by inhalation, aerosol delivery.

[0608] The pharmaceutical composition disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0609] Alternatively, one can administer one or more polypeptides in a local rather than systemic manner, for example by injecting the antibody directly into an arthritic joint or a pathogen-induced lesion characterized by immunopathology, typically in a depot or sustained release formulation. In addition, one can administer one or more polypeptides in a targeted drug delivery system (e.g., in liposomes coated with tissue-specific antibodies, targeted to, for example, an arthritic joint or a pathogen-induced lesion characterized by immunopathology). The liposomes will target the diseased tissue and be selectively taken up by the diseased tissue.

[0610] The administration regimen depends on several factors, including but not limited to the age, weight and physical condition of the dog being treated, serum or tissue turnover of the therapeutic antibody, symptom level, immunogenicity of one or more therapeutic polypeptides, and accessibility of target cells in the biomatrix. Preferably, the administration regimen delivers enough one or more therapeutic polypeptides to achieve improvement in the target disease state while minimizing undesirable side effects. Therefore, the amount of biological delivery depends in part on the specific therapeutic polypeptide and the severity of the disease being 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)).

[0611] One skilled in the art, for example, uses 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 is started with an amount slightly less than the optimal dosage and thereafter increased in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include those measures such as inflammatory symptoms or levels of inflammatory cytokines produced.

[0612] Nucleic acid, vector, host cell and preparation method

[0613] The present disclosure also encompasses 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.

[0614] One or more polypeptides described herein can be produced in bacteria or eukaryotic cells. Some polypeptides (e.g., Fab) can be produced in bacterial cells (e.g., E. 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) can be expressed in yeast cells such as Pichia (Pichia) (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)); Hanseula or Saccharomyces. 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 a suitable host cell. In order to improve expression, the nucleotide sequence of the gene can be recoded without changing (or minimally changing - for example, removing the C-terminal residue of the heavy chain or light chain) the amino acid sequence. Potentially recoded regions include regions associated with translation initiation, codon usage, and possible unintended mRNA splicing. Polynucleotides encoding the Fc region variants described herein are readily apparent to the skilled artisan.

[0615] Standard molecular biology techniques can be used to prepare one or more recombinant expression vectors, transfect the host cells, select for transformants, culture the host cells and recover the polypeptide (eg, antibody).

[0616] If the one or more polypeptides will be expressed in bacterial cells (e.g., Escherichia coli), the expression vector should have the feature of allowing the vector to be amplified in the bacterial cells. In addition, when Escherichia coli (such as JM109, DH5α, HB101 or XL1-Blue) is used as a host, the vector must have a promoter, such as lacZ promoter (Ward et al., 341:544-546 (1989)), araB promoter (Better et al., Science, 240:1041-1043 (1988)) or T7 promoter that can be allowed to be effectively expressed in Escherichia coli. The example of such a vector includes, for example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP and pET (when using this expression vector, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector can 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)) can be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0617] If the one or more polypeptides are expressed in animal cells such as CHO, COS and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, such as 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., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates the 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 such as G418, hygromycin or methotrexate to 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.

[0618] 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 (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, which are used with DHFR selection markers such as those 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 lines (e.g., NS0 myeloma cells and SP2 cells) and cells from transgenic animals (e.g., transgenic mammals). For example, the cells are mammary epithelial cells.

[0619] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of the antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain and light chain genes is operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, adenovirus, etc., 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 that have been transfected with the vector. The selected transformant host cells are cultured to allow expression of the antibody heavy chain and light chain, and the antibody is recovered from the culture medium.

[0620] Treatment

[0621] One or more polypeptides disclosed herein can be used to treat or prevent any disease or condition in a dog in need thereof. The invention is particularly useful for treating chronic conditions that require repeated dosing. Due to the increased half-life of protein therapeutics, less frequent dosing and / or reduced dosage levels may be possible.

[0622] In some embodiments, the disease, disorder, condition or symptom treated or prevented is allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, skeletal / musculoskeletal disease, cardiovascular disease, neurological disease, renal disease, metabolic disease, immune disease, gene / genetic disease, fertility-related disorder, infectious disease or cancer. In certain embodiments, the disease or condition treated or prevented is atopic dermatitis, allergic dermatitis, food allergy, osteoarthritis pain, perioperative pain, dental pain, cancer pain, arthritis, anemia, obesity or diabetes.

[0623] Antibodies can be used not only to treat or prevent disease, but also to regulate normal biological functions, such as managing fertility or behavior.

[0624] diagnosis

[0625] One or more polypeptides disclosed herein may also be used for various diagnostic purposes, such as determining whether a dog suffers from any particular disease or condition. In some embodiments, one or more polypeptides may comprise 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 further comprise a labeling group. Typically, labeling groups are divided into a variety of categories according to the assays for which they are to be detected: a) isotopic labels, which may be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by a second reporter molecule (e.g., leucine zipper pair sequences, binding sites of a second antibody, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used in the practice of the present invention.

[0626] In some embodiments, the labeling 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).

[0627] Specific labels may include optical dyes, including but not limited to chromophores, phosphors and fluorophores, the latter being specific in many cases. Fluorophores may be "small molecule" fluorophores or proteinaceous fluorophores.

[0628] Fluorescent label can be any molecule that can be detected by its intrinsic fluorescence property. Suitable fluorescent label includes but is not limited to fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescent yellow, cascade blue (Cascade BlueJ), Texas red (Texas Red), IAEDANS, EDANS, BODIPYFL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dye (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 (CascadeBlue), Cascade Yellow and R-phycoerythrin (PE) (Molecular Biotechnology, Inc., New York, NY). 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 Richard P. Haugland's Molecular Probes Handbook, which is incorporated herein by reference in its entirety.

[0629] Suitable proteinaceous fluorescent markers 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 No. 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. USA 85:2603-2607), 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.

[0630] Determination

[0631] Fc γ RI and FcγRIII binding:

[0632] Binding to FcγRI and FcγRIII is a measure of the ability of an antibody to mediate ADCC. To assess this property of an antibody, assays measuring binding of the antibody to FcγRI and FcγRIII can be performed using methods known in the art.

[0633] C1q binding:

[0634] 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 this property of an antibody, assays measuring antibody binding to CIq can be performed using methods known in the art.

[0635] half life:

[0636] Methods for measuring antibody half-life are well known in the art. See, for example, Booth et al., MAbs, 10(7): 1098-1110 (2018). Exemplary animal models include non-human primate models and transgenic mouse models. Transgenic mouse models (e.g., Tg32 or Tg276 transgenic mice) may be ineffective 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. For example, the half-life of canine antibodies can be measured by injecting the antibody into a dog model and measuring the level of the antibody in the serum over a certain period of time.

[0637] Example

[0638] Example 1: Alanine scanning mutagenesis of the CH2 and CH3 domains of canine IgGB

[0639] Alanine scanning mutagenesis (Morrison and Weiss, Curr. Opin. Chem. Biol. 5: 302-307 (2001)) 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. For this experiment, the wild-type (wt) sequence of the CH2 and CH3 domains of canine IgGB was synthesized and used as a template for mutagenesis. Each designated position except position 254 was individually changed to alanine by PCR mutagenesis using primers encoding the change. Position 254 in the wild-type sequence was alanine, and it was modified to serine. The PCR product was subcloned into the GenScript FASEBA plasmid, transformed into E. coli, and sequenced to verify the presence of the variant. Upstream of the CH2 domain is a SASA (single domain antibody to serum albumin) tag with pM affinity for albumin (see, e.g., US 2013 / 0129727A1). The PelB (pectate lyase B) signal peptide is located at the N-terminus to promote Fc secretion into the culture medium. The expression of the CH2-CH3 protein is regulated by the Lac promoter. The supernatant from the conditioned medium was analyzed using surface plasmon resonance (SPR) for binding to canine FcRn (UniProtKB-E2R0L6 [FcRn] and UniProtKB-E2RN10 [canine β2 microglobulin]) at pH 5.5.

[0640] 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 mixed 50mmol / L N-hydroxysuccinimide and 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride for 420s (10 μL / min). Then, BSA diluted in 10mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, while flow cell 1 was set to blank. After the amine coupling reaction, 1mM ethanolamine hydrochloride was injected for 420s to block the remaining active coupling sites on the chip surface. The running buffer used for the binding experiment was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 5.5) and was run at 25°C. Supernatants from alanine variants were injected onto the chip surface and captured via the SASA tag onto immobilized BSA for 60 seconds. 400 nM canine FcRn was injected for 120 seconds and dissociation was completed with running buffer for 120 seconds. The flow rate for the BSA stationary phase was 10 μl / min, and the flow rates for the binding and dissociation phases were 30 μl / min. All data were processed using Biacore 8K Evaluation Software version 1.1. Tabular data are shown in Table 3, where the last column contains the wild-type average KD divided by the variant KD. Sensorgrams are shown in Figure 7A-7U middle.

[0641] Table 3

[0642]

[0643]

[0644]

[0645] Example 2: Generation of NNK saturation mutagenesis libraries at selected positions and analysis of individual variants

[0646] The NNK saturation mutagenesis method is an effective strategy to produce 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 the specified position were used with the QuikChange site-directed mutagenesis kit (Agilent). The supernatant of 90 individual transformants from each library was determined for binding to canine FcRn at pH 5.5 using the Biacore method described in Example 1. The only difference is that the concentration of canine FcRn used in the assay is 200nM instead of 400nM. The sensorgrams of all NNK library variants are shown in Figures 8-15.

[0647] For the NNK library at position 250, none of the variants 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)).

[0648] Table 4

[0649] 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

[0650] For the NNK library at position 252, only variants L252Y and L252M had significantly higher affinity for canine FcRn at pH 5.5 (see Table 5 below). In the 90 transformants, the L252F variant was not present, so no binding data were obtained with this variant.

[0651] Table 5

[0652] 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

[0653] For the NNK library at position 254, none of the tested variants had significantly higher affinity for canine FcRn at pH 5.5. Data for the A254T variant are shown in Table 6, and the corresponding variants in human IgG1 have been used for the YTE variant (M252Y / S254T / T256E), which has increased affinity for human FcRn at pH 6.0 (Dall'Acqua et al., J. Immunol. 169:5171-5180 (2002)) and has been shown to increase the half-life of human IgG in preclinical models and in humans (Borrok et al., J. Biol. Chem. 290:4282-4290 (2015); Robbie et al., Antimicrob. Agents Ch. 57:6147-6153 (2013)). In 90 transformants, the A254H variant was not present, so no data were obtained with this variant.

[0654] Table 6

[0655] 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

[0656] For the NNK library at positions 309 and 311, none of the tested variants have significantly higher affinity to canine FcRn at pH 5.5. The data of variants G309P and Q311V are shown in Tables 7 and 8, and the corresponding human variants (L309P and Q311V) in human IgG1 and several combinations of other variants have been shown to have higher affinity to 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 Q311D were not identified in the NNK library, so FcRn binding was not tested.

[0657] Table 7

[0658] 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

[0659] Table 8

[0660] 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

[0661] For the NNK library at positions 378 and 380, none of the tested variants have significantly higher affinity to canine FcRn at pH 5.5. The data of variant D378V are shown in Table 9, and the corresponding variants in human IgG1 have been used in combination with other IgG variants to demonstrate that compared with wild-type Fc, at pH 6.0, human FcRn has a higher affinity, and extends the half-life of human IgG in transgenic human FcRn mice (Monnet et al., MABS.6: 422-436 (2014); Booth et al., 2018). Moreover, the data of variant E380A are shown in Table 10, and the corresponding variants in human IgG have been shown to have a higher binding affinity to 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.

[0662] Table 9

[0663] 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

[0664] Table 10

[0665] 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

[0666] 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 contain the N434F variant, so this variant was not tested for binding to canine FcRn.

[0667] Table 11

[0668]

[0669]

[0670] Example 3: Binding kinetics of L252Y, N434Y, N434W, N434R, N434H and YTE (L252Y / A254T / T256E) variants and wild-type Fc

[0671] The binding kinetics of several canine IgGB variants showing higher affinity for canine FcRn at pH 5.5 were further evaluated. In this study, the binding of variants (L252Y, N434Y, N434W, N434R, N434H), YTE variants (L252Y / A254T / T256E) and wild-type canine Fc to canine FcRn at pH 5.5 and pH 7.4 was evaluated. The Biacore method for pH 5.5 conditions was the same as described in Example 1, except that four FcRn concentrations (100nM, 200nM, 400nM, 800nM) that produced more accurate binding kinetics were tested. 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) as well as 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.

[0672] Table 12

[0673] 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

[0674] Example 4: Generation of NNK saturation mutagenesis libraries at selected positions and analysis of individual variants

[0675] The wild-type (wt) sequence (SEQ ID NO: 10) of the CH2 and CH3 domains of canine IgGB was synthesized and used as a template for NNK mutagenesis. The NNK saturation mutagenesis method is an effective strategy (Hogrefe et al., Biotechniques. 33: 1158-1165 (2002)) to produce all 20 possible amino acids at the desired position. A single NNK library at positions 286, 312, 426 and 436 (EU numbering) was generated. The NNK (N = A / C / G / T, K = G / T) primers at the specified position were used together with the QuikChange site-directed mutagenesis kit (Agilent). The PCR product was subcloned into the GenScript FASEBA plasmid, transformed into Escherichia coli, and the presence of the variant was verified by sequencing. Upstream of the CH2 domain is a SASA (single domain antibody to serum albumin) tag with pM affinity for albumin (Zhang, J.; Wu, S.; Liu, J. Methods and systems for increasing protein stability. U.S. Patent Application, 2013.). SASA antibody is able to capture Fc onto the surface of the sensor chip described below. PelB (pectate lyase B) signal peptide is located at the N-terminus to promote Fc secretion into the culture medium. The expression of CH2-CH3 protein is regulated by the Lac promoter. For variants at positions 426 and 312 at pH 5.5 and for variants at positions 286 and 436 at pH 6.0, surface plasmon resonance (SPR) was used to analyze the binding of supernatants from conditioned culture medium to canine FcRn (UniProtKB-E2R0L6 [FcRn] and UniProtKB-E2RN10 [canine β2 microglobulin]).

[0676] Supernatants of 90 individual transformants from each library were assayed for binding to canine FcRn at pH 5.5 for variants at positions 426 and 312 and at pH 6.0 for variants at positions 286 and 436 using the Biacore method as described below.

[0677] 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 mixed 50mmol / L N-hydroxysuccinimide and 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride for 420s (10 μL / min). Then, BSA diluted in 10mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, while flow cell 1 was set to blank. After the amine coupling reaction, 1mM ethanolamine hydrochloride was injected for 420s to block the remaining active coupling sites on the chip surface. The running buffer used for the binding experiment was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 5.5) and it was run at 25°C. The supernatant from the variant was injected onto the chip surface and captured onto the fixed BSA by the SASA tag for 60 seconds. 200 nM canine FcRn was injected for 120 seconds and dissociation was completed with running buffer for 120 seconds. The flow rate of the BSA fixed phase was 10 μl / min, and the flow rate of the binding and dissociation phases was 30 μl / min. All data were processed using Biacore 8K evaluation software version 1.1. See Figure 17 for Biacore sensorgrams.

[0678] When compared to wild-type canine IgGB Fc (SEQ ID NO: 10), the tested variants showed increased binding affinity to canine FcRn at pH 5.5 (for variants with amino acid substitutions at positions 312 or 426) and at pH 6.0 (for variants with amino acid substitutions at positions 286 or 436). The results are summarized in Tables 13 and 14 below.

[0679] Table 13:

[0680] Variants ka(1 / Ms) kd(1 / s) KD(M) A426Y 7.50E+05 1.09E-01 1.46E-07 A426Y 7.85E+05 1.12E-01 1.43E-07 A426H 8.43E+05 1.35E-01 1.60E-07 A426H 9.15E+05 1.40E-01 1.53E-07 A426F 7.86E+05 1.43E-01 1.82E-07 A426F 8.11E+05 1.47E-01 1.81E-07 D312P 3.07E+05 8.94E-02 2.91E-07 D312P 2.51E+05 1.10E-01 4.39E-07 wild type 1.94E+05 1.95E-01 1.01E-06 wild type 4.42E+05 2.97E-01 6.73E-07

[0681] Table 14:

[0682]

[0683]

[0684] Example 5: Binding kinetics of A426Y, A426H, A426F, T286Y, T286F, T286L, T286W, Y436H and wild-type Fc.

[0685] The binding kinetics of several canine IgGB variants showing higher affinity to canine FcRn were further evaluated. In this study, variants (A426Y, A426H, A426Y, T286Y, T286F, T286L, T286W, Y436H), YTE variants (L252Y / A254T / T256E) and wild-type canine IgGB Fc were evaluated for binding to canine FcRn at pH 5.5 or pH 6.0 and at pH 7.4. The Biacore method for pH 5.5 and pH 6.0 conditions was the same as described in Example 4 above, except that four FcRn concentrations (100nM, 200nM, 400nM, 800nM) that would produce more accurate binding kinetics were tested. 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. See Figure 18 for Biacore sensorgrams. Neither wild-type Fc nor any of the variants bound to FcRn at pH 7.4 using the conditions described.

[0686] The binding affinity data are shown in Tables 15 and 16 below.

[0687] Table 15

[0688]

[0689] Table 16

[0690]

[0691] Example 6: Canine Fc variants carrying an amino acid substitution at position 426 of canine IgGA Fc

[0692] Two canine Fc variants carrying amino acid modifications at position 426 (by EU numbering) and wild-type canine IgGA Fc (SEQ ID NO: 9) were synthesized using the variable domains described by Gearing DP et al. (2013, BMC Veterinary Research, 9: 226). Canine IgGA DNA was synthesized and subcloned into a pcDNA3.4 vector (ThermoFisher) and transfected into ExpiCHO-S cells using the ExpiCHO transfection method (ThermoFisher). Conditioned medium was purified 14 days after transfection of cells using GenScript protein G resin.

[0693] For pH 6.0 binding experiments, antibodies were directly coupled to a CM5 sensor chip, and canine FcRn was then flowed through in HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 6.0). The sensor chip surfaces of flow cells 1 and 2 were activated by freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 100 s (10 μL / min). The antibody was diluted in 10 mmol / L NaAC (pH 4.5) and injected into flow cell 2 to achieve conjugation of approximately 100 response units, while flow cell 1 was set as a blank. After the amine coupling reaction, the remaining active coupling sites on the chip surface were blocked with a 100 s injection of 1 mol / L ethanolamine hydrochloride. The running buffer for pH 6.0 binding experiments was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 6.0) and was run at 25°C. Canine FcRn (UniProtKB-E2R0L6 [FcRn] and UniProtKB-E2RN10 [canine β2 microglobulin]) was injected for 120 seconds, and dissociation was completed with running buffer for 120 seconds. The flow rate was 30 μL / min. The concentration of canine FcRn flowing through the sensor chip was 50 nM, 100 nM, 200 nM, 400 nM and 800 nM. All data were processed using Biacore 8K evaluation software version 1.1. Flow cell 1 and separate buffer injections in each cycle were used as references for response unit deduction. Table 17 below provides kinetic data for pH 6.0 binding experiments. It has been previously demonstrated that amine coupling of IgG to a Biacore CM5 biosensor chip reduces affinity for FcRn by 2-3 times compared to affinity determined by solution-based methods or directly coupled to a Biacore C1 chip (Abdiche et al., 2015. mAbs, 7:331). Therefore, the true affinity of these IgGs to FcRn may be at least 2 times higher at pH 6.0. However, this method is effective for comparing the relative FcRn binding affinity of different IgG Fc variants. The Biacore sensorgram is shown in Figure 19.

[0694] Table 17:

[0695]

[0696] Example 7: Screening for canine IgGB Fc variants with increased FcRn binding compared to wild-type canine IgGB Fc

[0697] Using the variable domains described by Gearing DP et al. (2013, BMC Veterinary Research, 9: 226), canine Fc variants carrying single amino acid substitutions or combinations of amino acid substitutions were synthesized as canine IgGB (SEQ ID NO: 10). Synthesize canine IgGB DNA and subclone it into pcDNA3.4 vector (ThermoFisher) and transfect it into ExpiCHO-S cells using the ExpiCHO transfection method (ThermoFisher). After 14 days of transfection of cells, the conditioned medium was purified using Monofinity A resin (GenScript). Use Biacore 8K to measure the binding of antibodies to canine FcRn under pH 6.0 and pH 7.4 conditions.

[0698] For pH 6.0 binding conditions, the sensor chip surfaces of flow cells 1 and 2 were activated by freshly mixed 50mmol / L N-hydroxysuccinimide (NHS) and 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 100s (10μL / min). The antibody was diluted in 10mmol / L NaAC (pH 4.5) and injected into flow cell 2 to achieve conjugation of approximately 100 response units, while flow cell 1 was set as a blank. After the amine coupling reaction, 1mol / L ethanolamine hydrochloride was injected for 100s to block the remaining active coupling sites on the chip surface. The running buffer for pH 6.0 binding experiments was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 6.0) and it was run at 25°C. Canine FcRn (UniProtKB-E2R0L6 [FcRn] and UniProtKB-E2RN10 [canine β2 microglobulin]) was injected for 120 seconds, and dissociation was completed with running buffer for 120 seconds. The flow rate was 30 μl / min. For wild-type IgG and A426H-N434R IgG variants, the concentration of canine FcRn flowing through the sensor chip was 200nM, 400nM, 800nM, 1600nM and 3200nM. For the remaining variants, the concentration of canine FcRn flowing through was 50nM, 100nM, 200nM, 400nM and 800nM. All data were processed using Biacore 8K evaluation software version 1.1.

[0699] Flow cell 1 and separate buffer injection in each cycle are used as references for response unit deduction. Table 18 below lists the kinetic data of pH 6.0 binding experiments. It has been previously demonstrated that the amine coupling of IgG and Biacore CM5 biosensor chip reduces the affinity to FcRn by 2-3 times compared with the affinity determined by solution-based methods or directly coupled with Biacore C1 chip (Abdiche et al., 2015.mAbs, 7:331). Therefore, the true affinity of these IgG to FcRn may be at least 2 times higher at pH 6.0. However, the method is effective for comparing the relative FcRn binding affinity of different IgG Fc variants. Biacore sensor diagrams are shown in Figures 20-23.

[0700] Table 18:

[0701]

[0702] For pH 7.4 binding conditions, the sensor chip surfaces of flow cells 1 and 2 were activated by freshly mixed 50mmol / L N-hydroxysuccinimide (NHS) and 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 420s (10μL / min). Then, canine FcRn diluted in 10mmol / L NaAC (pH 4.5) was injected into flow cell 2 to achieve conjugation of approximately 2000 response units, while flow cell 1 was set as blank. After the amine coupling reaction, 1mol / L ethanolamine hydrochloride was injected for 420s to block the remaining active coupling sites on the chip surface. The running buffer for pH 7.4 binding experiments was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween20, pH 7.4) and it was run at 25°C. The different antibodies were injected at 400 nM for 120 seconds and dissociation was completed with running buffer for 120 seconds. The flow rate was 30 μl / min. Table 19 below lists the kinetic data for the pH 7.4 binding experiments. The Biacore sensorgrams are shown in Figures 24-27.

[0703] Table 19:

[0704]

[0705] The affinity of canine IgG Fc interacting with canine FcRn at pH 7.4 is very weak and difficult to measure by SPR using most methods. In order to compare the pH 7.4 affinity of various canine Fc variants with canine FcRn, a sensor chip is coated with a high concentration of canine FcRn, and the variant IgG Fc is flowed through the chip to measure the interaction. In this format, there is an avidity effect, so the measured binding affinity is not an accurate measurement of a single canine IgG Fc variant-canine FcRn interaction, but can be used to compare the relative binding of variant IgG at pH 7.4. They should not be used for direct comparison with the binding affinity at pH 6.0.

[0706] Example 8: In vivo screening of canine IgGB Fc variants with increased FcRn binding compared to wild-type canine IgGB Fc.

[0707] Pharmacokinetic (PK) studies were performed with 16 male and female beagles. The canine IgGB Fc variants carrying a single amino acid substitution or a combination of amino acid substitutions were prepared by incorporating amino acid substitutions into canine IgGB (SEQ ID NO: 10) using the anti-NGF variable domains described by Gearing DP et al. (2013, BMC Veterinary Research, 9: 226; the contents of which are incorporated herein by reference in their entirety). The animals were randomly divided into eight groups, with one male and one female in each group. The average age of the beagles was >6 months, and their body weight was 8-10kg. A single intravenous dose of 2mg / kg of antibody was administered to each animal. Approximately 1.5ml of whole blood was collected at the following time points: 0 hours (before administration), 4 hours after injection, and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days. Serum was separated from whole blood and the presence of antibody variants was determined by ELISA specific for anti-NGF antibodies.

[0708] Non-compartmental PK analysis (NCA) of each individual serum antibody measurement was performed using an established Excel plug-in software tool for pharmacokinetic analysis ("PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel", Yong Zhang et al., Comput. Methods Programs Biomed.; September 2010; 99(3): 306-14. doi: 10.1016 / j.cmpb.2010.01.007; the contents of which are incorporated herein by reference in their entirety). PKSolver provides many options for determining PK parameters. In order to simplify use, model independence and increase analyst-analyst consistency, NCA was determined as the method of choice. For specific analysis, NCA IV Bolus in PKSolver was used to determine the terminal half-life (T1 / 2). After a set of initial dose-finding experiments, measurements far above the LLOQ were measured as far as 42 days after intravenous administration. Therefore, the terminal half-life was estimated from at least the last 4 weeks of antibody measurement to enable a robust estimate of the slope. No data points were discarded or excluded in any experiment. Fig.29 As shown, the combination of amino acid substitutions in the IgG Fc region significantly improved the terminal half-life of the anti-NGF IgGB antibody in dogs when compared to an anti-NGF IgGB antibody carrying (i) a wild-type canine IgGB Fc region or (ii) a canine IgGB Fc variant having only a single amino acid substitution.

[0709] Example 9: Binding kinetics of canine IgGB variants to canine FcRn using the C1 biosensor

[0710] The binding kinetics of several canine IgGB variants (A426Y, A426Y+T286L, A426Y+D312P, A426Y+Y436H, A426Y+T286L+Y436H, A426H, A426H+T286L, A426H+T286Y, A426H+D312P, A426H+Y436H and wild type) to canine FcRn (UniProtKB-E2R0L6 [canine large subunit FcRn] and UniProtKB-E2RN10 [canine β2 microglobulin]) were evaluated at pH 5.9. EU numbering was used to identify positions ( Fig.28In this study, canine Fc variants carrying single amino acid substitutions or combinations of amino acid substitutions were synthesized as canine IgGB (GENBANK accession number AAL35302.1) using the variable domains described by Gearing DP et al. (2013, BMC Veterinary Research, 9:226). Synthesized canine IgGB DNA was subcloned into a mammalian expression vector and transiently transfected into CHO cells. Conditioned medium was purified using protein A chromatography.

[0711] For canine FcRn binding experiments, all determinations were completed on the Biacore 8K+ system at 25°C. In the above examples (e.g., Examples 6 and 7), we measured the affinity of IgG variants to canine FcRn by coupling IgG to the amine of the Biacore CM5 biosensor chip, Abdiche et al., 2015 (mAbs, 7: 331) have demonstrated that the Biacore CM5 biosensor chip underestimates the affinity of Fc variants to FcRn compared to when using the S series C1 biosensor. In this group of experiments, in order to obtain a more accurate measurement of FcRn affinity, all antibodies were fixed to the S series C1 sensor chip using a standard amine coupling reagent. A mixture of 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 50mmol / L N-hydroxysuccinimide (NHS) was injected for 420 seconds to activate the surface. Then, the antibody was injected at a concentration of 0.5 to 2 μg / ml in 10 mM sodium acetate pH 5.0 for 120 seconds. Finally, 1 M ethanolamine was injected for 420 seconds. The running buffer was 1X PBS-P+ adjusted to pH 5.9 (Cytiva, catalog number 28995084).

[0712] To evaluate the binding affinity of canine IgGB variants to canine FcRn at pH 5.9, canine FcRn concentrations ranging from 1.56-2000 nM were selected and injected in single cycle mode. The concentrations of canine FcRn tested for each variant are shown in Table 20 below.

[0713] Table 20: Concentration of canine FcRn used for each IgGB variant

[0714] Variants FcRn concentration [nM] wild type 31.25，125，500，2000 A426Y 15.625，62.5，250，1000 A426Y+T286L 7.8125，31.25，125，500 A426Y+D312P 7.8125，31.25，125，500 A426Y+Y436H 7.8125，31.25，125，500 A426Y+T286L+Y436H 1.56，6.25，25，100 A426H 15.625，62.5，250，1000 A426H+T286L 3.9，15.625，62.5，250 A426H+T286Y 3.9，15.625，62.5，250 A426H+D312P 7.8125，31.25，125，500 A426H+Y436H 15.625，62.5，250，1000

[0715] Four concentrations of each antibody were injected at 5 μl / min for 90 seconds, followed by 180 seconds of dissociation. Each concentration series was injected three times in this format, with at least three buffer-only cycles for appropriate reference subtraction. The surface was regenerated by two injections of 1XPBS-P+ (pH 7.4) for 30 seconds, followed by a 60 second wait for command. Three priming cycles were included to stabilize the surface prior to analysis.

[0716] Data were evaluated using Insight Evaluation software by fitting to a 1:1 kinetic interaction model or by fitting to steady-state affinity. Acceptable parameters were selected using quality metrics including U and T values. For kinetic rate constants, U values ​​less than 15 were considered acceptable, while for kinetic rate constants, T values ​​greater than 100 were considered acceptable. In the case where these values ​​were outside the ranges, steady-state affinity parameters were considered acceptable.

[0717] The kinetic data for the 10 variants are shown in Table 21 below, and the sensorgrams are shown in Figures 30A-30K middle.

[0718] Table 21: Canine IgGB variants and canine FcRn binding kinetics

[0719] Variants ka kd <![CDATA[K D ]]> Methods used to fit the data wild type 9.51E-07 Steady-state affinity A426Y 8.78E+05 1.13E-01 1.29E-07 1∶1 kinetic interaction model A426Y+T286L 1.03E+06 4.36E-02 4.23E-08 1∶1 kinetic interaction model A426Y+D312P 1.10E+06 4.97E-02 4.51E-08 1∶1 kinetic interaction model A426Y+Y436H 1.06E+06 6.08E-02 5.77E-08 1∶1 kinetic interaction model A426Y+T286L+Y436H 5.67E+06 4.12E-02 7.26E-09 1∶1 kinetic interaction model A426H 1.24E+06 1.42E-01 1.15E-07 1∶1 kinetic interaction model A426H+T286L 1.16E+06 4.17E-02 3.58E-08 1∶1 kinetic interaction model A426H+T286Y 1.34E+06 2.86E-02 2.13E-08 1∶1 kinetic interaction model A426H+D312P 1.31E+06 5.32E-02 4.05E-08 1∶1 kinetic interaction model A426H+Y436H 8.08E+05 1.30E-01 1.61E-07 1∶1 kinetic interaction model

[0720] Example 10: Pharmacokinetic study of canine IgGB variants with increased FcRn binding and wild-type canine IgGB

[0721] Three pharmacokinetic (PK) studies were completed with male and female beagles. Using the anti-NGF variable domains described by Gearing DP et al. (2013, BMC Veterinary Research, 9: 226), canine Fc variants carrying a single amino acid substitution or a combination of amino acid substitutions were synthesized into canine IgGB (SEQ ID NO: 10) forms. For each study, animals were randomly divided into groups so that each group contained the same number of males and females. The number of males (M) and females (F) in each group and the IgGB variants evaluated in each study are shown below (Table 22).

[0722] Table 22: Description of the IgGB variants tested in each PK study

[0723]

[0724] The average age of the dogs was >6 months and the body weight was between 8-10 kg. Each animal was injected with a single intravenous dose of 1 mg / kg (Study 1) or 2 mg / kg (Studies 2 and 3) of the antibody. Approximately 1.5 ml of whole blood was collected at the following time points: 0 hours (pre-dose), 4 hours and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days after injection. Serum was extracted from the blood and the antibody variants were determined by ELISA specific for NGF antibodies.

[0725] A two-compartment pharmacokinetic (PK) model with linear clearance using nonlinear mixed effects modeling describes the serum concentration ( Fig.31 ). Population PK parameters were estimated using the maximum expected value stochastic approximation (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: Lixoft SAS, 2019). Each parameter was modeled as a random variable with a lognormal distribution. Population parameters were estimated from the combined data including all variants and studies. The study was a categorical covariate on clearance. mAb variants were distinguished by using categorical covariates on clearance, central and peripheral distribution volumes. The classification studies and variant covariates are described as follows:

[0726]

[0727] where if the individual covariates are in categories, then Ω i =1; otherwise Ω i = 0. The wild-type IgGB variant was used as reference.

[0728] Fig.29 The terminal half-lives observed in Study 2 for wild type, A426Y, and A426Y+Y436H variants are shown.

[0729] Data from all three studies were used to generate estimated PK parameters for each variant (Table 23).

[0730] Table 23: PK parameter estimates for dogs with 10 kg body weight

[0731]

[0732] Individual observed serum concentrations for wild type, A426Y, A426Y+Y436H, and A426Y+Y436H+T286L from Experiment 3 are shown in Fig.32B Individual observed serum concentrations of wild type, YTE, N434Y, and N434R from Experiment 1 are also shown in Fig.32A middle.

[0733] Simulations of predicted serum concentration profiles over a three-month period for anti-NGF antibodies carrying wild-type IgGB Fc or IgGB variants A426Y, A426Y+Y436H, A426Y+Y436H+T286L, N434R, N434Y, and YTE were determined and are shown in Fig.33 For this simulation, a 10 kg dog was used with a single intravenous dose of 2 mg / kg.

[0734] Example 11: Modeling of canine IgGB Fc variants binding to canine FcRn

[0735] To provide insights into the molecular mechanism of canine IgGB Fc variants binding to canine FcRn, a structural model of canine IgGB Fc in a complex with canine FcRn was created using MOE software (Molecular Operating Environment (MOE) _, 2020.09; Chemical Computing Group ULC, 1010 Sherbrooke St. West, Suite # 910, Montreal, QC, Canada, H3A 2R7, 2020) based on the co-crystal structure of human FcRn in a complex with YTE-Fc domain (PDBID: 4N0U). Mutations were incorporated into the structure of the model in MOE and energy was minimized with Amber14:EHT force field. Pymol software (The PyMOL Molecular Graphics System, version 1.2r3pre, LLC) to measure the canine Fc-FcRn interaction distance.

[0736] Canine Fc positions 286, 426 and 436 with variants that increase affinity for canine FcRn at low pH are shown in Fig.34 middle.

[0737] Canine IgGB A426H variant is shown in Fig.35 and position 426 is too far away to interact directly with FcRn. The model predicts that A426H causes a steric conflict with Y436, and Y436 replaces A426H in a more favorable conformation for binding to FcRn. Canine A426Y is shown in Fig.36 , and similar to A426H, it is too distal to directly interact with FcRn and shifts Y436 to a more favorable conformation for binding to FcRn.

[0738] The canine IgGB Y436H variant is shown in Fig.37 Changing the position 436 residue to His has a slight change in the predictions for the adjacent residues. The difference in charge may be what drives the tighter binding. The lack of charge on H436 at neutral pH is predicted to drive the adjacent residues to remain in an unfavorable binding environment, similar to the hydrophobic / aromatic Y436. However, the more hydrophilic / positively charged nature of the protonated His436 provides a more attractive interface for residues such as E135 in the FcRn large subunit.

[0739] The canine IgGB T286L variant is shown in Fig.38286Y, T286F and T286W variants have an increased affinity for canine FcRn at low pH, as shown in Examples 4 and 5.

[0740] The combination of A426Y, Y436H and T286L variants on canine IgGB Fc has been modeled ( Fig.39 ). It is predicted that steric clashes between the A426Y and Y436H variants result in 436 moving to a position more favorable for binding. This may have an additive effect with the pH-dependent effect of His. T286L is too far away to appear to be directly affected by the 426 and 436 mutations. The in vitro binding data of the triple variant (A426Y, Y436H and T286L) at low pH to canine FcRn in Example 9 are consistent with the model that the combination of the three variants increases FcRn affinity in an additive manner.

[0741] Other Implementations

[0742] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate rather than 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 following claims. Sequence Listing <110> Invetx Inc. <120> Compositions and methods of use for increasing the half-life of therapeutic agents in dogs <130> 47406-0015WO1 <150> 63 / 122,417 <151> 2020-12-07 <150> 63 / 023,083 <151> 2020-05-11 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 104 <212> PRT <213> Canine <400> 1 Gly Pro Ser Val Leu Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg 1 5 10 15 Ile Thr Arg Thr Pro Glu Val Thr Cys Val Val Leu Asp Leu Gly Arg 20 25 30 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Glu Val 35 40 45 His Thr Ala Lys Thr Gln Ser Arg Glu Gln Gln Phe Asn Gly Thr Tyr 50 55 60 Arg Val Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu Thr Gly 65 70 75 80 Lys Glu Phe Lys Cys Arg Val Asn His Ile Asp Leu Pro Ser Pro Ile 85 90 95 Glu Arg Thr Ile Ser Lys Ala Arg 100 <210> 2 <211> 104 <212> PRT <213> Canine <400> 2 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu 1 5 10 15 Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro 20 25 30 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met 35 40 45 Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr 50 55 60 Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly 65 70 75 80 Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile 85 90 95 Glu Arg Thr Ile Ser Lys Ala Arg 100 <210> 3 <211> 104 <212> PRT <213> Canine <400> 3 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Val 1 5 10 15 Thr Ala Arg Thr Pro Thr Val Thr Cys Val Val Val Asp Leu Asp Pro 20 25 30 Glu Asn Pro Glu Val Gln Ile Ser Trp Phe Val Asp Ser Lys Gln Val 35 40 45 Gln Thr Ala Asn Thr Gln Pro Arg Glu Glu Gln Ser Asn Gly Thr Tyr 50 55 60 Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Ser Gly 65 70 75 80 Lys Gln Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile 85 90 95 Glu Glu Ile Ile Ser Lys Thr Pro 100 <210> 4 <211> 104 <212> PRT <213> Canine <400> 4 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg 1 5 10 15 Ile Thr Arg Thr Pro Glu Ile Thr Cys Val Val Leu Asp Leu Gly Arg 20 25 30 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Glu Val 35 40 45 His Thr Ala Lys Thr Gln Pro Arg Glu Gln Gln Phe Asn Ser Thr Tyr 50 55 60 Arg Val Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu Thr Gly 65 70 75 80 Lys Glu Phe Lys Cys Arg Val Asn His Ile Gly Leu Pro Ser Pro Ile 85 90 95 Glu Arg Thr Ile Ser Lys Ala Arg 100 <210> 5 <211> 106 <212> PRT <213> Canine <400> 5 Lys Pro Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser 1 5 10 15 Ser Asp Thr Val Ser Ile Thr Cys Leu Ile Lys Asp Phe Tyr Pro Pro 20 25 30 Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Arg 35 40 45 Lys His Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 50 55 60 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp 65 70 75 80 Pro Phe Thr Cys Ala Val Met His Glu Thr Leu Gln Asn His Tyr Thr 85 90 95 Asp Leu Ser Leu Ser His Ser Pro Gly Lys 100 105 <210> 6 <211> 105 <212> PRT <213> Canine <400> 6 Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys 1 5 10 15 Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp 20 25 30 Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys 35 40 45 Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu 50 55 60 Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr 65 70 75 80 Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 85 90 95 Glu Ser Leu Ser His Ser Pro Gly Lys 100 105 <210> 7 <211> 105 <212> PRT <213> Canine <400> 7 Gln Pro Asn Val Tyr Val Leu Pro Pro Ser Arg Asp Glu Met Ser Lys 1 5 10 15 Asn Thr Val Thr Leu Thr Cys Leu Val Lys Asp Phe Phe Pro Pro Glu 20 25 30 Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys 35 40 45 Tyr Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu 50 55 60 Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr 65 70 75 80 Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 85 90 95 Ile Ser Leu Ser His Ser Pro Gly Lys 100 105 <210> 8 <211> 106 <212> PRT <213> Canine <400> 8 Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser 1 5 10 15 Ser Asp Thr Val Thr Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro 20 25 30 Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Pro Glu Pro Glu Ser 35 40 45 Lys Tyr His Thr Thr Ala Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 50 55 60 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp 65 70 75 80 Thr Phe Thr Cys Ala Val Met His Glu Ala Leu Gln Asn His Tyr Thr 85 90 95 Asp Leu Ser Leu Ser His Ser Pro Gly Lys 100 105 <210> 9 <211> 220 <212> PRT <213> Canine <400> 9 Val Pro Glu Pro Leu Gly Gly Pro Ser Val Leu Ile Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Ile Leu Arg Ile Thr Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Leu Asp Leu Gly Arg Glu Asp Pro Glu Val Gln Ile Ser Trp Phe 35 40 45 Val Asp Gly Lys Glu Val His Thr Ala Lys Thr Gln Ser Arg Glu Gln 50 55 60 Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His 65 70 75 80 Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His Ile 85 90 95 Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Arg 100 105 110 Ala His Lys Pro Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu 115 120 125 Ser Ser Ser Asp Thr Val Ser Ile Thr Cys Leu Ile Lys Asp Phe Tyr 130 135 140 Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro 145 150 155 160 Glu Arg Lys His Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser 165 170 175 Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln 180 185 190 Gly Asp Pro Phe Thr Cys Ala Val Met His Glu Thr Leu Gln Asn His 195 200 205 Tyr Thr Asp Leu Ser Leu Ser His Ser Pro Gly Lys 210 215 220 <210> 10 <211> 219 <212> PRT <213> Canine <400> 10 Ala Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe 35 40 45 Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu 50 55 60 Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His 65 70 75 80 Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys 85 90 95 Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln 100 105 110 Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu 115 120 125 Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro 130 135 140 Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu 145 150 155 160 Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr 165 170 175 Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly 180 185 190 Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr 195 200 205 Thr Gln Glu Ser Leu Ser His Ser Pro Gly Lys 210 215 <210> 11 <211> 219 <212> PRT <213> Canine <400> 11 Gly Cys Gly Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Ile Leu Val Thr Ala Arg Thr Pro Thr Val Thr Cys Val 20 25 30 Val Val Asp Leu Asp Pro Glu Asn Pro Glu Val Gln Ile Ser Trp Phe 35 40 45 Val Asp Ser Lys Gln Val Gln Thr Ala Asn Thr Gln Pro Arg Glu Glu 50 55 60 Gln Ser Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His 65 70 75 80 Gln Asp Trp Leu Ser Gly Lys Gln Phe Lys Cys Lys Val Asn Asn Lys 85 90 95 Ala Leu Pro Ser Pro Ile Glu Glu Ile Ile Ser Lys Thr Pro Gly Gln 100 105 110 Ala His Gln Pro Asn Val Tyr Val Leu Pro Pro Ser Arg Asp Glu Met 115 120 125 Ser Lys Asn Thr Val Thr Leu Thr Cys Leu Val Lys Asp Phe Phe Pro 130 135 140 Pro Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu 145 150 155 160 Ser Lys Tyr Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr 165 170 175 Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly 180 185 190 Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr 195 200 205 Thr Gln Ile Ser Leu Ser His Ser Pro Gly Lys 210 215 <210> 12 <211> 220 <212> PRT <213> Canine <400> 12 Val Pro Glu Ser Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Ile Leu Arg Ile Thr Arg Thr Pro Glu Ile Thr Cys Val 20 25 30 Val Leu Asp Leu Gly Arg Glu Asp Pro Glu Val Gln Ile Ser Trp Phe 35 40 45 Val Asp Gly Lys Glu Val His Thr Ala Lys Thr Gln Pro Arg Glu Gln 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His 65 70 75 80 Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His Ile 85 90 95 Gly Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln 100 105 110 Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu 115 120 125 Ser Ser Ser Asp Thr Val Thr Leu Thr Cys Leu Ile Lys Asp Phe Phe 130 135 140 Pro Pro Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Pro Glu Pro 145 150 155 160 Glu Ser Lys Tyr His Thr Thr Ala Pro Gln Leu Asp Glu Asp Gly Ser 165 170 175 Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln 180 185 190 Gly Asp Thr Phe Thr Cys Ala Val Met His Glu Ala Leu Gln Asn His 195 200 205 Tyr Thr Asp Leu Ser Leu Ser His Ser Pro Gly Lys 210 215 220 <210> 13 <211> 468 <212> PRT <213> Canine <400> 13 Met Glu Ser Val Phe Cys Trp Val Phe Leu Val Val Ile Leu Lys Gly 1 5 10 15 Val Gln Gly Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Tyr Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Gln Arg Val Ala His Ile Arg Gly Asp Gly Arg Thr Thr His Tyr Ala 65 70 75 80 Asp Ala Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Thr Val Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Val Lys Asp Ile Tyr Tyr Gly Val Gly Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val 145 150 155 160 Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser 180 185 190 Val Leu Gln Ser Ser Gly Leu His Ser Leu Ser Ser Met Val Thr Val 195 200 205 Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Val His 210 215 220 Pro Ala Ser Asn Thr Lys Val Asp Lys Pro Val Phe Asn Glu Cys Arg 225 230 235 240 Cys Thr Asp Thr Pro Pro Cys Pro Val Pro Glu Pro Leu Gly Gly Pro 245 250 255 Ser Val Leu Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg Ile Thr 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Leu Asp Leu Gly Arg Glu Asp 275 280 285 Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Glu Val His Thr 290 295 300 Ala Lys Thr Gln Ser Arg Glu Gln Gln Phe Asn Gly Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu Thr Gly Lys Glu 325 330 335 Phe Lys Cys Arg Val Asn His Ile Asp Leu Pro Ser Pro Ile Glu Arg 340 345 350 Thr Ile Ser Lys Ala Arg Gly Arg Ala His Lys Pro Ser Val Tyr Val 355 360 365 Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr Val Ser Ile 370 375 380 Thr Cys Leu Ile Lys Asp Phe Tyr Pro Pro Asp Ile Asp Val Glu Trp 385 390 395 400 Gln Ser Asn Gly Gln Gln Glu Pro Glu Arg Lys His Arg Met Thr Pro 405 410 415 Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asp Pro Phe Thr Cys Ala Val 435 440 445 Methionine Histidine Glutamic acid Threonine Leucine Glutamine Asparagine Histidine Tyrosine Threonine Aspartic acid Leucine Serine Leucine Serine Histidine 450 455 460 Serine Proline Glycine Lysine 465 <210> 14 <211> 96 <212> PRT <213> Canine <400> 14 Leucine Phenylalanine Threonine Arginine Threonine Lysine Arginine Arginine Serine Aspartic acid Valine Serine Tryptophan Glycine Asparagine Threonine 1 5 10 15 Glycine Serine Serine Glutamine Threonine Valine Isoleucine Arginine Alanine Serine Valine Alanine Serine Tryptophan Serine Arginine 20 25 30 Asparagine Glycine Aspartic acid Leucine Tyrosine Alanine Proline Lysine Proline Lysine Arginine Glutamic acid Asparagine Glycine Arginine Valine 35 40 45 Proline Arginine Proline Proline Aspartic acid Cysteine Lysine Alanine Methionine Phenylalanine Threonine Leucine Leucine Alanine Valine Aspartic acid 50 55 60 Proline Glutamine Methionine Glutamine Proline Glutamic acid Glycine Lysine Glutamine Threonine Lysine Asparagine Lysine Alanine Glutamine Glutamine 65 70 75 80 Arginine Glutamic acid Lysine Asparagine Leucine Phenylalanine Serine Tyrosine Threonine Arginine Threonine Isoleucine Alanine Histidine Glutamine Glutamic acid 85 90 95 <210> 15 <211> 105 <212> PRT <213> Canine <400> 15 Leu Tyr Ala Asp Cys Ala Ser Val Ser Pro Trp Thr Tyr Ser Asp Ile 1 5 10 15 Tyr Ser Val Arg Ala Val Ala Ala Pro Tyr Asp Ser His Tyr Met Pro 20 25 30 Ser Leu Phe Gln Ile Val Tyr Ala Thr Ala Lys Glu Lys Cys Asn Cys 35 40 45 Asn Asn Cys Gly Cys Gly Leu Phe Val Thr Ala Thr Val Asp Pro Asn 50 55 60 Ser Gln Gln Asn Pro Glu Ser Gly Ser Gln Lys Asn Lys Ala Glu Ile 65 70 75 80 Thr Pro Gln Gln Asn Arg Asp Met Lys Asn Thr Leu Val Phe Glu Ser 85 90 95 Tyr Met Arg Thr Ile Ala His Gln Ile 100 105 <210> 16 <211> 66 <212> PRT <213> Canine <400> 16 Leu Ser Asp Gly Ser Val Ser Trp Ala Val Ser Asn Arg Asp Tyr Ser 1 5 10 15 Lys Ala Ile His Val Thr Gly Val Trp Pro Arg His Met His Asn Ser 20 25 30 Leu Phe Tyr Thr Pro Lys Ser Thr Lys Cys Ile Ser Pro Glu Ser Phe 35 40 45 Ile Pro Ser Gly Gln Gln Thr Leu Phe Glu Pro Ser Tyr His Thr Ala 50 55 60 Thr Ala 65 <210> 17 <211> 17 <212> PRT <213> Canine <400> 17 Phe Asn Glu Cys Arg Cys Thr Asp Thr Pro Pro Cys Pro Val Pro Glu 1 5 10 15 Pro <210> 18 <211> 22 <212> PRT <213> Canine <400> 18 Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys 1 5 10 15 Cys Pro Ala Pro Glu Met 20 <210> 19 <211> 20 <212> PRT <213> Canine <400> 19 Ala Lys Glu Cys Glu Cys Lys Cys Asn Cys Asn Asn Cys Pro Cys Pro 1 5 10 15 Gly Cys Gly Leu 20 <210> 20 <211> 17 <212> PRT <213> Canine <400> 20 Pro Lys Glu Ser Thr Cys Lys Cys Ile Ser Pro Cys Pro Val Pro Glu 1 5 10 15 Ser <210> twenty one <211> 17 <212> PRT <213> Canine <400> twenty one Pro Lys Glu Ser Thr Cys Lys Cys Ile Pro Pro Cys Pro Val Pro Glu 1 5 10 15 Ser <210> twenty two <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> twenty two Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Ser Glu Arg 1 5 10 <210> twenty three <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> twenty three Ser Glu Arg Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr 1 5 10 <210> twenty four <211> 15 <212> PRT <213> Artificial <220> <223> synthetic peptide <400> 24 Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Ser Glu Arg 1 5 10 15 <210> 25 <211> 15 <212> PRT <213> Artificial <220> <223> synthetic peptide <400> 25 Ser Glu Arg Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr 1 5 10 15 <210> 26 <211> 18 <212> PRT <213> Artificial <220> <223> synthetic peptide <400> 26 Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Ser 1 5 10 15 Glu Arg <210> 27 <211> 18 <212> PRT <213> Artificial <220> <223> synthetic peptide <400> 27 Ser Glu Arg Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly 1 5 10 15 Leu Tyr <210> 28 <211> 21 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 28 Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly 1 5 10 15 Leu Tyr Ser Glu Arg 20 <210> 29 <211> 21 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 29 Ser Glu Arg Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Gly 1 5 10 15 Leu Tyr Gly Leu Tyr 20 <210> 30 <211> 202 <212> PRT <213> Canine <400> 30 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg 1 5 10 15 Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro 20 25 30 Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala 35 40 45 Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val 50 55 60 Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe 65 70 75 80 Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr 85 90 95 Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu 100 105 110 Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys 115 120 125 Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser 130 135 140 Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln 145 150 155 160 Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp 165 170 175 Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His 180 185 190 Glu Ala Leu His Asn His Tyr Thr Gln Glu 195 200 <210> 31 <211> 200 <212> PRT <213> human <400> 31 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 1 5 10 15 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 20 25 30 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 35 40 45 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 50 55 60 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 65 70 75 80 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 85 90 95 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 100 105 110 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 115 120 125 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 130 135 140 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 145 150 155 160 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 165 170 175 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 180 185 190 Leu His Asn His Tyr Thr Gln Lys 195 200

Claims

1. A polypeptide comprising a canine IgG.B Fc region variant, wherein the canine IgG.B Fc region variant is a variant of the canine IgG Fc region consisting of the amino acid sequence of SEQ ID NO: 10 and comprises an amino acid substitution selected from the group consisting of: (i) a combination of A426Y and any one of T286L, D312P, N434R and Y436H; (ii) a combination of A426H and any one of T286L, T286Y, D312P and Y436H; and (iii) T286L, A426Y and Y436H, wherein the amino acid substitutions are at positions corresponding to amino acid positions 286, 312, 426, 434 and 436 of the wild-type canine IgG.B Fc region, wherein the amino acid positions are based on EU numbering, and wherein the binding affinity of the polypeptide to canine FcRn is increased when compared to a polypeptide comprising a wild-type canine IgG.B Fc region consisting of the amino acid sequence of SEQ ID NO:

10.

2. The polypeptide of claim 1, wherein the amino acid substitution is selected from the group consisting of: (i) A426Y and T286L; (ii) A426Y and D312P; (iii) A426Y and Y436H; (iv) A426H and T286L; (v) A426H and T286Y; (vi) A426H and D312P; and (vii) T286L, A426Y and Y436H.

3. The polypeptide of claim 1, wherein the amino acid substitutions are A426Y and T286L.

4. The polypeptide of claim 1, wherein in a binding assay, the polypeptide binds to canine FcRn at a higher level at acidic pH than at neutral pH.

5. The polypeptide of claim 1, further comprising a binding domain comprising (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 pharmaceutical composition comprising (i) the polypeptide of claim 1 and (ii) a pharmaceutically acceptable excipient.

7. A polypeptide comprising a canine IgG.B Fc region variant, wherein the canine IgG.B Fc region variant is a canine IgG Fc region variant of the group consisting of the amino acid sequence of SEQ ID NO: 10 and comprises the following amino acid substitutions: A combination of N434R and any one of T286L, T286Y, D312P and Y436H, and wherein the amino acid substitutions are at positions corresponding to amino acid positions 286, 312, 426, 434 and 436 of the wild-type canine IgG.B Fc region, wherein the amino acid positions are based on EU numbering, and wherein the binding affinity of the polypeptide to canine FcRn is increased when compared to a polypeptide comprising a wild-type canine IgG.B Fc region consisting of the amino acid sequence of SEQ ID NO: 10.

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