Polypeptides with altered binding to neonatal FC receptor (FCRN) and methods of use

Abstract

Compositions and methods of use for increasing the half-life of a polypeptide or polypeptides in a canine are provided. The compositions comprise a variant canine IgG Fc region.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on December 19, 2023, is named "51682-006WO3_Sequence_Listing_12_19_23", and is 284,818 bytes in size.

[0002] The present disclosure generally relates to polypeptides (e.g., fusion polypeptides such as polypeptide-Fc region fusions, or binding molecules such as antibodies, antigen-binding antibody fragments, or ligand-binding portions of receptor-Fc fusions) that have an increased half-life in canines compared to their wild-type counterparts. [Background technology]

[0003] The Fc region of an antibody has many functional roles, including, but not limited to, protecting the antibody from degradation via the lysosomal pathway and mediating antibody effector functions. With the increasing use of canine antibodies as therapeutic agents, the focus has been not only on selecting the optimal antibody or antibody fragment (e.g., Fab), but also on combining it with an appropriate Fc for the desired half-life and effector function.

[0004] There is little guidance in the art regarding increasing the half-life of polypeptide therapeutics (e.g., antibodies) for use in dogs. Thus, there is a need for Fc region variants that improve the serum persistence of polypeptides (e.g., antibodies) in canines. Summary of the Invention

[0005] Provided herein are canine Fc regions (e.g., canine IgG Fc region variants) useful in therapeutic polypeptides, or canine FcRn-binding fragments thereof. For example, provided herein are polypeptides comprising canine IgG Fc region variants, which canine IgG Fc region variants have increased half-lives in canines compared to their wild-type counterparts.

[0006] In a first aspect, the invention provides a polypeptide comprising a canine IgG Fc region variant, wherein the canine IgG Fc region variant comprises at least one amino acid substitution at a position selected from the group consisting of: (i) Tyr or Met at a position corresponding to amino acid position 252 of wild-type canine IgG, and (ii) a position corresponding to amino acid position 286 of wild-type canine IgG and a position corresponding to amino acid position 426 of wild-type canine IgG; The polypeptides are characterized in that the amino acid positions are based on EU numbering, and the polypeptides have increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0007] In some embodiments, the polypeptide comprises a Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0008] In some embodiments, the polypeptide comprises a Leu, His, Phe, or Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0009] In some embodiments, the polypeptide is (i) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (ii) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iii) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iv) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (v) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vi) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vii) Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (viii) Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (ix) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (x) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (xi) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (xii) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (xiii) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (xiv) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (xv) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG, or (xvi) comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0010] In another aspect, the invention provides a polypeptide comprising a canine IgG Fc region variant, wherein the canine IgG Fc region variant comprises: (i) a His at a position corresponding to amino acid position 434 of wild-type canine IgG; and (ii) a position corresponding to amino acid position 286 of wild-type canine IgG, and at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The polypeptides are characterized in that the amino acid positions are based on EU numbering, and the polypeptides have increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0011] In some embodiments, the polypeptide comprises a Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0012] In some embodiments, the polypeptide comprises a Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0013] In some embodiments, the polypeptide is (i) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (ii) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iii) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iv) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (v) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vi) His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vii) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; or (viii) comprises a His at an amino acid position corresponding to amino acid position 434 of the wild-type canine IgG and a Trp at an amino acid position corresponding to amino acid position 426 of the wild-type canine IgG.

[0014] In some embodiments of any one of the aforementioned aspects, the wild-type canine IgG is a canine IgGA comprising an Fc domain having the amino acid sequence of SEQ ID NO: 9, a canine IgGB comprising an Fc domain having the amino acid sequence of SEQ ID NO: 10, a canine IgGC comprising an Fc domain having the amino acid sequence of SEQ ID NO: 11, or a canine IgGD comprising an Fc domain having the amino acid sequence of SEQ ID NO: 12. In some embodiments, the wild-type canine IgG is a canine IgGA comprising an Fc domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the wild-type canine IgG is a canine IgGB comprising an Fc domain having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the wild-type canine IgG is a canine IgGC comprising an Fc domain having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the wild-type canine IgG is a canine IgGD comprising an Fc domain having the amino acid sequence of SEQ ID NO: 12.

[0015] In some embodiments of any one of the aforementioned aspects, the polypeptide binds to canine FcRn to a higher level at acidic pH than at neutral pH.

[0016] In some embodiments, the polypeptide binds to canine FcRn to a higher level at a pH between 5.5 and 6.0 (eg, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0) than at pH 7.4.

[0017] In some embodiments of any one of the aforementioned aspects, 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.

[0018] In some embodiments of any one of the aforementioned aspects, the polypeptide further comprises a binding domain.

[0019] In some embodiments, the binding domain comprises an antibody, an antibody fragment, or a ligand-binding portion of a receptor.

[0020] In some embodiments, the antibody or antibody fragment comprises six complementarity determining regions (CDRs) of an immunoglobulin molecule.

[0021] In some embodiments, the antibody fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), an Fv, an Fab', an Fab'-SH, an F(ab')2, a nanobody, and a diabody.

[0022] In some embodiments, the ligand-binding portion of the receptor comprises the ligand-binding domain of a canine receptor protein, or the extracellular domain of a canine receptor protein.

[0023] 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 complexes of the complement system.

[0024] In another aspect, the invention features a pharmaceutical composition including (i) any one of the polypeptides disclosed herein and (ii) a pharmaceutically acceptable excipient.

[0025] In another aspect, the invention features a nucleic acid or nucleic acids that encode any one of the polypeptides disclosed herein.

[0026] In another aspect, the invention features an expression vector or vectors that include a nucleic acid or nucleic acids encoding any one of the polypeptides disclosed herein.

[0027] In another aspect, the invention features a host cell containing a nucleic acid or nucleic acids encoding any one of the polypeptides disclosed herein, or an expression vector or expression vectors that include a nucleic acid or nucleic acids encoding any one of the polypeptides disclosed herein.

[0028] In another aspect, the invention provides a method of making a polypeptide, comprising: (i) providing a nucleic acid or nucleic acids encoding any one of the polypeptides disclosed herein; (ii) expressing the nucleic acid or nucleic acids in a host cell culture, thereby producing a polypeptide; and optionally (iii) collecting the polypeptide produced in (ii) from the host cell culture.

[0029] In another aspect, the invention features a method of treating or preventing a canine disease or disorder in a dog in need thereof, comprising administering an effective amount of a composition comprising any one of the polypeptides disclosed herein, or a pharmaceutical composition comprising (i) any one of the polypeptides disclosed herein and (ii) a pharmaceutically acceptable excipient.

[0030] In some embodiments, the canine 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, an infertility-related disorder, an infectious disease, or cancer.

[0031] In other embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0032] In another aspect, the invention features any one of the polypeptides disclosed herein, or a pharmaceutical composition including (i) any one of the polypeptides disclosed herein and (ii) a pharmaceutically acceptable excipient, for use in a dog in need of treatment or prevention of a canine disease or disorder.

[0033] In some embodiments, the canine 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, an infertility-related disorder, an infectious disease, or cancer.

[0034] In other embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those 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 by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief explanation of the drawings]

[0037] [Figure 1]1 is an amino acid sequence alignment of canine IgG gamma chains. These chains contain VH, CH1, CH2, and CH3 domains, as well as the hinge region between CH1 and CH2. N-glycosylation sites are shown in bold and marked within blocks. These sequences for IgGA, IgGB, IgGC, and IgGD are assigned SEQ ID NOs: 13, 14, 15, and 16, respectively. [Figure 2] Amino acid sequence alignment of the CH2 region of canine IgG gamma chains. These sequences for IgGA, IgGD, IgGB, and IgGC are assigned SEQ ID NOs: 1, 4, 2, and 3, respectively. Residues substituted to increase half-life are identified by underlining. [Figure 3] Amino acid sequence alignment of the CH3 region of canine IgG gamma chains. These sequences for IgGA, IgGD, IgGB, and IgGC are assigned SEQ ID NOs: 5, 8, 6, and 7, respectively. Residues substituted to increase half-life are identified by underlining. [Figure 4] 1 is an amino acid sequence alignment of the Fc region of canine IgG gamma chains. These sequences for IgGA, IgGD, IgGB, and IgGC are assigned SEQ ID NOs: 9, 12, 10, and 11, respectively. Residues substituted to increase half-life are identified by underlining. [Figure 5-1] 1 is a table providing EU numbering for the CH2 region of canine IgG. hIgG1 = human IgG1; cIgG.A = canine IgGA; cIgG.B = canine IgGB; cIgG.C = canine IgGC; cIgG.D = canine IgGD; SM = mutation capable of extending antibody half-life. [Figure 5-2] continuation. [Figure 5-3] continuation. [Figure 6-1] 1 is a table providing EU numbering for the CH3 region of canine IgG. [Figure 6-2] continuation. [Figure 6-3] continuation. DETAILED DESCRIPTION OF THE INVENTION

[0038] With the increasing use of polypeptides (e.g., antibodies, antigen-binding antibody fragments, ligand-binding domains of receptors, enzymes, ligands, and peptides) as therapeutic agents for the prevention and treatment of a wide variety of canine diseases, it is important to develop polypeptides with extended half-lives, especially for the prevention or treatment of chronic diseases where repeated administration of the polypeptide is required.

[0039] Accordingly, the present disclosure features canine immunoglobulin Fc regions, or canine FcRn-binding regions thereof, containing mutations that improve the half-life of a polypeptide or polypeptides containing these sequences. Also disclosed are polypeptides containing these domains and methods of use thereof. These polypeptides can be used for a variety of therapeutic and diagnostic purposes.

[0040] For example, the disclosure features polypeptides that have increased binding to canine FcRn relative to a control polypeptide (e.g., a wild-type counterpart IgG canine Fc region). In some cases, these polypeptides can bind to canine FcRn at a higher level (i.e., with greater affinity) at, for example, an acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5) than at a neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some cases, these polypeptides bind to canine FcRn at a higher level at pH 5.5 and / or 6.0 than at pH 7.4. The disclosure also relates, in part, to polypeptides that have increased half-life in canines relative to their wild-type counterparts. For example, provided are polypeptides (e.g., binding molecules such as antibodies, antigen-binding antibody fragments, or ligand-binding portions of receptors) that have increased half-lives compared to versions of these polypeptides that do not bind to the Fc region disclosed herein or the canine FcRn-binding region thereof. Also provided are enzyme-Fc region fusions, ligand-Fc region fusions, nanobody-Fc fusions, and peptide-Fc region fusions, which fusions have increased half-lives compared to their wild-type counterparts. In addition to having one or more substitutions that increase half-life (compared to a wild-type canine Fc region), the Fc region can also include other substitutions that, for example, increase effector function, decrease effector function, increase binding to Protein A, and / or decrease polypeptide heterogeneity (e.g., by removing one or more post-translational modifications within the Fc region). The canine Fc region sequence can be derived from any canine antibody. In some cases, the canine Fc region sequence is derived from a canine IgG (e.g., IgGA, IgGB, IgGC, or IgGD).

[0041] When values ​​are described as ranges, the description should be understood to include disclosure of all possible subranges within such ranges and specific numerical values ​​falling within such ranges, whether or not a specific numerical value or specific subrange is explicitly stated.D , temperatures, times, concentrations, and molecular weights include (+) or (-) approximation ranges that vary by increments of 1.0 or 0.1, or alternatively by a variance of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%, as appropriate. It is understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about," and that the numerical designation may include values ​​that are rounded to the nearest significant figure. It is also understood, although not always explicitly stated, that the reagents described herein are merely exemplary and equivalents thereof are known in the art.

[0042] Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include the plural and plural terms shall include the singular. In the event of conflicts in definitions among various sources or references, the definitions provided herein shall prevail.

[0043] Embodiments of the invention described herein are understood to include aspects and embodiments such as "comprising," "consisting of," and "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural references (e.g., at least one, one or more) unless otherwise indicated. The use of the term "or" herein means "and / or" and does not imply that alternatives are mutually exclusive unless otherwise specified. In the context of multiple dependent claims, the use of "or" when returning to other claims refers to those claims in the alternative only.

[0044] In this application, the use of "or" means "and / or" unless expressly stated or understood by one of ordinary skill in the art. In the context of multiple dependent claims, the use of "or" refers back to more than one preceding independent or dependent claim.

[0045] 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.

[0046] As used herein, "percent (%) amino acid sequence identity," "% identity," and "homology" with respect to nucleic acid or polypeptide sequences are defined as the percentage of nucleotides or amino acid residues in a reference sequence that are identical to those in a particular nucleic acid or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any parameters necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, a variant has at least 50% sequence identity with a reference nucleic acid molecule or polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least 50% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a reference nucleic acid or polypeptide sequence.

[0047] The term "amino acid substitution" refers to the replacement of one amino acid in a polypeptide with another amino acid. In some embodiments, the amino acid substitution is a conservative substitution. The amino acid substitution may be introduced into a polypeptide that has been screened for a desired activity, such as retention or improvement of FcRn binding, retention or improvement of antigen binding, reduced immunogenicity, improvement of antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or improved pharmacokinetics.

[0048] As used herein, the term "conservative substitution" refers to the substitution of one amino acid residue for another amino acid residue having similar properties, such as charge, hydrophobicity, and size. For example, amino acids can be grouped according to common side chain properties: (i) Hydrophobic: Norleucine (Nle), Met, Ala, Val, Leu, Ile; (ii) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (iii) Acidic: Asp, Glu; (iv) basic: His, Lys, Arg; (v) Stiffness: Gly, Pro; (vi) Aromatic: Trp, Tyr, Phe. Conservative substitutions involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions involve exchanging a member of one of these classes for another class. In some embodiments, a conservative amino acid substitution refers to a substitution that results in similar properties or functions as another amino acid substitution. For example, a conservative amino acid substitution for A426Y can be A426F, A426T, or A426W. Additional non-limiting examples of conservative amino acid substitutions are shown in Table 1. [Table 1]

[0049] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or receptor) and its binding partner (e.g., an antigen or ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and antigen, receptor and ligand). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common protein-protein interaction tools known in the art, such as, for example, immunoblot, enzyme-linked immunosorbent assay (ELISA), binding equilibrium exclusion assay (KinExA), biolayer interferometry (BLI), or surface plasmon resonance (SPR) devices. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0050] "Surface plasmon resonance (SPR)" refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al., 1993, Ann. Biol. Clin. 51:19-26.

[0051] The term "amino acid sequence" refers to the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, with no minimum length restriction. Such polymers of amino acid residues may contain natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins containing modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), so long as the protein maintains the desired activity. These modifications may be deliberate, via site-directed mutagenesis, or may be accidental, via mutations of hosts producing the protein or errors due to PCR amplification.

[0052] The term "antibody" herein is used in the broadest sense and refers to a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab), so long as they exhibit the desired antigen-binding activity.

[0053] The term "antibody fragment" refers to a molecule other than a full-length antibody that contains a portion of a full-length antibody that binds to the antigen bound by the full-length antibody. In some embodiments, antibody fragments include, but are not limited to, Fab, single-chain variable fragments (e.g., scFv), Fv, Fab', Fab'-SH, F(ab'), nanobodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0054] The terms "full length antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0055] The terms "nanobody," "VHH," "VHH antibody fragment," and "single domain antibody," used interchangeably herein, refer to the variable domain of a single heavy chain of an antibody of the type found in the Camelidae family, which are typically found devoid of light chains in their native form. Suitable nanobodies are well known to those skilled in the art, and illustrative examples include camel, dromedary, llama, and alpaca nanobodies. However, single domain antibodies can also be derived from non-camelidae sources.

[0056] The term "binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab, scFv, Fv, Fab', Fab'-SH, F(ab')2, nanobodies, and diabodies), receptors or fragments thereof (e.g., the extracellular domains of canine receptor proteins), ligands, aptamers, and other molecules with identified binding partners.

[0057] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0058] The terms "Fc region," "Fc domain," and "Fc polypeptide" refer to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term "wild-type canine IgG Fc domain" refers to the native Fc region of a canine antibody. The term "canine IgG Fc region variant" refers to a variant of the Fc region of a canine antibody that has one or more substitutions relative to the wild-type canine Fc region. In some embodiments, the canine Fc region sequence is derived from a canine IgG (e.g., IgGA, IgGB, IgGC, or IgGD). In some embodiments, an IgG Fc polypeptide comprises a hinge, CH2, and CH3, but does not comprise a CH1 or CL. In some embodiments, an IgG Fc polypeptide comprises a CH2 and CH3, but does not comprise a CH1, hinge, or CL. In some embodiments, an IgG Fc polypeptide comprises a CH1, hinge, CH2, and CH3, with or without a CLI. In some embodiments, an Fc polypeptide, such as an IgG Fc polypeptide, lacks one or more C-terminal amino acids, e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 2, while retaining biological activity. In some embodiments, the biological activity of the Fc polypeptide is the ability to bind to FcRn. Unless otherwise specified herein, numbering of amino acid residues in an Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0059] The term "wild-type" refers to a non-mutated form of a naturally occurring polypeptide, or a fragment thereof. A wild-type polypeptide may be recombinantly produced. In some embodiments, a wild-type IgG Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 9-12.

[0060] The term "disorder" refers to any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0061] The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, myeloma, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma (e.g., angiosarcoma, osteosarcoma, soft tissue sarcoma, histiocytic sarcoma), leukemia, head and neck squamous cell carcinoma, salivary gland carcinoma, breast cancer, mast cell tumor, melanoma, lung cancer (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal carcinoma, hepatocellular carcinoma, squamous cell carcinoma, meningioma, glioma, gastric cancer, intestinal cancer, colon cancer, colorectal cancer, pancreatic adenocarcinoma, glioblastoma, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, bladder cancer, prostate cancer, kidney cancer, vulvar cancer, thyroid cancer, and transitional cell carcinoma.

[0062] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0063] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0064] An "effective amount" of a composition, e.g., a polypeptide of the present disclosure, or a composition thereof (e.g., a pharmaceutical composition), refers to at least the minimum amount necessary to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder (e.g., any disorder affecting canines, e.g., a cell proliferative disorder, e.g., cancer). An effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the animal, and the ability of the antibody to elicit a desired response in the animal. An effective amount also is one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutic beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from a disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effectiveness of other drugs, such as through targeting, slowing disease progression, and / or extending survival. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical field, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in relation to the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0065] The terms "host cell" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include bacteria (e.g., E. coli cells) and eukaryotic cells. In some embodiments, host cells include yeast cells (e.g., Pichia (see, e.g., Powers et al., 2001, J Immunol Methods. 251:123-135), Hanseula, or Saccharomyces). In some embodiments, host cells also include "transformants" and "transformed cells," including the primary transformed cell line (e.g., CHO, 293E, COS, 293T, and HeLa) and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0066] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0067] The term "pharmaceutical composition" refers to a preparation that is in a form such that the biological activity of the active ingredient contained in the preparation is effective, and that does not contain any additional components that are unacceptably toxic to the subject to whom the formulation will be administered.

[0068] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0069] As used herein, the term "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or primary palliation of the disease state, and remission or improved prognosis. In some embodiments, the polypeptides of the invention are used to delay the onset of disease or slow the progression of the disease.

[0070] As used herein, the term "delaying progression" of a disorder or disease means to prolong, hinder, delay, prevent, stabilize, and / or postpone the onset of a disease or disorder (e.g., a cell proliferative disorder, e.g., cancer). The delay may be of varying duration depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay may, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, may be delayed.

[0071] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds. For example, an epitope can be a linear epitope or a conformational epitope.

[0072] As used herein, the terms "reduce" and "inhibit" refer to the ability to cause an overall decrease, e.g., of 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more, as compared to a reference or control.

[0073] The terms "increase" and "improve" refer to the ability to cause an overall increase, e.g., of 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more, as compared to a reference or control.

[0074] The terms "variable region" and "variable domain" refer to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., 2007, Kuby Immunology, 6th ed. W.H. Freeman and Co., p. 91.) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains using a VH or VL domain, respectively, from an antibody that binds to the antigen. See, e.g., Portolano et al., 1993, J. Immunol. 150:880-887, and Clarkson et al., 1991, Nature 352:624-628.

[0075] A "variant" is a polypeptide that differs from a reference polypeptide by single or multiple non-natural amino acid substitutions, deletions, and / or additions. In some embodiments, a variant retains at least one biological activity of the reference polypeptide. In some embodiments, a variant has a biological activity that the reference polypeptide substantially lacks. A "canid IgG Fc region variant" comprises an amino acid sequence that differs from that of a wild-type canid IgG Fc region by at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, a canid IgG Fc region variant has at least one amino acid substitution compared to the wild-type canid IgG Fc region, e.g., 1 to 10 amino acid substitutions, preferably 1 to 5 amino acid substitutions in the wild-type canid IgG Fc region. A canid IgG Fc region variant herein preferably has at least 80% homology to the wild-type canid IgG Fc region, most preferably at least 90% homology, and more preferably at least 95% homology. In some embodiments, the canine IgG Fc region is a canine IgGA Fc region variant, a canine IgGB Fc region variant, a canine IgGC Fc region variant, or a canine IgGD Fc region variant.

[0076] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0077] As used herein, "administration" refers to a method of providing a subject with a dosage of a compound (e.g., a polypeptide of the present disclosure) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a polypeptide of the present disclosure). Compositions used in the methods described herein can be administered, for example, parenterally, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, by catheter, by lavage, in a cream, or in a lipid composition. Administration can be local or systemic. Methods of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0078] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential or sequential administration in any order. The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent occurs within a short period of time compared to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered approximately a certain number of minutes or less apart. The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, where the administration of one or more agents continues after the administration of one or more other agents is discontinued, or where the administration of one or more agents begins before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is administered approximately a certain number of minutes or more apart. As used herein, "in conjunction with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to the administration of one therapeutic modality to an animal before, during, or after the administration of another therapeutic modality.

[0079] Canine Polypeptides Dogs have four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgGA, IgGB, IgGC, and IgGD. The amino acid and DNA sequences of these heavy chains can be obtained from Tang et al., 2001, Vet. Immunol. Immunopathol., 80:259-270 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 contain two types of light chains: kappa and lambda. The DNA and amino acid sequences of these light chains can also be obtained from the GENBANK database. For example, the canine kappa light chain amino acid sequence has accession number ABY57289.1, and the canine lambda light chain has accession number ABY55569.1.

[0080] CH2 region of canine Fc domain: The CH2 region of a canine antibody comprises or consists of amino acids 237-340 (according to EU numbering) of a canine IgG antibody. It is understood that the CH2 region can include 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0081] The amino acid sequence of the CH2 region of canine IgGA is provided below: GPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKAR (SEQ ID NO: 1)

[0082] The amino acid sequence of the CH2 domain of canine IgGB is provided below: GPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKAR (SEQ ID NO: 2)

[0083] The amino acid sequence of the CH2 domain of canine IgGC is provided below: GPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTP (SEQ ID NO: 3)

[0084] The amino acid sequence of the CH2 domain of canine IgGD is provided below: GPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTAKTQ PREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR (SEQ ID NO: 4)

[0085] CH3 region of canine Fc domain: 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 is understood that the CH3 region can include 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0086] The amino acid sequence of the CH3 domain of canine IgGA is provided below: KPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK (SEQ ID NO: 5)

[0087] The amino acid sequence of the CH3 domain of canine IgGB is provided below: QP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK (SEQ ID NO: 6)

[0088] The amino acid sequence of the CH3 domain of canine IgGC is provided below: Q PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK (SEQ ID NO: 7)

[0089] The amino acid sequence of the CH3 domain of canine IgGD is provided below: QPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW QQGDTFTCAV MHEALQNHYT DLSLSHSPGK (SEQ ID NO: 8)

[0090] Canine Fc region: 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.

[0091] The amino acid sequence of the Fc domain of canine IgGA is provided below: VPEPLGGPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKARGR AHKPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK (SEQ ID NO: 9)

[0092] The amino acid sequence of the Fc domain of canine IgGB is provided below: APEMLGGPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKARGQAHQP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK (SEQ ID NO: 10)

[0093] The amino acid sequence of the Fc domain of canine IgGC is provided below: GCGLLGGPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTPGQAHQ PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK (SEQ ID NO: 11)

[0094] The amino acid sequence of the Fc domain of canine IgGD is provided below: VPESLGGPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTakTQ PREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR GQAHQPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW QQGDTFTCAV MHEALQNHYT DLSLSHSPGK (SEQ ID NO: 12)

[0095] Substitutions in canine IgG Fc that improve half-life Increased serum persistence is a beneficial property for therapeutic polypeptides. The present disclosure features substitutions in wild-type canine IgGA, IgGB, IgGC, and IgGD Fc regions that increase the half-life of a polypeptide or polypeptides comprising these Fc regions in dogs compared to a control polypeptide or polypeptides that are identical to the polypeptide or polypeptides except for having the corresponding wild-type canine IgG Fc region instead of the IgG Fc region variant. Substitutions to increase half-life can be made in the context of a canine CH2 region, one or more of a canine CH3 region, or a canine Fc (e.g., CH2+CH3) region.

[0096] The present disclosure provides a polypeptide comprising a canine IgG Fc region variant, wherein the canine IgG Fc region variant comprises: (i) an amino acid substitution (e.g., Tyr or Met) at a position corresponding to amino acid position 252 of wild-type canine IgG; and (ii) a position corresponding to amino acid position 286 of wild-type canine IgG, and at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The present invention provides polypeptides, wherein the amino acid positions are based on EU numbering, and wherein the polypeptides have increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG. In some examples, the amino acid substitution at the position corresponding to amino acid position 252 of wild-type canine IgG is a conservative amino acid substitution of Tyr or Met.

[0097] For example, the disclosure provides a polypeptide comprising a canine IgG Fc region variant, wherein the canine IgG Fc region variant comprises: (i) a Tyr or Met at a position corresponding to amino acid position 252 of wild-type canine IgG; and (ii) a position corresponding to amino acid position 286 of wild-type canine IgG, and at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The present invention provides polypeptides, wherein the amino acid positions are based on EU numbering, and wherein the polypeptides have increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG. In some examples, the IgG Fc region variants comprise a Tyr at a position corresponding to amino acid position 252 of wild-type canine IgG. In other examples, the IgG Fc region variants comprise a Met at a position corresponding to amino acid position 252 of wild-type canine IgG.

[0098] In some embodiments, the polypeptide comprises Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Leu at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Trp at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canid IgG is a conservative amino acid substitution of Tyr, Phe, Leu, or Trp.

[0099] In some embodiments, the polypeptide comprises Leu, His, Phe, or Tyr at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Leu at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises His at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of the wild-type canid IgG is a conservative amino acid substitution for Leu, His, or Tyr.

[0100] In some embodiments, the polypeptide is (i) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (ii) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iii) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iv) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (v) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vi) a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vii) Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (viii) Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (ix) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (x) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (xi) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (xii) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (xiii) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (xiv) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (xv) Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG, or (xvi) comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0101] In another aspect, the invention provides a polypeptide comprising a canine IgG Fc region variant, wherein the canine IgG Fc region variant comprises: (i) an amino acid substitution (e.g., His) at a position corresponding to amino acid position 434 of wild-type canine IgG; and (ii) a position corresponding to amino acid position 286 of wild-type canine IgG, and at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The polypeptides are characterized by having increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG, where amino acid positions are based on EU numbering. In some examples, the amino acid substitution at a position corresponding to amino acid position 434 of wild-type canine IgG is a conservative amino acid substitution of His.

[0102] For example, the invention provides a polypeptide comprising a canine IgG Fc region variant, wherein the canine IgG Fc region variant comprises: (i) a His at a position corresponding to amino acid position 434 of wild-type canine IgG; and (ii) a position corresponding to amino acid position 286 of wild-type canine IgG, and at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The polypeptides are characterized in that the amino acid positions are based on EU numbering, and the polypeptides have increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0103] In some embodiments, the polypeptide comprises Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Leu at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises Trp at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canid IgG is a conservative amino acid substitution of Tyr, Phe, Leu, or Trp.

[0104] In some embodiments, the polypeptide comprises Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Phe at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Leu at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises Trp at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of the wild-type canid IgG is a conservative amino acid substitution of Tyr, Phe, Leu, or Trp.

[0105] In some embodiments, the polypeptide is (i) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (ii) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iii) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (iv) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (v) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vi) His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; (vii) a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG; or (viii) comprises a His at an amino acid position corresponding to amino acid position 434 of the wild-type canine IgG and a Trp at an amino acid position corresponding to amino acid position 426 of the wild-type canine IgG.

[0106] In some embodiments of any one of the aforementioned aspects, the wild-type canine IgG is a canine IgGA comprising an Fc domain having the amino acid sequence of SEQ ID NO: 9, a canine IgGB comprising an Fc domain having the amino acid sequence of SEQ ID NO: 10, a canine IgGC comprising an Fc domain having the amino acid sequence of SEQ ID NO: 11, or a canine IgGD comprising an Fc domain having the amino acid sequence of SEQ ID NO: 12. In some embodiments, the wild-type canine IgG is a canine IgGA comprising an Fc domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the wild-type canine IgG is a canine IgGB comprising an Fc domain having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the wild-type canine IgG is a canine IgGC comprising an Fc domain having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the wild-type canine IgG is a canine IgGD comprising an Fc domain having the amino acid sequence of SEQ ID NO: 12.

[0107] In some embodiments, the polypeptide comprises at least one amino acid substitution at a position corresponding to one or more of amino acid positions 252, 286, 426, and 434 of wild-type canine IgG, where amino acid positions are based on EU numbering, and the polypeptide has increased binding to canine FcRn compared to the Fc domain of wild-type canine IgG. The at least one amino acid substitution encompassed by the present disclosure can include one or more (e.g., one, two, three, or four) of those disclosed in Table 2. [Table 2]

[0108] In some embodiments of any one of the aforementioned aspects, the polypeptide binds to canine FcRn to a higher level at an acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5) than at a neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5).

[0109] In some embodiments, the polypeptide binds to canine FcRn at a higher level at a pH of 5.5 to 6.0 than at pH 7.4. In some embodiments, the polypeptide binds to canine FcRn at a higher level at a pH of 5.5 than at pH 7.4. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4.

[0110] Any of the polypeptides disclosed herein may include one or more additional amino acid substitutions, including any amino acid substitutions as disclosed in U.S. Patent Application Publication Nos. 2020 / 0216536 and 2020 / 0362035, U.S. Patent Application No. 17 / 875,934, and U.S. Patent No. 11,434,276, each of which is incorporated by reference in its entirety.

[0111] 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: (i) a position corresponding to amino acid position 286 of wild-type canine IgG; (ii) a position corresponding to amino acid position 426 of wild-type canine IgG, and (iii) at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 436 of wild-type canine IgG; Provided are polypeptides, wherein the amino acid substitution at a position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of Tyr, Phe, Leu, and Trp, where the amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to canine FcRn, compared to the Fc domain of wild-type canine IgG. In some embodiments, the polypeptide has increased binding affinity to canine FcRn in a binding assay at a pH of about 5.0 to about 6.5 (e.g., about 5.5 or about 6.0), compared to the Fc domain of wild-type canine IgG. In some embodiments, a binding assay refers, for example, to an assay in which the binding affinity of a polypeptide variant described herein to canine FcRn at a pH of about 5.0 to about 6.5 is compared to the binding affinity of wild-type canine IgG to canine FcRn at the same pH (e.g., a pH of about 5.0 to about 6.5). In some embodiments, the binding assay is performed using equivalent conditions. In some embodiments, the binding assay is a surface plasmon resonance (SPR) assay.

[0112] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises a Tyr, His, or Phe at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG. In some embodiments, the polypeptide comprises a Phe at an amino acid position corresponding to amino acid position 426 of the wild-type canid IgG.

[0113] 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.

[0114] 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.

[0115] In some embodiments, the present disclosure provides canine IgG CH2 region variants comprising an amino acid sequence 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: 1-4. Also provided are canine IgG CH2 region variants comprising an amino acid sequence that varies 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids from any one of SEQ ID NOs: 1-4.

[0116] In other embodiments, the disclosure features canine IgG CH3 region variants comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-8. Also featured are canine IgG CH3 region variants comprising an amino acid sequence that varies 1-15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids from any one of SEQ ID NOs: 5-8.

[0117] In certain embodiments, the disclosure features canine IgG Fc region variants comprising an amino acid sequence 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 varies 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 from any one of SEQ ID NOs: 9 to 12.

[0118] In some embodiments, the following regions in the canine IgG Fc CH2 region variant: amino acid positions 250–256; Amino acid positions 285 to 288, and at least one (e.g., one, two, or three) of amino acid positions 307-315 are identical to the corresponding region in a wild-type canine IgG Fc CH2 region; Amino acid positions are based on EU numbering. In some embodiments, 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.

[0119] In some embodiments, the following regions in the canine IgG Fc CH3 region variant: amino acid positions 376 to 380, and at least one (e.g., one or two) of amino acid positions 428-436 is identical to the corresponding region in a wild-type canine IgG Fc CH3 region; Amino acid positions are based on EU numbering. In some embodiments, all of the above regions in the variant canine IgG Fc CH3 region are identical to the corresponding regions in the wild-type canine IgG Fc CH3 region.

[0120] In some embodiments, the following regions in the canine IgG Fc variant: amino acid positions 250–256; amino acid positions 285–288; amino acid positions 307–315; amino acid positions 376 to 380, and at least one (e.g., one, two, three, four, or five) of amino acid positions 428-436 are identical to the corresponding region in a wild-type canine IgG Fc; Amino acid positions are based on EU numbering. In some embodiments, all of the following regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc:

[0121] In some embodiments, the following regions in the canine IgG Fc variant: amino acid positions 250–256; amino acid positions 285, 287, and 288; amino acid positions 307–315; amino acid positions 376 to 380, and at least one (e.g., one, two, three, four, or five) of amino acid positions 428-436 are identical to the corresponding region in a wild-type canine IgG Fc; Amino acid positions are based on EU numbering. In some embodiments, all of the following regions in the canine IgG Fc variant are identical to the corresponding regions in the wild-type canine IgG Fc:

[0122] In some embodiments, a polypeptide or 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 set forth in any one of SEQ ID NOs: 1-4.

[0123] Some embodiments feature a polypeptide or polypeptides comprising 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 set forth in any one of SEQ ID NOs:5-8.

[0124] Some embodiments feature a polypeptide or polypeptides comprising a canine IgG Fc region variant, wherein the Fc 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 set forth in any one of SEQ ID NOs:9-12.

[0125] In some embodiments, the polypeptide is (i) Tyr, His, or Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG, and / or (ii) contains Tyr, Phe, Leu, and Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0126] In some embodiments, the polypeptide is (i) a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG, and / or (ii) contains Tyr, Phe, Leu, and Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0127] In some embodiments, the polypeptide is (i) a His at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG, and / or (ii) contains Tyr, Phe, Leu, and Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0128] As described 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, where amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0129] In some embodiments, the polypeptide or polypeptides are: an amino acid other than the wild-type amino acid occurring at amino acid position 250; an amino acid other than the wild-type amino acid occurring at amino acid position 251; an amino acid other than the wild-type amino acid occurring at amino acid position 252; an amino acid other than the wild-type amino acid occurring at amino acid position 254; an amino acid other than the wild-type amino acid occurring at amino acid position 256; an amino acid other than the wild-type amino acid occurring at amino acid position 285; an amino acid other than the wild-type amino acid occurring at amino acid position 286; an amino acid other than the wild-type amino acid occurring at amino acid position 307; an amino acid other than the wild-type amino acid occurring at amino acid position 308; an amino acid other than the wild-type amino acid occurring at amino acid position 309; an amino acid other than the wild-type amino acid occurring at amino acid position 311, or a canine IgG CH2 region(s) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acids other than the wild-type amino acid occurring at amino acid position 315; Amino acid positions are based on EU numbering for canine IgGA, IgGB, IgGC, and IgGD antibodies.

[0130] In some embodiments, the polypeptide or polypeptides are: an amino acid other than the wild-type amino acid occurring at amino acid position 378; an amino acid other than the wild-type amino acid occurring at amino acid position 380; an amino acid other than the wild-type amino acid occurring at amino acid position 428; an amino acid other than the wild-type amino acid occurring at amino acid position 430; an amino acid other than the wild-type amino acid occurring at amino acid position 433; an amino acid other than the wild-type amino acid occurring at amino acid position 434; an amino acid other than the wild-type amino acid occurring at amino acid position 435, or a canine IgG CH3 region(s) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) amino acids other than the wild-type amino acid occurring at amino acid position 436; Amino acid positions are based on EU numbering for canine IgGA, IgGB, IgGC, and IgGD antibodies.

[0131] In certain embodiments, the polypeptide or polypeptides are: an amino acid other than the wild-type amino acid occurring at amino acid position 250; an amino acid other than the wild-type amino acid occurring at amino acid position 251; an amino acid other than the wild-type amino acid occurring at amino acid position 252; an amino acid other than the wild-type amino acid occurring at amino acid position 254; an amino acid other than the wild-type amino acid occurring at amino acid position 256; an amino acid other than the wild-type amino acid occurring at amino acid position 285; an amino acid other than the wild-type amino acid occurring at amino acid position 286; an amino acid other than the wild-type amino acid occurring at amino acid position 307; an amino acid other than the wild-type amino acid occurring at amino acid position 308; an amino acid other than the wild-type amino acid occurring at amino acid position 309; an amino acid other than the wild-type amino acid occurring at amino acid position 311; an amino acid other than the wild-type amino acid occurring at amino acid position 315; an amino acid other than the wild-type amino acid occurring at amino acid position 378; an amino acid other than the wild-type amino acid occurring at amino acid position 380; an amino acid other than the wild-type amino acid occurring at amino acid position 428; an amino acid other than the wild-type amino acid occurring at amino acid position 430; an amino acid other than the wild-type amino acid occurring at amino acid position 433; an amino acid other than the wild-type amino acid occurring at amino acid position 434; an amino acid other than the wild-type amino acid occurring at amino acid position 435; a canine IgG Fc region(s) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids other than the wild-type amino acid occurring at amino acid position 436; Amino acid positions are based on EU numbering for canine IgGA, IgGB, IgGC, and IgGD antibodies.

[0132] 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 wild-type canine IgG, where amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding to canine FcRn compared to the Fc domain of wild-type canine IgG. 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 3. [Table 3]

[0133] In some embodiments, the at least one additional amino acid substitution encompassed by this disclosure includes one or more (eg, one, two, three, or four) of those substitutions disclosed in Table 4. [Table 4]

[0134] All possible combinations and permutations of the substitutions disclosed above are encompassed by the present disclosure. In some embodiments, the polypeptide comprises: (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256; (ii) Leu at amino acid position 428 and Ser at amino acid position 434; (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311; (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378; (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; (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315; (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; (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378; (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378; (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378; (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; (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; (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378; (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; (xvi) Gln at amino acid position 307, Ala at amino acid position 380, and Ser or Ala at amino acid position 434; (xvii) Leu at amino acid position 428 and Ser or Ala at amino acid position 434; (xviii) Gln at amino acid position 250 and Leu at amino acid position 428; (xix) Glu at amino acid position 250 and Glu at amino acid position 251; (xx) Phe at amino acid position 256 and Phe at amino acid position 309; (xxi) Ala at amino acid position 430 and Lys at amino acid position 433; (xxii) Phe at amino acid position 434 and His at amino acid position 436, and (xxiii) comprises at least one (e.g., two or more, three or more, four or more, five or more) additional amino acid substitution selected from the group consisting of Tyr at amino acid position 435 and His at amino acid position 436.

[0135] In some embodiments, the substitution does not include the combination of Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256.

[0136] In some embodiments, the at least one additional amino acid substitution is (i) an amino acid position corresponding to amino acid position 250 of wild-type canine IgG; (ii) an amino acid position corresponding to amino acid position 251 of wild-type canine IgG; (iii) an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (iv) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (v) an amino acid position corresponding to amino acid position 256 of wild-type canine IgG; (vi) an amino acid position corresponding to amino acid position 285 of wild-type canine IgG; (vii) an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (viii) an amino acid position corresponding to amino acid position 307 of wild-type canine IgG; (ix) an amino acid position corresponding to amino acid position 308 of wild-type canine IgG; (x) an amino acid position corresponding to amino acid position 309 of wild-type canine IgG; (xi) an amino acid position corresponding to amino acid position 311 of wild-type canine IgG; (xii) an amino acid position corresponding to amino acid position 315 of wild-type canine IgG; (xiii) an amino acid position corresponding to amino acid position 378 of wild-type canine IgG; (xiv) an amino acid position corresponding to amino acid position 380 of wild-type canine IgG; (xv) an amino acid position corresponding to amino acid position 428 of wild-type canine IgG; (xvi) an amino acid position corresponding to amino acid position 430 of wild-type canine IgG; (xvii) an amino acid position corresponding to amino acid position 433 of wild-type canine IgG; (xviii) an amino acid position corresponding to amino acid position 434 of wild-type canine IgG; (xix) an amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and (xx) at a position selected from the group consisting of an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0137] In some embodiments, the polypeptide is (i) Glu or Gln at an amino acid position corresponding to amino acid position 250 of wild-type canine IgG; (ii) Asp or Glu at an amino acid position corresponding to amino acid position 251 of wild-type canine IgG; (iii) Tyr or Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (iv) Thr or Ser at an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (v) Asp, Glu, or Phe at an amino acid position corresponding to amino acid position 256 of wild-type canine IgG; (vi) Asn or Asp at an amino acid position corresponding to amino acid position 285 of wild-type canine IgG; (vii) Asp, Tyr, Phe, Leu, or Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG; (viii) Arg, Gln, or Ala at an amino acid position corresponding to amino acid position 307 of wild-type canine IgG; (ix) Pro at an amino acid position corresponding to amino acid position 308 of wild-type canine IgG; (x) Pro at an amino acid position corresponding to amino acid position 309 of wild-type canine IgG; (xi) Val at an amino acid position corresponding to amino acid position 311 of wild-type canine IgG; (xii) Asp at an amino acid position corresponding to amino acid position 315 of wild-type canine IgG; (xiii) Val at an amino acid position corresponding to amino acid position 378 of wild-type canine IgG; (xiv) Ala at an amino acid position corresponding to amino acid position 380 of wild-type canine IgG; (xv) Leu at an amino acid position corresponding to amino acid position 428 of wild-type canine IgG; (xvi) Ala or Lys at an amino acid position corresponding to amino acid position 430 of wild-type canine IgG; (xvii) Lys at an amino acid position corresponding to amino acid position 433 of wild-type canine IgG; (xviii) Trp, Tyr, Arg, His, Ser, Ala, or Phe at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG; (xix) Tyr at an amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and / or (xx) Contains a His at an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0138] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canid IgG. In some embodiments, the polypeptide comprises (i) an amino acid position corresponding to amino acid position 250 of wild-type canine IgG; (ii) an amino acid position corresponding to amino acid position 251 of wild-type canine IgG; (iii) an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (iv) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (v) an amino acid position corresponding to amino acid position 256 of wild-type canine IgG; (vi) an amino acid position corresponding to amino acid position 285 of wild-type canine IgG; (vii) an amino acid position corresponding to amino acid position 307 of wild-type canine IgG; (viii) an amino acid position corresponding to amino acid position 308 of wild-type canine IgG; (ix) an amino acid position corresponding to amino acid position 309 of wild-type canine IgG; (x) an amino acid position corresponding to amino acid position 311 of wild-type canine IgG; (xi) an amino acid position corresponding to amino acid position 315 of wild-type canine IgG; (xii) an amino acid position corresponding to amino acid position 378 of wild-type canine IgG; (xiii) an amino acid position corresponding to amino acid position 380 of wild-type canine IgG; (xiv) an amino acid position corresponding to amino acid position 428 of wild-type canine IgG; (xv) an amino acid position corresponding to amino acid position 430 of wild-type canine IgG; (xvi) an amino acid position corresponding to amino acid position 433 of wild-type canine IgG; (xvii) an amino acid position corresponding to amino acid position 434 of wild-type canine IgG; (xviii) an amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and (xix) at least one additional amino acid substitution at a position selected from the group consisting of the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0139] In some embodiments, the polypeptide is (i) Glu or Gln at an amino acid position corresponding to amino acid position 250 of wild-type canine IgG; (ii) Asp or Glu at an amino acid position corresponding to amino acid position 251 of wild-type canine IgG; (iii) Tyr or Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (iv) Thr or Ser at an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (v) Asp, Glu, or Phe at an amino acid position corresponding to amino acid position 256 of wild-type canine IgG; (vi) Asn or Asp at an amino acid position corresponding to amino acid position 285 of wild-type canine IgG; (vii) Arg, Gln, or Ala at an amino acid position corresponding to amino acid position 307 of wild-type canine IgG; (viii) Pro at an amino acid position corresponding to amino acid position 308 of wild-type canine IgG; (ix) Pro at an amino acid position corresponding to amino acid position 309 of wild-type canine IgG; (x) Val at an amino acid position corresponding to amino acid position 311 of wild-type canine IgG; (xi) Asp at an amino acid position corresponding to amino acid position 315 of wild-type canine IgG; (xii) Val at an amino acid position corresponding to amino acid position 378 of wild-type canine IgG; (xiii) Ala at an amino acid position corresponding to amino acid position 380 of wild-type canine IgG; (xiv) Leu at an amino acid position corresponding to amino acid position 428 of wild-type canine IgG; (xv) Ala or Lys at an amino acid position corresponding to amino acid position 430 of wild-type canine IgG; (xvi) Lys at an amino acid position corresponding to amino acid position 433 of wild-type canine IgG; (xvii) Trp, Tyr, Arg, His, Ser, Ala, or Phe at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG; (xviii) Tyr at an amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and / or (xix) Contains a His at an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0140] In some embodiments, the at least one additional amino acid substitution is (i) an amino acid position corresponding to amino acid position 250 of wild-type canine IgG; (ii) an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (iii) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (iv) an amino acid position corresponding to amino acid position 256 of wild-type canine IgG; (v) an amino acid position corresponding to amino acid position 285 of wild-type canine IgG; (vi) an amino acid position corresponding to amino acid position 307 of wild-type canine IgG; (vii) an amino acid position corresponding to amino acid position 309 of wild-type canine IgG; (viii) an amino acid position corresponding to amino acid position 311 of wild-type canine IgG; (ix) an amino acid position corresponding to amino acid position 315 of wild-type canine IgG; (x) an amino acid position corresponding to amino acid position 433 of wild-type canine IgG; (xi) an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and (xii) at a position selected from the group consisting of an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0141] In some embodiments, the polypeptide is (i) Glu or Gln at an amino acid position corresponding to amino acid position 250 of wild-type canine IgG; (ii) Tyr or Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (iii) Thr or Ser at an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (iv) Asp, Glu, or Phe at an amino acid position corresponding to amino acid position 256 of wild-type canine IgG; (v) Asn or Asp at an amino acid position corresponding to amino acid position 285 of wild-type canine IgG; (vi) Arg, Gln, or Ala at an amino acid position corresponding to amino acid position 307 of wild-type canine IgG; (vii) Pro at an amino acid position corresponding to amino acid position 309 of wild-type canine IgG; (viii) Val at an amino acid position corresponding to amino acid position 311 of wild-type canine IgG; (ix) Asp at an amino acid position corresponding to amino acid position 315 of wild-type canine IgG; (x) Lys at an amino acid position corresponding to amino acid position 433 of wild-type canine IgG; (xi) Trp, Tyr, Arg, His, Ser, Ala, or Phe at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and (xii) contains a His at an amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0142] In some embodiments, the at least one additional amino acid substitution is (i) an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (ii) an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (iii) an amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and (iv) at an amino acid position selected from the group consisting of: an amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0143] In some embodiments, the polypeptide is (i) Tyr or Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (ii) Thr or Ser at an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (iii) Asp, Glu, or Phe at an amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or (iv) contains Trp, Tyr, Arg, His, Ser, Ala, or Phe at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0144] In some embodiments, the polypeptide is (i) Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG; (ii) Thr at an amino acid position corresponding to amino acid position 254 of wild-type canine IgG; (iii) Glu at an amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or (iv) contains Trp, Tyr, Arg, or His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0145] Substitutions may be made on one or both chains of the CH2 domain, the CH3 domain, or the Fc domain. In some embodiments, the substitutions on both chains of the CH2 domain, the CH3 domain, or the Fc domain are identical. In some embodiments, the substitutions on both chains of the CH2 domain, the CH3 domain, or the Fc domain are not identical. In some embodiments, the Fc region contains one or more additional substitutions that result in increased or decreased effector function and / or improved product heterogeneity.

[0146] The present disclosure also provides a polypeptide comprising a canine IgG Fc region variant, or a canine FcRn-binding region thereof, comprising: (i) a position corresponding to amino acid position 286 of wild-type canine IgG; (ii) a position corresponding to amino acid position 426 of wild-type canine IgG; (iii) a position corresponding to amino acid position 434 of wild-type canine IgG, and (iv) amino acid substitutions at two or more (e.g., 2, 3, 4, or 5) positions selected from the group consisting of: a position corresponding to amino acid position 436 of wild-type canine IgG; Provided are polypeptides in which amino acid positions are based on EU numbering, and in which the polypeptides have increased binding affinity to canine FcRn when compared to the Fc domain of wild-type canine IgG.

[0147] 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, so long as the polypeptide has increased binding affinity to canine FcRn when compared to a polypeptide containing only one of the two or more amino acid substitutions.

[0148] In some embodiments, the polypeptides described herein comprise at least one additional amino acid substitution at a position other than those corresponding to wild-type canine IgG positions 286, 426, 434, and 436. For example, the polypeptides described herein can comprise about 1, 2, 3, 4, or 5, up to about 30, additional amino acid substitutions of canine IgG.

[0149] In some embodiments, the polypeptide has increased binding affinity to 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 are well known to those of skill in the art, and illustrative examples are provided elsewhere herein.

[0150] Differences in FcRn-binding activity may be suitably determined using an equivalent or similar assay for each variable (e.g., pH value, number of amino acid substitutions, location of amino acid substitutions, type of amino acid substitution, etc.). Comparable assays in this context refer to assays that operate in substantially the same or similar manner to minimize or otherwise avoid unnecessary variables that may substantially affect the results independent of how the assay is performed and the variable being evaluated. However, it will be understood that the conditions required to perform an assay to determine FcRn binding at, for example, pH 6.0 may differ from the conditions required to perform a similar assay at pH 7.4, taking into account, for example, the effect of pH on how the assay is performed. In some embodiments, the polypeptide has increased binding affinity to 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 an equivalent assay.

[0151] In some embodiments, the polypeptide binds to canine FcRn at a higher level at acidic pH than at neutral pH in an equivalent assay. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 5.5 than at pH 7.4 in an equivalent assay. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4 in an equivalent assay.

[0152] In some embodiments, 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 T286L, T286Y, and any of the foregoing conservative amino acid substitutions.

[0153] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG is selected from the group consisting of A426Y, A426H, and any of the foregoing conservative amino acid substitutions.

[0154] 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.

[0155] 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.

[0156] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG.

[0157] In some embodiments, the polypeptide is (i) positions corresponding to amino acid positions 426 and 286 of wild-type canine IgG; (ii) positions corresponding to amino acid positions 426 and 434 of wild-type canine IgG, and (iii) comprises amino acid substitutions at two or more positions selected from the group consisting of positions corresponding to amino acid positions 426 and 436 of wild-type canine IgG.

[0158] In some embodiments, the two or more amino acid substitutions are (i) A426Y in combination with one or more of T286L, T286Y, N434R, and Y436H; (ii) A426H in combination with one or more of T286L, T286Y, N434R, and Y436H; and (iii) selected from the group consisting of N434R in combination with one or more of T286L, T286Y, and Y436H.

[0159] In some embodiments, the polypeptide is (i) A426Y and T286L, (ii) A426Y and Y436H, (iii) A426H and T286L, and (iv) contains an amino acid substitution selected from the group consisting of A426H and T286Y.

[0160] In some embodiments, the polypeptide includes any of the foregoing substitutions, or any combination thereof, and an amino acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to an amino acid sequence selected from the group consisting of SEQ ID NOs:9-12 (e.g., SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12).

[0161] Other substitutions that can be combined with half-life-enhancing substitutions The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process requiring the coordination of a series of intricate tasks 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 production and purification methods. The present disclosure encompasses substitutions at one or more additional amino acid positions in Fc region variants that promote any one or more of the above goals.

[0162] In some embodiments, the Fc region variants comprise amino acid substitutions at one or more additional amino acid positions that result in increased or decreased effector function and / or improved product heterogeneity.

[0163] In some embodiments, substitutions are introduced to reduce 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: 238, 265, 297, 298, 299, 327, and 329 (numbered according to EU numbering). The substitution(s) may be at any of the other 19 amino acids. In some embodiments, the substitutions are conservative. In specific, non-limiting embodiments, 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 embodiments, the variant Fc region is derived from a canine IgGB or IgGC antibody. In some embodiments, the variant Fc region is derived from a canine IgGB antibody.

[0164] In some embodiments, substitutions are introduced into a wild-type canine IgG Fc region to improve binding to Protein A to facilitate purification by Protein A chromatography. Such substitutions may be at one or both (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions of canine IgG: 252 and 254 (numbered according to EU numbering). The substitution(s) may be with any of the other 19 amino acids. In some embodiments, the substitutions are conservative. In a specific, non-limiting embodiment, the substituted amino acid at position 252 is Met and the substituted amino acid at position 254 is Ser.

[0165] In some embodiments, substitutions may be made to alter binding affinity to FcRn (e.g., to increase or decrease binding affinity to FcRn) compared to the parent or wild-type polypeptide. In some variations, the modifications may be one, two, three, or four modifications selected from the group consisting of 308F, 428L, 434M, and 434S, where numbering is according to EU numbering. In some embodiments, the Fc variant comprises 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, where numbering is according to EU numbering. In some embodiments, the Fc variant comprises one or more modifications selected from the group consisting of 428L / 434S, 308F / 428L / 434S, where numbering is according to EU numbering. In some embodiments, the Fc variant comprises one or more modifications selected from the group consisting of 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S, and 308F / 319L / 434S, where numbering is according to EU numbering. A detailed description of these modifications is described, for example, in US8883973B2, which is incorporated herein by reference in its entirety.

[0166] In some embodiments, the polypeptide comprises a canine antibody hinge region. In some embodiments, a modification is made to the canine antibody hinge region to increase half-life. In some embodiments, the modification is 228P according to EU numbering.

[0167] In some embodiments, binding to FcRn is pH-dependent. H310 and H435 (EU numbering) may be essential for pH-dependent binding. Thus, in some embodiments, the amino acid at position 310 (EU numbering) is histidine. In some embodiments, the amino acid at position 435 (EU numbering) is histidine. In some embodiments, the amino acids at both positions are histidine.

[0168] In some embodiments, the Fc region has a MALA mutation (M234A and L235A mutations according to EU numbering) or a MALA-PG mutation (M234A, L235A, P329G mutations according to EU numbering). In some embodiments, the Fc region has a P234A, M234A, or S234A mutation. 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.

[0169] Polypeptides containing canine IgG Fc variants The present disclosure encompasses any polypeptide that would benefit from having an increased half-life in dogs, wherein the polypeptide is designed to include an Fc region variant as described above (e.g., a CH2 region, a CH3 region, or a CH2+CH3 region) to increase half-life.

[0170] In some embodiments, the polypeptide of the present disclosure comprises 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 embodiments, the hinge region is linked to the C-terminus of a cytokine, growth factor, enzyme, or peptide, and the hinge region is linked to the N-terminus of the Fc region variant. Exemplary hinge region sequences are provided below. IgGA: FNECRCTDTPPCPVPEP (SEQ ID NO: 17), IgGB:PKRENGRVPRPPDCPKCPAPEM (SEQ ID NO: 18), IgGC:AKECECKCNCNNCPCPGCGL (SEQ ID NO: 19), IgGD:PKESTCKCISPCPVPES (SEQ ID NO: 20), and IgGDmut:PKESTCKCIPPCPVPES (SEQ ID NO: 21).

[0171] When used in recombinant proteins of the present disclosure, the hinge region can contain 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 17-21. In some embodiments, the hinge region used in a 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.

[0172] In some embodiments, a linker sequence may be used in place 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 is composed of 1 to 20 amino acids linked by peptide bonds, where the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those of skill in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is composed predominantly of 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 Gly Gly Gly (SEQ ID NO: 25); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 26); Ser Gly Gly Gly Gly Gly (SEQ ID NO: 27); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 28); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 29); (Gly Gly Gly Gly Ser) n (SEQ ID NO: 24) (n is an integer of 1 or more (e.g., 1, 2, 3, 4, 5)); and (Ser Gly Gly Gly Gly) n (SEQ ID NO: 25) (n is an integer of 1 or greater (e.g., 1, 2, 3, 4, 5)).

[0173] Non-peptide linkers can also be used to link a polypeptide or polypeptides of interest to the Fc region variants disclosed herein, for example, -NH(CH2) where n=2-20. nAlkyl linkers such as C(O)- can be used, which can be further substituted with any non-sterically hindering group such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0174] A polypeptide or polypeptides of the present disclosure may comprise a binding domain. The binding domain is capable of specifically binding to a protein, subunit, domain, motif, and / or epitope of a selected target described herein. In some embodiments, the binding domain comprises an antibody, antibody fragment, or the ligand-binding portion of a receptor. In some embodiments, the antibody or antibody fragment comprises the six complementarity-determining regions (CDRs) of an immunoglobulin molecule. In other embodiments, the antibody fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), an Fv, a Fab', a Fab'-SH, a F(ab')2, a nanobody, and a diabody. In other embodiments, the ligand-binding portion of the receptor comprises the ligand-binding domain of a canine receptor protein or the extracellular domain of a canine receptor protein. In some embodiments, a polypeptide or polypeptides (e.g., a fusion polypeptide) may comprise a protein, wherein the protein is a therapeutic protein described herein. In some embodiments, the target (e.g., target of the binding domain) or therapeutic protein (e.g., 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 RIBALK-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, APRIL, AR, IgE, angiotensin type 1 (AT1) receptor , angiotensin type 2 (AT2) receptor, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), 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 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, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19 ...10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23 CL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, C D11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2 , CXCL3, CXCL4, CXCL5, CXCL6, 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 promoting factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA- A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, 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-alpha1, GFR-alpha2, GFR-alpha3, 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, 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-31 R, IL-33R), interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin A LFA6, integrin beta 1, integrin beta 2, integrin gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP(TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase Proteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, 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, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NAV 1.7, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, oncostatin M receptor (OSMR), 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 receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta R1 (ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic CK-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, 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(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF R1CD120a, p55-60), TNFRSF1B(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNFRIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB) CD137, ILA), TNFRSF21(DR6), TNFRSF22(DCTRAIL R2 TNFRH2), TNFRST23(DCTRAIL R1TNFRH1), TNFRSF25(DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β 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, transferrin receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigens expressing Lewis Y-related glycans, TWEAK, TXB2, Ung, UPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand 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 hormone and growth factor receptors.

[0175] In some embodiments, the antibody or antibody fragment comprises one or more complementarity determining regions (CDRs) having an amino acid sequence selected from Table 5 below. For example, the antibody or antibody fragment may comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 5. For example, the antibody or antibody fragment may comprise all six CDRs for an antibody listed in Table 5 as binding to a particular target. In some embodiments, the antibody or antibody fragment is selected from the group consisting of antibodies and antibodies disclosed in U.S. Patent Application Publication Nos. US2020 / 0062840, US2022 / 0251209, US2021 / 0040223, US2022 / 0204615, US2022 / 0251230, US2021 / 0163618, US2021 / 0253722, US2022 / 0119513, US2022 / 0106391, or US2021 / 0163618. The antibody or antibody fragment may be any antibody or antibody fragment disclosed in U.S. Pat. Nos. 20020022 / 0177594, U.S. Pat. Nos. US11,091,556, US11,447,561, US10,040,849, or US9,951,128, and International Patent Application Publication Nos. WO2020 / 056393, WO2022 / 079138, WO2021 / 123092, WO2022 / 029447, or WO2023 / 097275. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] Table 5-7 Table 5-8 Table 5-9

[0176] In some embodiments, the binding domain specifically binds to one or more therapeutic targets or antigens in a canine, such as, but not limited to, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (myostatin), GLP1, GLP2, GnRH, growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, 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, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1 bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OSMR, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta R1(ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, and VEGFR-3 (flt-4).

[0177] In some embodiments, the polypeptide or polypeptides can comprise a protein, and the protein is a therapeutic protein, such as EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonist, or thrombopoietin-binding peptide. In some embodiments, the therapeutic protein is ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (myostatin), GLP1, GLP2, GnRH, growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, 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, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1 bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OSMR, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta R1(ALK-5), TGF-beta R11, TGF-betaRIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, or VEGFR-3 (flt-4).

[0178] For example, a polypeptide or polypeptides of the present disclosure may comprise a binding domain comprising six CDRs of an immunoglobulin molecule. In some embodiments, the binding domain specifically binds to NGF. In some embodiments, the binding domain is an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 304, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 305, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 306, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 307, a CDR-L2 comprising the amino acid sequence ATS, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 309.

[0179] In some embodiments, the polypeptide comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to any one of SEQ ID NOs: 274-298, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0180] In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 274, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0181] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 275, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0182] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG, and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 276, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0183] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 277, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0184] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 278, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0185] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 279, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0186] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 280, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0187] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 281, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0188] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 282, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0189] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 283, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0190] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 284, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0191] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 285, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0192] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 286, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0193] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 287, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0194] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 288, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0195] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of wild-type canine IgG and a His at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 289, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0196] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 290, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0197] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Tyr at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 291, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0198] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Phe at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 292, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0199] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 293, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0200] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Trp at an amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 294, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0201] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Leu at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 295, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0202] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Trp at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 296, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0203] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Tyr at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 297, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0204] In some embodiments, the polypeptide comprises a His at an amino acid position corresponding to amino acid position 434 of wild-type canine IgG and a Phe at an amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 298, and the light chain comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, and 100%) sequence identity to SEQ ID NO: 273.

[0205] In some embodiments, the therapeutic protein is any protein described herein. In some embodiments, the polypeptide or polypeptides further comprise a canine IgG CH2 domain, an IgG CH3 domain, or an IgG Fc region described herein. The modified canine IgG CH2 domain, IgG CH3 domain, or IgG Fc region can improve the half-life of the therapeutic protein in vivo.

[0206] Pharmaceutical Compositions In one aspect, the invention features a pharmaceutical composition including (i) any of the polypeptides disclosed herein and (ii) a pharmaceutically acceptable excipient.

[0207] To prepare pharmaceutical or sterile compositions of a polypeptide or polypeptides described herein, the polypeptide or polypeptides can 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)).

[0208] Formulations of therapeutic and diagnostic agents can be prepared, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension, by mixing with an acceptable carrier, excipient, or stabilizer (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: (See Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, a polypeptide or polypeptides of the present invention are diluted to an appropriate concentration with sodium acetate solution at pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0209] Toxicity and therapeutic efficacy of polypeptide compositions administered alone or in combination with other agents can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (lethal dose for 50% of the population) and ED 50The dose ratio between toxic and therapeutic effects is the therapeutic index (LD ). 50 / ED 50 In certain embodiments, a polypeptide or polypeptides that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in canines. The dosage of such compounds is preferably within the ED range with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form and route of administration employed.

[0210] Any suitable administration mode can be used.Exemplary suitable administration routes include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial.In some embodiments, one or more polypeptides can be administered by an invasive route such as injection.In further embodiments, one or more polypeptides are administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation or aerosol delivery.

[0211] The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implant and module forms 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 an implantable variable flow rate infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0212] Alternatively, a polypeptide or polypeptides may be administered locally rather than systemically, for example, by directly injecting an antibody, often in a depot or sustained-release formulation, into an arthritic joint or pathogen-induced lesion characterized by immunopathology. Furthermore, a polypeptide or polypeptides may be administered in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody that targets an arthritic joint or pathogen-induced lesion characterized by immunopathology. The liposome is targeted to and selectively taken up by the affected tissue.

[0213] The administration regimen depends on several factors, including, but not limited to, the age, weight, and physical condition of the canine being treated, the serum or tissue conversion rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic polypeptide(s), and the accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers enough therapeutic polypeptide(s) to result in improvement of the targeted disease state while minimizing undesirable side effects. Thus, the amount of biologic delivered will depend in part on the particular therapeutic polypeptide(s) and the severity of the condition being treated. Guidance on 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)).

[0214] The appropriate dosage of a polypeptide or a plurality of polypeptides can be determined by those skilled in the art, for example, by using the parameters or factors that are known or estimated to affect treatment in the art.Generally, administration is started from a slightly lower amount than the optimal dose, and then increased by small increments until desired or optimal effect is achieved against any negative side effects.Important diagnostic indicators include, for example, the symptoms of inflammation or the level of inflammatory cytokines produced.

[0215] Nucleic acids, vectors, host cells, and methods of production The present disclosure also encompasses a nucleic acid or nucleic acids encoding a polypeptide or polypeptides described herein, a vector or vectors comprising the nucleic acid or nucleic acids, and host cells comprising the nucleic acid or nucleic acids or vector or vectors.

[0216] In one aspect, the invention features a nucleic acid or nucleic acids that encode any of the polypeptides disclosed herein.

[0217] In another aspect, the invention features an expression vector or vectors that include a nucleic acid or nucleic acids encoding any of the polypeptides disclosed herein.

[0218] In another aspect, the invention features a host cell containing a nucleic acid or nucleic acids encoding any of the polypeptides disclosed herein, or an expression vector or vectors that include a nucleic acid or nucleic acids encoding any of the polypeptides disclosed herein.

[0219] In another aspect, the invention provides a method of making a polypeptide, comprising: (i) providing a nucleic acid or nucleic acids encoding any of the polypeptides disclosed herein; (ii) expressing the nucleic acid or nucleic acids in a host cell culture, thereby producing a polypeptide; and optionally (iii) collecting the polypeptide produced in (ii) from the host cell culture.

[0220] The polypeptide or polypeptides described herein can be produced in bacterial cells or eukaryotic cells. Some polypeptides, such as Fabs, can be produced in bacterial cells, such as 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., scFvs) can be expressed in yeast cells, such as Pichia (e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the desired antibody, a polynucleotide or polynucleotides encoding the polypeptide or polypeptides are constructed, introduced into an expression vector or vectors, and then expressed in a suitable host cell. To improve expression, the nucleotide sequence of the gene can be recoded without changing the amino acid sequence (or with minimal changes, such as removing the C-terminal residue of the heavy or light chain). Regions that may be targeted for recoding include those associated with translation initiation, codon usage, and potential unintended mRNA splicing. Polynucleotides encoding the Fc region variants described herein will be readily apparent to those skilled in the art.

[0221] Standard molecular biology techniques can be used to prepare the recombinant expression vector(s), transfect the host cells, select for transformants, culture the host cells, and recover the polypeptide (e.g., an antibody).

[0222] If the polypeptide or polypeptides are expressed in bacterial cells (eg, E. coli), the expression vector may have properties that allow for amplification of the vector in the bacterial cell. Additionally, when E. coli, such as JM109, DH5α, HB101, or XL1-Blue, is used as a host, the vector contains a promoter such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter, which can enable efficient expression in E. coli. Examples of such vectors include M13 vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21, which expresses T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., 2004) is used. al., J. Bacteriol., 169:4379 (1987)) can be used as a signal sequence for antibody secretion. For bacterial expression, the calcium chloride method or electroporation can be used to introduce the expression vector into bacterial cells.

[0223] When one or more polypeptides are expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector may contain a promoter for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)) (e.g., the early simian virus 40 promoter), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter (e.g., the 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 regulating replication of the vector in host cells (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Examples of vectors containing selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0224] In some embodiments, the polypeptide or polypeptides are produced in mammalian cells. Exemplary mammalian host cells for expressing the polypeptide or 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, e.g., used with the DHFR selection marker, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601 621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphoid cell 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.

[0225] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy and light chains of an antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to enhancer / promoter regulatory elements (e.g., those derived from SV40, CMV, adenovirus, etc., e.g., the CMV enhancer / AdMLP promoter regulatory element or the SV40 enhancer / AdMLP promoter regulatory element) to drive high gene transcription levels. The recombinant expression vector also carries a DHFR gene, which allows CHO cells transfected with the vector to be selected using methotrexate selection / amplification. The selected transformed host cells are cultured to express the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0226] Treatment method A polypeptide or polypeptides disclosed herein can be used to treat or prevent any disease or disorder in a dog in need thereof. The present invention is particularly useful for treating chronic conditions that require repeated administration. The increased half-life of the protein therapeutic may allow for less frequent administration and / or reduced dosage levels.

[0227] In one aspect, the invention features a method of treating or preventing a canine disease or disorder in a dog in need thereof, comprising administering an effective amount of a composition comprising any of the polypeptides disclosed herein, or a pharmaceutical composition comprising (i) any of the polypeptides disclosed herein and (ii) a pharmaceutically acceptable excipient.

[0228] In another aspect, the invention features any of the polypeptides disclosed herein, or a pharmaceutical composition including (i) any of the polypeptides disclosed herein and (ii) a pharmaceutically acceptable excipient, for use in a dog in need of treatment or prevention of a canine disease or disorder.

[0229] Any suitable canine disease or disorder may be treated, hi some embodiments, the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, infertility-related disorder, infectious disease, or cancer.

[0230] In other embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0231] In some embodiments, the disease, disorder, condition, or symptom being treated or prevented is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, skeletal / musculoskeletal disease, cardiovascular disease, neurological disease, renal disease, metabolic disease, immunological disease, genetic / hereditary disease, infertility-related disease, infectious disease, or cancer. In certain embodiments, the disease or disorder being treated or prevented is atopic dermatitis, allergic dermatitis, food allergy, osteoarthritic pain, perioperative pain, dental pain, cancer pain, arthritis, anemia, obesity, or diabetes.

[0232] Antibodies can be used not only to treat or prevent disease, but also to regulate normal biological functions, for example, to control fertility or behavior.

[0233] In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered parenterally, by subcutaneous administration, intravenous infusion, or intramuscular injection.In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered as a bolus injection or by continuous infusion over a period of time.In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation.

[0234] In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered in an amount ranging from 0.01 mg / kg to 50 mg / kg of body weight per dose. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered at a dose ranging from 0.01 to 55 mg / kg, 0.01 to 50 mg / kg, 0.01 to 45 mg / kg, 0.01 to 40 mg / kg, 0.01 to 35 mg / kg, 0.01 to 30 mg / kg, 0.01 to 25 mg / kg, 0.01 to 20 mg / kg, 0.01 to 15 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, or 0.01 to 1 mg / kg daily, weekly, monthly, every two months, every three months, every four months, every five months, or every six months. An exemplary antibody dosage ranges from 0.01 mg / kg to 15 mg / kg. Thus, one or more doses of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 10 mg / kg, or 15 mg / kg (or any combination thereof) may be administered to an animal. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered at 2 mg / kg body weight per dose.

[0235] In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered within 1, 2, 3, 4, 5, or 6 months, or within 1, 2, or 3 weeks of each other. In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered weekly. In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered every two weeks. In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered every three weeks. In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered monthly. In some embodiments, a polypeptide or polypeptides disclosed herein or pharmaceutical compositions comprising a polypeptide or polypeptides disclosed herein are administered every two months. In some embodiments, a polypeptide or polypeptides disclosed herein or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein is administered every three months. In some embodiments, a polypeptide or polypeptides disclosed herein or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein is administered every four months. In some embodiments, a polypeptide or polypeptides disclosed herein or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein is administered every five months.In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered every six months. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered to a dog once or over a series of treatments. In some embodiments, a dose is administered once a week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally with an interval of one week or more between treatments.

[0236] In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered simultaneously, sequentially, or in combination with one or more additional therapeutic agents. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered in combination with one or more additional therapeutic agents. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered simultaneously with one or more additional therapeutic agents. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered sequentially with one or more additional therapeutic agents. In some embodiments, a polypeptide or polypeptides disclosed herein, or a pharmaceutical composition comprising a polypeptide or polypeptides disclosed herein, is administered in combination with one or more additional therapeutic agents. Any suitable additional therapeutic agent may be used.

[0237] diagnosis The polypeptide or polypeptides disclosed herein can also be used for various diagnostic purposes, for example, to determine whether a dog has any particular disease or disorder. In some embodiments, the polypeptide or polypeptides can include a binding domain. The binding domain can specifically bind to a protein, subunit, domain, motif, and / or epitope (e.g., a marker for cancer cells) described herein. In some embodiments, the polypeptide or polypeptides further include a labeling group. Generally, labeling groups are classified into various classes depending on the assay in which they are detected: a) isotopic labels, which can be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags, etc.). In some embodiments, the labeling group is attached to the antibody via spacer arms of various lengths to reduce potential steric hindrance. A variety of methods for labeling proteins are known in the art and can be used in practicing the present invention.

[0238] 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).

[0239] Specific labels also include optical dyes, including, but not limited to, chromophores, fluorophores, and fluorophores, the latter being specific in many embodiments. Fluorophores can be either "small molecule" fluorophores or proteinaceous fluorophores.

[0240] The fluorescent label can be any molecule that can be detected through its inherent fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Sciences, Pittsburgh, Pa.) Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland, which is incorporated herein by reference in its entirety.

[0241] Suitable proteinaceous fluorescent labels also include, but are not limited to, GFP from Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1994, Science 263:802-805), and erythrocyte-derived fluorescent protein (YFP, Clontech Laboratories, Inc.). al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA. 85:2603-2607), and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019, U.S. Patent 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 references cited above in this paragraph are expressly incorporated herein by reference in their entirety.

[0242] Assay Fc γ RI and FcγRIII binding: Binding to FcγRI and FcγRIII is indicative of the ability of an antibody to mediate ADCC. To assess this property of an antibody, assays that measure antibody binding to FcγRI and FcγRIII can be performed using methods known in the art.

[0243] C1q binding: Binding to the first component of complement, C1q, is an indicator of an antibody's ability to mediate CDC. To assess this property of an antibody, an assay measuring antibody binding to C1q can be performed using methods known in the art.

[0244] Half-life: Methods for measuring the half-life of antibodies are well known in the art. See, for example, Booth et al., MAbs, 10(7):1098-1110 (2018). As an example, the half-life of an antibody (e.g., a feline antibody) can be measured by injecting the antibody into an animal model (e.g., a canine model) and measuring the level of the antibody in the serum over a period of time. Exemplary animal models include non-human primate models and transgenic mouse models. The transgenic mouse model lacks the mouse FcRn alpha chain and can express a canine FcRn alpha transgene (e.g., under the control of a constitutive promoter). The canine FcRn alpha chain can pair in vivo with the mouse β2-microglobulin protein to form a functional chimeric FcRn heterodimer. As an example, the half-life of a canine antibody can be measured by injecting the antibody into a canine model and measuring the level of the antibody in the serum over a period of time. [Example]

[0245] Example 1: Surface Plasmon Resonance (SPR) Analysis Using Biacore™ 8K For SPR analysis using a Biacore™ 8K, bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. The sensor chip surface in flow cells 1 and 2 was activated with freshly mixed 50 mmol / L N-hydroxysuccinimide and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for 420 seconds (10 μL / min). Subsequently, BSA diluted in 10 mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, while flow cell 1 was set as a blank. After the amine coupling reaction, remaining active coupling sites on the chip surface were blocked with a 420-second injection of 1 mM ethanolamine hydrochloride. The running buffer for binding experiments was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 5.5) and was run at 25°C. Supernatants from the variants were injected onto the chip surface and captured via the SASA (single-domain antibody against serum albumin) tag (see, e.g., US2013 / 0129727A1) onto immobilized BSA for 60 seconds. 400 nM canine FcRn (UniProtKB-E2R0L6 (FcRn) and UniProtKB-E2RN10 (canine beta-2-microglobulin)) was injected for 120 seconds, and dissociation was completed over 120 seconds using running buffer. The flow rate for the BSA stationary phase was 10 μl / min, and the flow rates for the association and dissociation phases were 30 μl / min. All data were processed using Biacore™ 8K Evaluation software version 1.1.

[0246] Example 2: Binding kinetics of L252Y, L252M, and N434H variants and wild-type Fc Several canine IgGB variants were evaluated for their binding kinetics with canine FcRn. In this study, the binding of variants (L252Y, L252M, and N434H) and wild-type canine Fc to canine FcRn was evaluated at pH 5.5 and pH 7.4. The Biacore™ method for pH 5.5 conditions was the same as that described in Example 1, except that four concentrations of FcRn (100 nM, 200 nM, 400 nM, and 800 nM) were tested to obtain more accurate binding kinetics. For pH 7.4 Biacore™ conditions, 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. Like the wild-type, all variants did not bind to canine FcRn at pH 7.4, and the variants tested were expected to show increased affinity for canine FcRn at acidic pH (e.g., pH 5.5 or pH 6.0) compared to the wild-type Fc.

[0247] Example 3: Binding kinetics of A426Y, A426H, A426F, A426L, A426W, T286Y, T286F, T286L, T286W, and wild-type Fc. Several canine IgGB variants were evaluated for binding kinetics with canine FcRn. In this study, the binding of variants (A426Y, A426H, A426F, A426L, A426W, T286Y, T286F, T286L, and T286W) and wild-type canine IgGB Fc to canine FcRn was evaluated at either pH 5.5 or pH 6.0 and at pH 7.4. The Biacore™ method for the pH 5.5 and pH 6.0 conditions was the same as that described in Example 1, except that four concentrations of FcRn (100 nM, 200 nM, 400 nM, and 800 nM) were tested to obtain more accurate binding kinetics. For Biacore™ conditions at pH 7.4, the running buffer used was 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4, and the concentration of canine FcRn tested was 200 nM. Like the wild-type, all variants did not bind to canine FcRn at pH 7.4 under the conditions described, and it was expected that the tested variants would show increased affinity for canine FcRn at acidic pH (e.g., pH 5.5 or pH 6.0) compared to the wild-type Fc.

[0248] Example 4: Screening for canine IgGB Fc variants with increased FcRn binding compared to wild-type canine IgGB Fc Single amino acid substitutions (e.g., L252Y, L252M, T286Y, T286F, T286L, T286W, A426L, A426H, N434H, A426W, A426Y, and A426F), or combinations of amino acid substitutions (e.g., L252Y+T286Y, L252Y+T286F, L252Y+T286L, L252Y+T286W, L252Y+A426L, L252Y+A426H, L252M+T Canine Fc variants bearing the following variable domains were synthesized in a canine IgGB (SEQ ID NO: 10) format using the variable domains described in Gearing DP et al. (2013, BMC Veterinary Research, 9:226). Canine IgGB DNA was synthesized, subcloned into the pcDNA3.4 vector (ThermoFisher), and transfected into ExpiCHO-S™ cells using the ExpiCHO transfection method (ThermoFisher). 14 days after transfection, conditioned medium was purified using Monofinity A resin (GenScript). Binding of the antibodies to canine FcRn was measured at both pH 6.0 and pH 7.4 using a Biacore™ 8K.

[0249] For pH 6.0 binding conditions, the sensor chip surface of flow cells 1 and 2 was activated with freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 100 seconds (10 μL / min). The antibody was diluted with 10 mmol / L NaAC (pH 4.5) and injected into flow cell 2 to achieve conjugation of approximately 100 response units, with flow cell 1 set as the blank. After the amine coupling reaction, remaining active coupling sites on the chip surface were blocked with a 100-second injection of 1 mol / L ethanolamine hydrochloride. The pH 6.0 running buffer was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 6.0), which was run at 25°C. Canine FcRn (UniProtKB-E2R0L6 (FcRn) and UniProtKB-E2RN10 (canine beta-2-microglobulin)) was injected for 120 seconds, and dissociation was completed with running buffer for 120 seconds. The flow rate was 30 μl / min. The concentrations of canine FcRn flowed over the sensor chip were 200 nM, 400 nM, 800 nM, 1600 nM, and 3200 nM for wild-type IgG and IgG variants. The concentrations of canine FcRn flowed for the remaining variants were 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM. All data were processed using Biacore™ 8K Evaluation Software version 1.1.

[0250] Flow cell 1 and buffer-only injections at each cycle were used as the reference for response unit subtraction. Previously, amine coupling of IgG to a Biacore™ CM5 biosensor chip was shown to reduce FcRn affinity by 2-3 fold compared to the affinity determined by solution-based methods or direct coupling to a Biacore™ C1 chip (Abdiche et al., 2015. mAbs, 7:331). Therefore, the true affinity of these IgGs for FcRn at pH 6.0 is likely at least 2-fold higher. However, this method is useful for comparing the relative FcRn binding affinities of different IgG Fc variants. The tested variants were expected to show increased affinity for canine FcRn at acidic pH (e.g., pH 5.5 or 6.0) compared to the wild-type Fc.

[0251] For pH 7.4 binding conditions, the sensor chip surfaces of flow cells 1 and 2 were activated with freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 420 seconds (10 μL / min). Canine FcRn diluted with 10 mmol / L NaAC (pH 4.5) was then injected into flow cell 2 to achieve conjugation of approximately 2000 response units, with flow cell 1 set as the blank. After the amine coupling reaction, the remaining active coupling sites on the chip surface were blocked with a 420-second injection of 1 mol / L ethanolamine hydrochloride. The running buffer at pH 7.4 was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) and was run at 25°C. 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. Similar to the wild type, all variants were expected to have low or no binding to canine FcRn at pH 7.4.

[0252] The affinity of the interaction between canine IgG Fc and canine FcRn at pH 7.4 is very weak and difficult to measure by SPR using most methods. To compare the pH 7.4 affinity of various canine Fc variants to canine FcRn, a sensor chip is coated with a high concentration of canine FcRn, and the variant IgG Fc is flowed over the chip to measure the interaction. In this format, due to avidity effects, the measured binding affinity is not an accurate measure of individual canine IgG Fc variant-canine FcRn interactions, but it can be used to compare the relative binding of variant IgGs at pH 7.4. These should not be used for direct comparison with binding affinities at pH 6.0.

[0253] Example 5: In vivo screening of canine IgGB Fc variants with increased FcRn binding compared to wild-type canine IgGB Fc. Pharmacokinetic (PK) studies were conducted using male and female beagles. Canine IgGB Fc variants bearing single or combinations of amino acid substitutions were prepared by incorporating amino acid substitution(s) into canine IgGB (SEQ ID NO: 10) using the anti-NGF variable domain described in Gearing DP et al. (2013, BMC Veterinary Research, 9:226; the contents of which are incorporated herein by reference in their entirety). Animals were randomized into eight groups, with males and females in each group. The average age of the beagles was >6 months and their body weight was 8-10 kg. Each animal received a single intravenous dose of 2 mg / kg of antibody. Approximately 1.5 ml of whole blood was collected at time 0 (pre-dose), 4 hours after injection, and 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42 days after injection. Serum was separated from the whole blood and assayed for the presence of antibody variants by ELISA specific for anti-NGF antibodies.

[0254] Noncompartmental pharmacokinetic analysis (NCA) of individual serum antibody measurements was performed using a well-documented 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.; 2010 Sep; 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 offers many options for determining PK parameters. NCA is recognized as the method of choice due to its ease of use, model independence, and improved inter-analyst consistency. For specific analysis, the terminal half-life (T1 / 2) was determined using PKSolver's NCA IV Bolus. Following an initial series of dose-finding experiments, measurements well above the LLOQ could be measured up to 42 days after intravenous administration. Therefore, terminal half-lives are estimated based on at least the last 4 weeks of antibody measurement to allow for robust estimation of the slope. No data points were discarded or excluded from any experiment. Combinations of amino acid substitutions in the IgG Fc region are expected to significantly improve the terminal half-life of anti-NGF IgGB antibodies in canines in vivo compared to anti-NGF IgGB antibodies with (i) wild-type canine IgGB Fc regions or (ii) canine IgGB Fc variants with only a single amino acid substitution.

[0255] Example 6: Binding kinetics of canine IgGB variants to canine FcRn using C1 biosensor Several canine IgGB variants (L252Y, L252M, T286Y, T286F, T286L, T286W, A426L, A426H, N434H, A426W, A426Y, A426F, L252Y+T286Y, L252Y+T286F, L252Y+T286L, L252Y+T286W, L252Y+A426L, L252Y+A426H, L252M+T286Y, L252M+T286F, L252M+T286L, L252M+T286W, L252M+A426 The following fragments (L, L252M+A426H, N434H+T286Y, N434H+T286F, N434H+T286L, N434H+T286W, N434H+A426L, N434H+A426W, N434H+A426Y, N434H+A426F, and wild-type) were evaluated for binding kinetics to canine FcRn (UniProtKB-E2R0L6 (canine large subunit FcRn) and UniProtKB-E2RN10 (canine beta-2 microglobulin)) at pH 5.9. EU numbering was used to identify positions. In this study, canine Fc variants carrying single or combinations of amino acid substitutions were synthesized in a canine IgGB (GENBANK accession number AAL35302.1) format using the variable domains described in Gearing DP et al. (2013, BMC Veterinary Research, 9:226). The 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.

[0256] For canine FcRn binding experiments, all assays were completed on a Biacore™ 8K+ system at 25°C. In Example 7, the affinity of IgG variants for canine FcRn was measured by amine coupling of IgG to a Biacore™ CM5 biosensor chip, which has been demonstrated by Abdiche et al., 2015 (mAbs, 7:331) to underestimate the affinity of Fc variants for FcRn compared to using an S C1 biosensor chip. In this series of experiments, all antibodies were immobilized to an S C1 sensor chip using standard amine coupling reagents to obtain a more accurate measurement of FcRn affinity. The surface was activated by injecting a mixture of 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mmol / L N-hydroxysuccinimide (NHS) for 420 seconds. Next, antibodies were injected at concentrations of 0.5–2 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 phosphate-buffered saline (PBS)-P+ (Cytiva, catalog number 28995084), adjusted to pH 5.9.

[0257] To assess the binding affinity of canine IgGB variants to canine FcRn at pH 5.9, a concentration range of 1.56-2000 nM canine FcRn was selected and injected in single cycle mode.

[0258] Four concentrations per antibody were injected at 5 μl / min for 90 seconds, followed by a 180-second dissociation period. Each concentration series was injected three times in this format, with at least three buffer-only cycles for proper reference subtraction. The surface was regenerated with two 30-second injections of 1x PBS-P+, pH 7.4, followed by a 60-second wait command. Three start-up cycles were included to allow the surface to stabilize before analysis.

[0259] The data were evaluated using Insight Evaluation software by fitting a 1:1 kinetic interaction model or by fitting a steady-state affinity. Quality metrics including U and T values ​​were used to select acceptable parameters. U values ​​less than 15 were considered acceptable for kinetic rate constants, and T values ​​greater than 100 were considered acceptable for kinetic rate constants. If these values ​​were outside the range, the steady-state affinity parameters were considered acceptable.

[0260] The variants tested were expected to show increased affinity for canine FcRn compared to wild-type Fc under the conditions tested.

[0261] Example 7: Pharmacokinetic study of canine IgGB variants with increased FcRn binding and wild-type canine IgGB Three pharmacokinetic (PK) studies were completed using male and female beagles. Canine Fc variants carrying single or combinations of amino acid substitutions were synthesized in a canine IgGB (SEQ ID NO: 10) format using the anti-NGF variable domain described by DP et al. (2013, BMC Veterinary Research, 9:226). For each study, animals were randomized so that each group contained an equal number of males and females.

[0262] The dogs were an average age of >6 months and weighed 8-10 kg. Each animal received a single intravenous dose of antibody at 1 mg / kg (Study 1) or 2 mg / kg (Studies 2 and 3). Approximately 1.5 ml of whole blood was collected at time 0 (pre-dose), 4 hours post-injection, and 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42 days. Serum was extracted from the blood and assayed for NGF antibodies and antibody variants by specific ELISA.

[0263] Serum concentrations were described using a two-compartment pharmacokinetic (PK) model with linear clearance using nonlinear mixed-effects modeling. Population PK parameters were predicted using the stochastic approximation of expectation maximization (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: Lixoft SAS, 2019). Individual parameters were modeled as random variables with log-normal distribution. Population parameters were estimated from pooled data including all variants and studies. Study is a classification covariate for clearance. mAb variants were differentiated using classification covariates for clearance, central, and peripheral distribution volumes. Classification study and variant covariates are described as follows:

number

[0264] Data from all three studies are used to generate putative PK parameters for each of the variants. Antibodies containing the canine IgGB Fc variants are predicted to have increased terminal half-lives compared to those containing wild-type canine IgGB Fc.

[0265] Example 8: Binding kinetics of canine IgG Fc variants to canine FcRn A set of canine Fc variants was expressed and purified in IgG format. The IgG contained a light chain comprising the amino acid sequence of SEQ ID NO: 273 and a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 274-298. The variable domains of the heavy and light chains of the IgG are described in International Patent Application Publication No. WO 2023 / 97275, which is incorporated herein by reference in its entirety. The canine IgG B constant domain contained MALA mutations (M234A and L244A according to EU numbering) that reduce potential effector activity (ADCC and CDC). The heavy and light chains were synthesized and subcloned into the PCDNA™ 3.4 vector (Thermo Fisher Scientific) with signal sequences at the N-terminus of the chains. The heavy and light chain constructs were co-transfected into EXPICHO™ cells and incubated for 7 days, after which the conditioned medium was purified using MABSELECT™ SURE™ Protein A resin. The purified antibody was buffer-exchanged into PBS, pH 7.4. The FcRn complex consists of a large subunit (p51) and a small subunit (β2-microglobulin, p14), and canine FcRn protein was produced by coexpressing these two proteins in CHO cells. The soluble portion of the large subunit p51 isoform X2 of canine FcRn (NCBI Reference SEQ ID NO: XP_038512242.1), which has a 6xHis tag (HHHHHH, SEQ ID NO: 301) at the C-terminus and a signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 302) at the N-terminus, is set forth in SEQ ID NO: 299. Canine β2-microglobulin (NCBI Reference SEQ ID NO: NP_001271408.1), which has a signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 302) at the N-terminus and STREP-TAG® II (WSHPQFEK, SEQ ID NO: 303) at the C-terminus, is set forth in SEQ ID NO: 300. Conditioned medium from transfected CHO cells was purified using HISTRAP™ FF chromatography and formulated in PBS, pH 7.2. Canine FcRn was >95% pure by size-exclusion chromatography analysis using a TSKGEL® G3000SWxi column.

[0266] Canine FcRn binding experiments at pH 5.9 and pH 7.4 were completed on a BIACORE™ T200 instrument. A Series S Protein L sensor chip (Cytiva, catalog number BR29205137) was used to capture antibody variants via the kappa (κ) light chain. Canine variants were captured on the Protein L chip at a flow rate of 10 μL / min for 60 seconds. Approximately 250 RU of variant was captured. For low pH binding conditions, 1XPBX-P+ (Cytiva, catalog number 28995084) was adjusted to pH 5.9. Canine FcRn was flowed over the sensor chip at 30 μL / min with a contact time of 120 seconds and a dissociation time of 600 seconds. Flow cell regeneration was completed by flowing 10 mM glycine, pH 1.7, at 30 μL / min for 30 seconds. The data were evaluated with BIACORE™ T200 evaluation software (version 3.2.1) by fitting to a 1:1 kinetic interaction model. The kinetic binding data for the canine IgG variants at pH 5.9 are shown in Table 6 below. [Table 6]

[0267] A subset of Fc variants was tested for binding to canine FcRn at pH 7.4. For these experiments, 1XPBX-P+ (Cytiva, catalog no. 28995084) was adjusted to pH 7.4. Binding conditions were the same as above, except that the concentrations of canine FcRn used to flow over the sensor chip were 2,000 nM, 1,000 nM, 500 nM, 250 nM, and 125 nM. Additionally, the pH 7.4 data were evaluated using BIACORE™ T200 evaluation software (version 3.2.1) by fitting to a steady-state affinity model. Kinetic binding data for the variants at pH 7.4 and the binding data for these variants at pH 5.9 are shown in Table 7 below. [Table 7]

[0268] In conclusion, these data demonstrate that the canine Fc variants tested have superior FcRn binding properties compared to the wild-type canine Fc.

[0269] Other embodiments While the present invention has been described in conjunction with a detailed description thereof, the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A polypeptide comprising a canine IgG Fc region variant, said canine IgG Fc region variant comprising: (i) a Tyr or Met at a position corresponding to amino acid position 252 of wild-type canine IgG; and (ii) (i) a position corresponding to amino acid position 286 of wild-type canine IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The polypeptide, wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has an increased binding affinity to canine FcRn compared to the Fc domain of the wild-type canine IgG.

2. 2. The polypeptide of claim 1, wherein the polypeptide comprises Tyr, Phe, Leu, or Trp at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG.

3. 3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises Leu, His, Phe, or Tyr at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG.

4. the polypeptide (i) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Tyr at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (ii) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Phe at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (iii) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Leu at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (iv) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Trp at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (v) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Leu at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; (vi) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a His at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; (vii) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Phe at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; (viii) a Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Tyr at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (ix) a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Tyr at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (x) a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Phe at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (xi) a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Leu at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (xii) a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Trp at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (xiii) a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Leu at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; (xiv) a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a His at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (xv) Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and Phe at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; or (xvi) the polypeptide of any one of claims 1-3, comprising a Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, and a Tyr at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG.

5. 1. A polypeptide comprising a canine IgG Fc region variant, said canine IgG Fc region variant comprising: (i) a His at a position corresponding to amino acid position 434 of wild-type canine IgG; and (ii) (i) a position corresponding to amino acid position 286 of wild-type canine IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of: a position corresponding to amino acid position 426 of wild-type canine IgG; The polypeptide, wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has an increased binding affinity to canine FcRn compared to the Fc domain of the wild-type canine IgG.

6. 6. The polypeptide of claim 5, wherein the polypeptide comprises Tyr, Phe, Leu, or Trp at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG.

7. 7. The polypeptide of claim 5 or 6, wherein the polypeptide comprises Tyr, Phe, Leu, or Trp at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG.

8. the polypeptide (i) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Tyr at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (ii) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Phe at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (iii) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Leu at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (iv) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Trp at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG; (v) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Tyr at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (vi) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Phe at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; (vii) a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Leu at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG; or (viii) the polypeptide of any one of claims 5-7, comprising a His at the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and a Trp at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG.

9. 9. The polypeptide of any one of claims 1 to 8, wherein the wild-type canine IgG is a canine IgGA comprising an Fc domain having the amino acid sequence of SEQ ID NO:9, a canine IgGB comprising an Fc domain having the amino acid sequence of SEQ ID NO:10, a canine IgGC comprising an Fc domain having the amino acid sequence of SEQ ID NO:11, or a canine IgGD comprising an Fc domain having the amino acid sequence of SEQ ID NO:

12.

10. 10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide binds to the canine FcRn to a higher level at acidic pH than at neutral pH.

11. 11. The polypeptide of claim 10, wherein the polypeptide binds to the canine FcRn to a higher level at a pH of 5.5 to 6.0 than at pH 7.

4.

12. 12. The polypeptide of any one of claims 1 to 11, wherein 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.

13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide further comprises a binding domain.

14. The polypeptide of claim 13 , wherein the binding domain comprises an antibody, an antibody fragment, or a ligand-binding portion of a receptor.

15. 15. The polypeptide of claim 14, wherein the antibody or antibody fragment comprises six complementarity determining regions (CDRs) of an immunoglobulin molecule.

16. The antibody fragment may be Fab, single chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab') ２ 15. The polypeptide of claim 14, wherein the polypeptide is selected from the group consisting of a nucleotide sequence, ...

17. 15. The polypeptide of claim 14, wherein the ligand-binding portion of a receptor comprises a ligand-binding domain of a canine receptor protein or an extracellular domain of a canine receptor protein.

18. 18. The polypeptide of any one of claims 13 to 17, wherein the binding domain specifically binds to an antigen selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and complexes of the complement system.

19. A pharmaceutical composition comprising: (i) a polypeptide according to any one of claims 1 to 18; and (ii) a pharmaceutically acceptable excipient.

20. A nucleic acid or nucleic acids encoding the polypeptide according to any one of claims 1 to 18.

21. 21. An expression vector or vectors comprising the nucleic acid or nucleic acids of claim 20.

22. 22. A host cell comprising the nucleic acid or nucleic acids of claim 20 or the expression vector or vectors of claim 21.

23. 1. A method of making a polypeptide, comprising: (i) providing one or more nucleic acids according to claim 20; (ii) expressing said nucleic acid or nucleic acids in a host cell culture, thereby producing said polypeptide; and optionally (iii) harvesting the polypeptide produced in (ii) from the host cell culture.

24. A method of treating or preventing a canine disease or disorder in a dog in need thereof, the method comprising administering to the dog a composition comprising an effective amount of a polypeptide of any one of claims 1 to 18, or a pharmaceutical composition of claim 19.

25. 25. The method of claim 24, wherein the canine 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, an infertility-related disorder, an infectious disease, or cancer.

26. 25. The method of claim 24, wherein the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

27. 20. A polypeptide according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 19, for use in a dog in need of treatment or prevention of a canine disease or disorder.

28. 28. The polypeptide for use or pharmaceutical composition for use of claim 27, wherein the disease or disorder of the canine is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, infertility-related disorder, infectious disease, or cancer.

29. 28. The polypeptide for use or pharmaceutical composition for use of claim 27, wherein the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

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