Polypeptides with altered binding to neonatal Fc receptor (FcRn) and methods of use

Abstract

Compositions for extending the half-life of one or more polypeptides in cats and methods of using the same are provided. The composition relates to a variant feline IgG Fc region.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy was created on December 11, 2023, with the file name "51682-007WO3_Sequence_Listing_12_11_23.xml" and is 164,984 bytes in size. Technical Field

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

[0004] The Fc region of an antibody plays a variety of 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 feline antibodies as therapeutic agents, people are paying more attention not only to selecting the best antibody or antibody fragment (e.g., Fab), but also to combining the antibody or antibody fragment with the appropriate Fc to achieve the desired half-life and effector function.

[0005] There is little guidance in the art regarding extending the half-life of polypeptide therapeutics (e.g., antibodies) for cats. Therefore, there is a need for Fc region variants that can improve the serum persistence of polypeptides (e.g., antibodies) in cats. Summary of the Invention

[0006] Provided herein are feline Fc regions (e.g., feline IgG Fc region variants) or their feline FcRn binding fragments that can be used for therapeutic polypeptides. For example, provided herein are polypeptides comprising feline IgG Fc region variants, wherein the feline IgG Fc region variants have an extended half-life in cats compared to their wild-type counterparts.

[0007] In a first aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) a Tyr at a position corresponding to amino acid position 252 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0008] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0009] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0010] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp or Val;

[0011] (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0012] (v) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0013] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0014] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

[0015] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

[0016] In some embodiments, the polypeptide comprises a Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

[0017] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0018] In some embodiments, the polypeptide comprises:

[0019] (i) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0020] (ii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0021] (iii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0022] (iv) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0023] (v) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0024] (vi) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0025] (vii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0026] (viii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0027] (ix) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0028] (x) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0029] (xi) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0030] In another aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) a Met at a position corresponding to amino acid position 252 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0031] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0032] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0033] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp or Val;

[0034] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Val;

[0035] (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0036] (vi) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0037] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0038] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

[0039] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

[0040] In some embodiments, the polypeptide comprises a Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG.

[0041] In some embodiments, the polypeptide comprises a Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

[0042] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0043] In some embodiments, the polypeptide comprises:

[0044] (i) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0045] (ii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0046] (iii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0047] (iv) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0048] (v) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0049] (vi) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0050] (vii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0051] (viii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG;

[0052] (ix) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0053] (x) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0054] (xi) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0055] (xii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0056] In another aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) a Met at a position corresponding to amino acid position 428 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0057] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0058] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0059] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG;

[0060] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Val;

[0061] (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0062] (vi) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0063] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0064] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

[0065] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

[0066] In some embodiments, the polypeptide comprises Asp, Glu, or Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG.

[0067] In some embodiments, the polypeptide comprises a Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG.

[0068] In some embodiments, the polypeptide comprises a Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

[0069] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0070] In some embodiments, the polypeptide comprises:

[0071] (i) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0072] (ii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0073] (iii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0074] (iv) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0075] (v) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0076] (vi) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0077] (vii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0078] (viii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0079] (ix) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG;

[0080] (x) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0081] (xi) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0082] (xii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0083] (xiii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0084] In another aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) Leu at a position corresponding to amino acid position 428 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0085] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0086] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0087] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp;

[0088] (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0089] (v) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0090] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0091] In some embodiments, the polypeptide comprises an Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

[0092] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

[0093] In some embodiments, the polypeptide comprises an Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG.

[0094] In some embodiments, the polypeptide comprises a Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

[0095] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0096] In some embodiments, the polypeptide comprises:

[0097] (i) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0098] (ii) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0099] (iii) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0100] (iv) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0101] (v) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0102] (vi) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0103] (vii) Leu at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0104] (viii) Leu at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0105] (ix) Leu at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0106] In some embodiments of any of the aforementioned aspects, the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO: 1, a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO: 2, or a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the wild-type feline IgG is a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the wild-type feline IgG is a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO: 3.

[0107] In some embodiments of any of the aforementioned aspects, the polypeptide binds to the feline FcRn at a higher level at acidic pH than at neutral pH.

[0108] In some embodiments, the polypeptide binds to the feline FcRn at a pH of 5.5 to 6.0 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0) at a higher level than at pH 7.4.

[0109] In some embodiments of any 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.

[0110] In some embodiments of any of the aforementioned aspects, the polypeptide further comprises a binding domain.

[0111] In some embodiments, the binding domain comprises an antibody, antibody fragment, or a ligand binding portion of a receptor.

[0112] In some embodiments, the antibody or antibody fragment comprises six complementarity determining regions (CDRs) of an immunoglobulin molecule.

[0113] In some embodiments, the antibody fragment is selected from the group consisting of: Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, nanobody, and diabody.

[0114] In some embodiments, the ligand binding portion of the receptor comprises the ligand binding domain of a feline receptor protein or the extracellular domain of a feline receptor protein.

[0115] In some embodiments, the binding domain specifically binds to an antigen selected from the group consisting of: NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52 and complement system complexes.

[0116] In another aspect, the invention features a pharmaceutical composition comprising (i) any of the polypeptides disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0117] In another aspect, the invention features one or more nucleic acids encoding any of the polypeptides disclosed herein.

[0118] In another aspect, the invention features one or more expression vectors containing one or more nucleic acids encoding any of the polypeptides disclosed herein.

[0119] In another aspect, the invention features a host cell comprising one or more nucleic acids encoding any of the polypeptides disclosed herein, or one or more expression vectors comprising one or more nucleic acids encoding any of the polypeptides disclosed herein.

[0120] In another aspect, the present invention provides a method for preparing a polypeptide, the method comprising:

[0121] (i) providing one or more nucleic acids encoding any of the polypeptides disclosed herein;

[0122] (ii) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; and, optionally,

[0123] (iii) collecting the polypeptide produced in (ii) from the host cell culture.

[0124] In another aspect, the invention features a method of treating or preventing a feline disease or condition in a cat in need thereof, the method 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.

[0125] In some embodiments, the feline disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, a fertility-related disorder, an infectious disease, or cancer.

[0126] In other embodiments, the feline disease or condition is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

[0127] In another aspect, the invention features any of the polypeptides disclosed herein, or a pharmaceutical composition comprising (i) any of the polypeptides disclosed herein, and (ii) a pharmaceutically acceptable excipient, for use in treating or preventing a feline disease or disorder in a cat in need thereof.

[0128] In some embodiments, the feline disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, a fertility-related disorder, an infectious disease, or cancer.

[0129] In other embodiments, the feline disease or condition is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present application, including definitions, shall prevail. The materials, methods and examples are illustrative only and are not intended to be limiting.

[0131] Other features and advantages of the present invention will be understood from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1Shown is an alignment of the amino acid sequences of wild-type feline IgG1a Fc region (SEQ ID NO: 1) and wild-type feline IgG1b Fc region (SEQ ID NO: 2) with a putative wild-type feline IgG2 Fc region (SEQ ID NO: 3). The hinge region is located between the triangles. Arrows indicate cysteine ​​residues in the hinge region that may participate in the disulfide bridge between the two heavy chains (from Strietzel et al., 2014, Vet. Immunol. Immunopathol., 158: 214-223).

[0133] Figure 2 An alignment of the amino acid sequences of wild-type feline IgG1a Fc (SEQ ID NO: 1) and human IgG1 Fc regions based on EU numbering is shown. DETAILED DESCRIPTION

[0134] 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 variety of feline diseases, the development of polypeptides with extended half-lives is of great importance, especially for the prevention or treatment of chronic diseases where repeated administration of the polypeptide is necessary.

[0135] Thus, the present disclosure features feline immunoglobulin Fc regions, or their feline FcRn-binding regions, comprising mutations that extend the half-life of one or more polypeptides comprising these sequences. Also disclosed are polypeptides comprising these domains and methods of using them. These polypeptides can be used for a variety of therapeutic and diagnostic purposes.

[0136] For example, the present disclosure is characterized in that there is increased polypeptide with cat FcRn binding that can be used for therapeutic polypeptides, or their cat FcRn binding fragments. For example, provided herein are polypeptides with increased cat FcRn binding compared to a control polypeptide (e.g., a wild-type counterpart IgG cat Fc region). In some cases, for example, compared to at a neutral pH (e.g., pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4 or pH 7.5), these polypeptides can bind to cat FcRn at a higher level (in other words, with stronger affinity) at an acidic pH (e.g., pH 5.5, pH 6.0 or pH 6.5). In some cases, compared to at pH 7.4, these polypeptides have a higher binding level to cat FcRn at pH 5.5 and / or pH 6.0. The present disclosure also relates, in part, to polypeptides with an extended half-life in cats compared to wild-type polypeptides. For example, there is provided such a polypeptide (for example, a binding molecule, such as an antibody, an antigen-binding antibody fragment or the ligand binding portion of a receptor), which has an extended half-life relative to the form of the polypeptides not attached to the Fc district disclosed herein or cat FcRn binding regions. Enzyme-Fc district fusions, ligand-Fc district fusions, nanobody-Fc fusions and peptide-Fc district fusions are also provided, wherein the fusions have an extended half-life compared to their wild-type counterparts. The Fc district, in addition to having one or more replacements (relative to wild-type cat Fc district) that extend the half-life, can also include other replacements (for example, by removing one or more post-translational modifications in the Fc district) that increase effector functions, reduce effector functions, increase the combination with protein A and / or reduce the heterogeneity of the polypeptide. Cat Fc district sequences can be from any cat antibody. In some cases, cat Fc district sequences are from cat IgG (for example, IgG1a, IgG1b, IgG2).

[0137] Where values ​​are described in ranges, it should be understood that the description includes disclosure of all possible subranges within these ranges, as well as specific values ​​falling within these ranges, regardless of whether a specific value or subrange is explicitly stated. D , temperatures, times, concentrations, and molecular weights, including ranges, are approximate and may be varied in increments of (+) or (-) 1.0 or 0.1, or in increments of + / - 15%, or 10%, or 5%, or 2%. Although not always expressly stated, it is understood that all numerical designations are preceded by the term "about" and that numerical designations may include values ​​rounded to the nearest significant figure. Although not always expressly stated, it is also understood that the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0138] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires or clearly indicates otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. For conflicts in definitions between different sources or references, the definitions provided herein shall prevail.

[0139] It should be understood that embodiments of the invention described herein include "including aspects and embodiments," "consisting of aspects and embodiments," and "consisting essentially of aspects and embodiments." As used herein, unless otherwise indicated, 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 is not meant to imply that alternatives are mutually exclusive. In the context of multiple dependent claims, when referencing other claims above, the use of "or" refers only to those claims in the alternative.

[0140] In this application, unless explicitly stated or understood by one skilled in the art, the use of "or" means "and / or." In the context of multiple dependent claims, the use of "or" refers to more than one preceding independent or dependent claim.

[0141] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, etc., is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0142] 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 the nucleotides or amino acid residues in a particular nucleic acid or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percent sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN. TM(DNASTAR) software is realized. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including realizing any parameters required for maximum comparison on the full length of the compared sequences. In some embodiments, after comparing sequences and introducing gaps to realize maximum sequence identity percentage when necessary, and not considering any conservative substitution as a part for sequence identity, variant has at least 50% sequence identity with the reference nucleic acid molecule or polypeptide. Such variants include, for example, polypeptides wherein one or more amino acid residues are added at the N-terminus or C-terminus of the polypeptide or the deletion of one or more amino acid residues. In some embodiments, 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 with the sequence of reference nucleic acid or polypeptide.

[0143] 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. Amino acid substitutions can be introduced into polypeptides screened for a desired activity, such as maintained or improved binding to FcRn, maintained or improved antigen binding, reduced immunogenicity, improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or enhanced pharmacokinetics.

[0144] As used herein, the term "conservative substitution" refers to the substitution of one amino acid residue by another amino acid residue having similar properties (such as charge, hydrophobicity, and size). For example, amino acids can be grouped according to the following common side chain properties:

[0145] (i) Hydrophobicity: norleucine (Nle), Met, Ala, Val, Leu, Ile;

[0146] (ii) neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0147] (iii) acidic: Asp, Glu;

[0148] (iv) Basic: His, Lys, Arg;

[0149] (v) Rigidity: Gly, Pro;

[0150] (vi) Aromatic: Trp, Tyr, Phe.

[0151] Conservative substitutions will entail exchanging a member of one of these categories with another member of the same category. Non-conservative substitutions will entail exchanging a member of one of these categories with another category. In some embodiments, a conservative amino acid substitution refers to a substitution that produces a property or function similar to that of another amino acid substitution. For example, a conservative amino acid substitution for A426Y can be A426F, A426T, or A426W. Other non-limiting examples of conservative amino acid substitutions are shown in Table 1 below.

[0152] Table 1

[0153]

[0154]

[0155] The term "affinity" refers to the strength of the sum of all 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, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and antigen, a receptor and ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K D ). Affinity can be measured by common protein-protein interaction tools known in the art, such as immunoblotting, enzyme-linked immunosorbent assay (ELISA), kinetic exclusion assay (KinExA), biolayer interferometry (BLI), or surface plasmon resonance (SPR) devices. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0156] "Surface Plasmon Resonance (SPR)" means allowing for example the use of BIAcore TM The BIAcore system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ) analyzes optical phenomena of biospecific interactions in real time by detecting changes in protein concentration within a biosensor matrix. For further description, see Jonsson et al., 1993, Ann. Biol. Clin. 51: 19-26.

[0157] 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 and refer to polymers of amino acid residues, and are not limited to a minimum length. This polymer of amino acid residues can contain natural or non-natural amino acid residues, and includes, but is not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition encompasses both full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In addition, for the purposes of this disclosure, "polypeptide" refers to a protein comprising modifications of the native sequence, such as deletions, additions, and substitutions (usually conservative in nature), as long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0158] The term "antibody" herein is used in the broadest sense and refers to various 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.

[0159] 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 an antigen to which the full-length antibody binds. 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')2; nanobodies; diabodies; and multispecific antibodies formed from antibody fragments.

[0160] The terms "full length antibody" and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.

[0161] 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 in their native form generally lack light chains. Suitable Nanobodies are familiar to those skilled in the art, examples of which include those of camels, dromedaries, llamas and alpacas. However, single domain antibodies may also be derived from non-Camelidae sources.

[0162] 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 antibodies, polyclonal antibodies, recombinant antibodies, 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 domain of a cat receptor protein), ligands, aptamers, and other molecules with identified binding partners.

[0163] 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.

[0164] 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 a constant region. The term "Fc domain of a wild-type cat IgG" refers to the natural Fc region of a cat antibody. The term "cat IgG Fc region variant" refers to a variant of the Fc region of a cat antibody that has one or more substitutions relative to the wild-type cat Fc region. In some embodiments, the cat Fc region sequence is from a cat IgG (e.g., IgG1a, IgG1b, or IgG2). In some embodiments, the IgG Fc polypeptide comprises a hinge, CH2, and CH3, but does not comprise CH1 or CL. In some embodiments, the IgG Fc polypeptide comprises CH2 and CH3, but does not comprise CH1, hinge, or CL. In some embodiments, the IgG Fc polypeptide comprises CH1, hinge, CH2, and CH3, and may or may not comprise CLI. In some embodiments, the Fc polypeptide, such as an IgG Fc polypeptide, lacks one or more C-terminal amino acids, such as 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 2 amino acids, while still maintaining biological activity. In some embodiments, the biological activity of the Fc polypeptide is the ability to bind to FcRn. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0165] The term "wild-type" refers to the unmutated form of a polypeptide found in nature, or a fragment thereof. A wild-type polypeptide can be produced recombinantly. In some embodiments, the wild-type IgG Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0166] The term "disorder" refers to any condition that would benefit from treatment and includes, but is not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0167] The term "cancer" refers to or describes the physiological condition in mammals that is generally characterized by dysregulated 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, and histiocytic sarcoma), leukemia, head and neck squamous cell carcinoma, salivary gland cancer, breast cancer, mast cell tumor, melanoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, squamous cell carcinoma, meningioma, glioma, gastric cancer, intestinal cancer, colon cancer, colorectal cancer, pancreatic adenocarcinoma, glioblastoma, cervical cancer, endometrial cancer or uterine cancer, ovarian cancer, bladder cancer, prostate cancer, kidney cancer or renal cancer, vulvar cancer, thyroid cancer, and transitional cell carcinoma.

[0168] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. As mentioned herein, the terms "cancer," "cancerous," "cell proliferative disease," "proliferative disease," and "tumor" are not mutually exclusive.

[0169] The term "effector function" refers to those biological activities caused by the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0170] An "effective amount" of a composition, e.g., a polypeptide of the present invention or a composition thereof (e.g., a pharmaceutical composition) refers to at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular condition (e.g., any condition affecting cats, e.g., a cell proliferative disease, e.g., cancer). The effective amount herein can vary depending on a number of factors, such as the disease state, the age, sex, and weight of the animal, and the ability of the antibody to elicit the desired response in the animal. An effective amount is also an amount in which the beneficial effects of the treatment outweigh any toxic or deleterious effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing risk, reducing severity, or delaying the onset of a disease (including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that arise during the course of the disease). For therapeutic use, beneficial or desired results include clinical results, such as reducing one or more symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dose of other drugs required to treat the disease, such as enhancing the effect of another drug by targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount can be administered in one or more administrations. For purposes of the present invention, the effective amount of a medicine, compound or pharmaceutical composition is the amount that is enough to directly or indirectly realize preventive or therapeutic treatment. As understood in clinical context, the effective amount of a medicine, compound or pharmaceutical composition can be realized by co-administered with another medicine, compound or pharmaceutical composition, or is not realized by co-administered. Therefore, in the context of using one or more therapeutic agents, it is possible to consider "effective amount", and if it is possible to realize or have realized the desired result in combination with one or more other medicaments, it can be considered that a single medicament is given with an effective amount.

[0171] The terms "host cell" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include bacterial cells (e.g., Escherichia coli (E.coli) cells) and eukaryotic cells. In some embodiments, host cells include yeast cells (e.g., Pichia pastoris (Pichia) (see, e.g., Powers et al., 2001, J Immunol Methods.251:123-135), Hansen yeast (Hanseula) or yeast (Saccharomyces)). In some embodiments, host cells also include "transformants" and "transformed cells", which include primary transformed cell lines (e.g., CHO, 293E, COS, 293T and HeLa) and progeny derived therefrom without considering passage number. The nucleic acid content of progeny may not be identical to that of the parental cell but may contain mutations. Screened or selected mutant progeny with the same function or biological activity as the initially transformed cell are included herein.

[0172] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, wherein the substantially homogeneous antibodies, i.e., the individual antibodies forming the population, are identical and / or bind to the same epitope, except for possible mutant antibodies, e.g., mutations occurring during the generation of a monoclonal antibody preparation containing naturally occurring mutations or monoclonal antibody preparations, such mutations typically existing in trace amounts. Contrary to polyclonal antibody preparations typically comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" represents the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody used in accordance with the present invention can be prepared 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 a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are as described herein.

[0173] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the preparation would be administered.

[0174] The term "pharmaceutically acceptable carrier" refers to ingredients other than the active ingredient in a pharmaceutical formulation that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0175] As used herein, the term "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated and can be performed for prevention or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the polypeptides of the present invention are used to delay the development of a disease or slow the progression of a disease.

[0176] As used herein, the term "delaying the progression of a condition or disease" means to postpone, hinder, slow, slow, stabilize, and / or delay the development of a disease or condition (e.g., a cell proliferative disorder, e.g., cancer). The length of time for such a delay may vary, depending on the history of the disease and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually encompass prevention, in that the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

[0177] The term "epitope" refers to one or more specific sites on an antigen molecule to which an antibody binds. For example, an epitope can be a linear epitope or a conformational epitope.

[0178] As used herein, the terms "reduce" and "inhibit" refer to the ability to reduce an overall amount, for example, by 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more, for example, compared to a reference or control.

[0179] The terms "increase" and "enhance" refer to the ability to increase overall, for example, by 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more, for example, compared to a reference or control.

[0180] The terms "variable region" and "variable domain" refer to the domains of the heavy or light chains of an antibody that are involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., 2007, Kuby Immunology, 6th edition WH Freeman and Co., p. 91.) A single VH domain or VL domain is sufficient to confer antigen binding specificity. In addition, using VH or VL domains from antibodies that bind to a specific antigen to screen for complementary VL or VH domain libraries, respectively, antibodies that bind to that antigen can be isolated. See, e.g., Portolano et al., 1993, J. Immunol. 150: 880-887; and Clarkson et al., 1991, Nature 352: 624-628.

[0181] " Variant " is a polypeptide that the difference with the reference polypeptide is one or more non-natural amino acid substitutions, deletions and / or additions. In some embodiments, variant maintains at least one biological activity of the reference polypeptide. In some embodiments, variant has the biological activity that the reference polypeptide substantially lacks. " Cat IgG Fc region variant " comprises an amino acid sequence that has the difference of at least one amino acid modification (preferably one or more amino acid substitutions) with the wild-type cat IgG Fc region. Preferably, compared with the wild-type cat IgG Fc region, the cat IgG Fc region variant has at least one amino acid substitution, for example, in the wild-type cat IgG Fc region, there is one to ten amino acid substitutions, and preferably one to five amino acid substitutions. The cat IgG Fc region variant herein will preferably have at least 80% homology with the wild-type cat IgG Fc region, and most preferably at least 90% homology with it, more preferably at least 95% homology with it. In some embodiments, the cat IgG Fc region is a cat IgG1a Fc region variant, a cat IgG1b Fc region variant or a cat IgG2 Fc region variant.

[0182] 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 that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0183] As used herein, "administering" means giving a subject a certain dose of a compound (e.g., polypeptide of the present disclosure) or a method for a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a polypeptide of the present disclosure). The composition utilized in the methods described herein can be, for example, administered in the following manner: parenteral, intramuscular, intravenous, intradermal, transcutaneous, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical (topically), intratumor, peritoneum, subcutaneous, subconjunctival, intravascular, mucosal, pericardial, umbilical, intraocular, oral, topical (topically), local (locally), by inhalation, by injection, by infusion, by continuous infusion, by local perfusion of directly bathing target cells, by catheter, by lavage, in a cream or in a lipid composition. Administration can be local or systemic. The method of administration may vary depending on various factors, such as the compound or composition being administered and the severity of the disorder, disease or condition being treated.

[0184] Administration "in combination" with one or more other therapeutic agents includes simultaneous (synchronous) and continuous or sequential administration in any order. As used herein, the term "synchronous" refers to the administration of two or more therapeutic agents, wherein at least a portion of the administration overlaps in time, or wherein the administration of one therapeutic agent falls within a shorter time period relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered at a time interval of no more than about a specified number of minutes. As used herein, the term "sequential" refers to the administration of two or more therapeutic agents, wherein the administration of one or more medicaments continues after stopping the administration of one or more other medicaments, or wherein the administration of one or more medicaments begins before the administration of one or more other medicaments. For example, the administration of two or more therapeutic agents is administered at a time interval of no more than about a number of minutes. As used herein, "combination" refers to the combined administration of a treatment modality and another treatment modality. Therefore, "combination" refers to administering a treatment modality to an animal before, during, or after administering another treatment modality to the animal.

[0185] Cat polypeptide

[0186] Cats typically have three IgG heavy chains, designated IgG1a, IgG1b, and IgG2. These heavy chains represent three different subclasses of feline IgG. The amino acid and DNA sequences of these heavy chains can be found in Tang et al., 2001, Vet. Immunol. Immunopathol., 80:259-270, and the GENBANK database. For example, the GENBANK accession number for the amino acid sequence of the feline IgG1a heavy chain is BAA32229.1, the GENBANK accession number for the amino acid sequence of the feline IgG1b heavy chain is BAA32230.1, and the GENBANK accession number for the amino acid sequence of the feline IgG2 heavy chain is KF811175.1. Feline antibodies also contain two types of light chains: κ and λ. The DNA and amino acid sequences of these light chains can also be found in the GENBANK database. For example, the accession number for the amino acid sequence of the feline κ light chain is AF198257.1, and the accession number for the feline λ light chain is E07339.1.

[0187] CH2 region of the feline Fc region:

[0188] The CH2 region of a feline antibody comprises or consists of amino acids 231 to 340 (according to EU numbering) of a feline IgG antibody. It will be appreciated that the CH2 region may comprise one to six (e.g., 1, 2, 3, 4, 5, or 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0189] The amino acid sequence of the CH2 region of feline IgG1a is provided below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAK (SEQ ID NO: 4)

[0190] The amino acid sequence of the CH2 domain of feline IgG1b is provided below:

[0191] PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDK(SEQ ID NO:5)

[0192] The amino acid sequence of the CH2 domain of feline IgG2 is provided below:

[0193] VPEIPGAPSVFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAK(SEQ ID NO:6)

[0194] CH3 region of cat Fc region:

[0195] The CH3 region of a feline antibody comprises or consists of amino acids 341 to 447 (according to EU numbering) of a feline IgG antibody. It will be appreciated that the CH3 region may comprise one to six (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0196] The amino acid sequence of the CH3 domain of feline IgG1a is provided below:

[0197] GQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:7)

[0198] The amino acid sequence of the CH3 domain of feline IgG1b is provided below:

[0199] GQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:8)

[0200] The amino acid sequence of the CH3 domain of feline IgG2 is provided below:

[0201] GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:9)

[0202] Fc region of a feline Fc region:

[0203] The Fc region of a feline IgG antibody comprises or consists of amino acids 231 to 447 (according to EU numbering) of a feline IgG antibody.

[0204] The amino acid sequence of the Fc domain of feline IgG1a is provided below:

[0205] PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKG QPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:1)

[0206] The amino acid sequence of the Fc domain of feline IgG1b is provided below:

[0207] PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKG QPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:2)

[0208] The amino acid sequence of the Fc domain of feline IgG2 is provided below:

[0209] VPEIPGAPSVFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKG QPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:3)

[0210] Table 2 below compares the amino acid sequences of the CH2 domain and CH3 domain of human IgG1, feline IgG1a, feline IgG1b, and feline IgG2 based on EU numbering:

[0211] Table 2

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] Replacement of feline IgG Fc to extend half-life

[0220] Increasing serum persistence is a beneficial property of therapeutic polypeptides. The disclosure is characterized in that the displacement in wild-type cat IgG1a, IgG1b and IgG2 Fc district, relative to one or more control polypeptides, the displacement extends the half-life of one or more polypeptides comprising these Fc districts in cat, wherein except having corresponding wild-type cat IgG Fc district in the position of IgG Fc district variants, one or more control polypeptides are identical with one or more polypeptides. The displacement extending the half-life can be in one or more of cat CH2 district, cat CH3 district, or in the context of cat Fc (for example, CH2+CH3) district, carry out.

[0221] The present disclosure provides a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) an amino acid substitution (e.g., Tyr) at a position corresponding to amino acid position 252 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0222] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0223] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0224] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG;

[0225] (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0226] (v) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0227] Wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of the wild-type feline IgG. In some instances, the amino acid substitution at the position corresponding to amino acid position 252 of the wild-type feline IgG is a conservative amino acid substitution of Tyr. In some instances, the amino acid substitution at the position corresponding to amino acid position 309 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Val.

[0228] For example, the present disclosure provides a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) Tyr at a position corresponding to amino acid position 252 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0229] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0230] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0231] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp or Val;

[0232] (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0233] (v) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0234] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of the wild-type feline IgG. In some instances, the IgG Fc region variant comprises an Asp at a position corresponding to amino acid position 309 of wild-type feline IgG. In some instances, the IgG Fc region variant comprises a Val at a position corresponding to amino acid position 309 of wild-type feline IgG.

[0235] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0236] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 301 of the wild-type feline IgG is a conservative amino acid substitution of Leu, Tyr, or Val.

[0237] In some embodiments, the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 377 of the wild-type feline IgG is a conservative amino acid substitution of Leu or Tyr.

[0238] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 392 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0239] In some embodiments, the polypeptide comprises:

[0240] (i) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0241] (ii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0242] (iii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0243] (iv) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0244] (v) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0245] (vi) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0246] (vii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0247] (viii) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0248] (ix) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0249] (x) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0250] (xi) Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0251] In another aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) an amino acid substitution at a position corresponding to amino acid position 252 of wild-type feline IgG (e.g., Met), and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0252] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0253] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0254] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG;

[0255] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG;

[0256] (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0257] (vi) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0258] Wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to cat FcRn compared to the Fc domain of the wild-type cat IgG. In some instances, the amino acid replacement at the position corresponding to amino acid position 252 of wild-type cat IgG is a conservative amino acid replacement of Met. In some instances, the amino acid replacement at the position corresponding to amino acid position 309 of wild-type cat IgG is a conservative amino acid replacement of Asp or Val. In some instances, the amino acid replacement at the position corresponding to amino acid position 311 of wild-type cat IgG is a conservative amino acid replacement of Val.

[0259] For example, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) a Met at a position corresponding to amino acid position 252 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0260] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0261] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0262] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp or Val;

[0263] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Val;

[0264] (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0265] (vi) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0266] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of the wild-type feline IgG. In some instances, the IgG Fc region variant comprises an Asp at a position corresponding to amino acid position 309 of wild-type feline IgG. In some instances, the IgG Fc region variant comprises a Val at a position corresponding to amino acid position 309 of wild-type feline IgG.

[0267] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0268] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 301 of the wild-type feline IgG is a conservative amino acid substitution of Leu, Tyr, or Val.

[0269] In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 311 of the wild-type feline IgG is a conservative amino acid substitution of Val.

[0270] In some embodiments, the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 377 of the wild-type feline IgG is a conservative amino acid substitution of Leu or Tyr.

[0271] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 392 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0272] In some embodiments, the polypeptide comprises:

[0273] (i) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0274] (ii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0275] (iii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0276] (iv) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0277] (v) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0278] (vi) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0279] (vii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0280] (viii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG;

[0281] (ix) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0282] (x) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0283] (xi) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0284] (xii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0285] In another aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) an amino acid substitution at a position corresponding to amino acid position 428 of wild-type feline IgG (e.g., Met), and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0286] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0287] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0288] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG;

[0289] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG;

[0290] (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0291] (vi) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0292] Wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of the wild-type feline IgG. In some instances, the amino acid substitution at the position corresponding to amino acid position 311 of wild-type feline IgG is a conservative amino acid substitution of Val. In some instances, the amino acid substitution at the position corresponding to amino acid position 428 of wild-type feline IgG is a conservative amino acid substitution of Met.

[0293] For example, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) a Met at a position corresponding to amino acid position 428 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0294] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0295] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0296] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG;

[0297] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Val;

[0298] (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0299] (vi) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0300] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0301] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0302] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 301 of the wild-type feline IgG is a conservative amino acid substitution of Leu, Tyr, or Val.

[0303] In some embodiments, the polypeptide comprises Asp, Glu, or Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 309 of the wild-type feline IgG is a conservative amino acid substitution of Asp, Glu, or Val.

[0304] In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 311 of the wild-type feline IgG is a conservative amino acid substitution of Val.

[0305] In some embodiments, the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 377 of the wild-type feline IgG is a conservative amino acid substitution of Leu or Tyr.

[0306] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 392 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0307] In some embodiments, the polypeptide comprises:

[0308] (i) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0309] (ii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0310] (iii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0311] (iv) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0312] (v) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0313] (vi) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0314] (vii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0315] (viii) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0316] (ix) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG;

[0317] (x) Met at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0318] (xi) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0319] (xii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0320] (xiii) Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0321] In another aspect, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) an amino acid substitution (e.g., Leu) at a position corresponding to amino acid position 428 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0322] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0323] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0324] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG;

[0325] (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0326] (v) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0327] Wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of the wild-type feline IgG. In some instances, the amino acid substitution at the position corresponding to amino acid position 309 of wild-type feline IgG is a conservative amino acid substitution of Asp. In some instances, the amino acid substitution at the position corresponding to amino acid position 428 of wild-type feline IgG is a conservative amino acid substitution of Leu.

[0328] For example, the invention features a polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (i) Leu at a position corresponding to amino acid position 428 of wild-type feline IgG, and (ii) at least one amino acid substitution at a position selected from the group consisting of:

[0329] (i) a position corresponding to amino acid position 286 of wild-type feline IgG;

[0330] (ii) a position corresponding to amino acid position 301 of wild-type feline IgG;

[0331] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp;

[0332] (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and

[0333] (v) a position corresponding to amino acid position 392 of wild-type feline IgG;

[0334] wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0335] In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of the wild-type feline IgG is a conservative amino acid substitution of Asp.

[0336] In some embodiments, the polypeptide comprises Leu, Tyr, or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 301 of the wild-type feline IgG is a conservative amino acid substitution of Leu, Tyr, or Val.

[0337] In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 309 of wild-type feline IgG is a conservative amino acid substitution of Asp.

[0338] In some embodiments, the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 377 of the wild-type feline IgG is a conservative amino acid substitution of Leu or Tyr.

[0339] In some embodiments, the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the polypeptide comprises Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG. In some embodiments, the amino acid substitution at the position corresponding to amino acid position 392 of the wild-type feline IgG is a conservative amino acid substitution of Asp or Glu.

[0340] In some embodiments, the polypeptide comprises:

[0341] (i) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG;

[0342] (ii) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0343] (iii) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0344] (iv) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG;

[0345] (v) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG;

[0346] (vi) Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and Leu at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0347] (vii) Leu at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG;

[0348] (viii) Leu at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG; or

[0349] (ix) Leu at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

[0350] In some embodiments of any of the aforementioned aspects, the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO: 1, a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO: 2, or a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the wild-type feline IgG is a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the wild-type feline IgG is a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO: 3.

[0351] In some embodiments, the polypeptide comprises at least one amino acid replacement at a position corresponding to one or more of amino acid positions 252, 286, 301, 309, 311, 377, 392, and 428 of wild-type feline IgG, wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding to feline FcRn compared to the Fc domain of wild-type feline IgG. The at least one amino acid replacement encompassed by the present disclosure can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) amino acid replacements of those disclosed in Table 3.

[0352] Table 3

[0353]

[0354] In some embodiments of any of the aforementioned aspects, the polypeptide binds to the feline FcRn at a higher level at acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5) than at neutral pH (e.g., pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, or pH 7.5).

[0355] In some embodiments, the polypeptide binds to the feline FcRn at a higher level at pH 5.5 to pH 6.0 than at pH 7.4. In some embodiments, the polypeptide binds to the feline FcRn at a higher level at pH 5.5 than at pH 7.4. In some embodiments, the polypeptide binds to the feline FcRn at a higher level at pH 6.0 than at pH 7.4.

[0356] Any of the polypeptides disclosed herein may comprise one or more additional amino acid substitutions, including any amino acid substitutions disclosed in U.S. Patent Application Publication No. 2022 / 0259282, U.S. Patent Application No. 18 / 046,082, and U.S. Patent No. 11,498,953, each of which is incorporated herein by reference in its entirety.

[0357] The present disclosure provides a polypeptide comprising a feline IgG Fc region variant, or a feline FcRn binding region thereof, wherein the polypeptide comprises an amino acid substitution at at least one position selected from the group consisting of:

[0358] (i) a position corresponding to amino acid position 252 of wild-type feline IgG, wherein the amino acid substitution is S252W;

[0359] (ii) a position corresponding to amino acid position 254 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: S254R and S254K;

[0360] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309Y;

[0361] (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: Q311R, Q311V, Q311L, and Q311K;

[0362] (v) a position corresponding to amino acid position 428 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: S428M, S428Y, S428H, and S428R; and

[0363] (vi) one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG;

[0364] wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0365] In some embodiments, the polypeptide has increased binding affinity to feline FcRn 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 feline IgG.

[0366] In some embodiments, the polypeptide comprises the amino acid substitution at a position corresponding to amino acid position 252 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 252 of the wild-type feline IgG is S252W.

[0367] In some embodiments, the polypeptide comprises the amino acid substitution at a position corresponding to amino acid position 254 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 254 of the wild-type feline IgG is S254R. In some embodiments, the amino acid substitution at position 254 of the wild-type feline IgG is S254K.

[0368] In some embodiments, the polypeptide comprises the amino acid substitution L309V or L309Y.

[0369] In some embodiments, the polypeptide comprises the amino acid substitution at a position corresponding to amino acid position 311 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 311 of the wild-type feline IgG is Q311R. In some embodiments, the amino acid substitution at position 311 of the wild-type feline IgG is Q311V. In some embodiments, the amino acid substitution at position 311 of the wild-type feline IgG is Q311K. In some embodiments, the amino acid substitution at position 311 of the wild-type feline IgG is Q311L.

[0370] In some embodiments, the polypeptide comprises the amino acid substitution at a position corresponding to amino acid position 428 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 428 of the wild-type feline IgG is S428M.

[0371] In some embodiments, the polypeptide comprises at least the amino acid substitution S428Y. In some embodiments, the amino acid substitution at position 428 of the wild-type feline IgG is S428Y. In some embodiments, the amino acid substitution at position 428 of the wild-type feline IgG is S428R. In some embodiments, the amino acid substitution at position 428 of the wild-type feline IgG is S428H.

[0372] In another embodiment, the polypeptide comprises an amino acid replacement at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG. In some embodiments, the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293H, E293K, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L, and conservative amino acid substitutions of any of the foregoing. In some embodiments, the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293H, E293K, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L.

[0373] In another aspect, the present disclosure provides a polypeptide comprising a feline IgG Fc region variant, or a feline FcRn binding region thereof, wherein the polypeptide comprises two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of:

[0374] (i) an amino acid substitution at a position corresponding to amino acid position 252 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: S252W, S252Y, S252F, and S252R;

[0375] (ii) an amino acid substitution at a position corresponding to amino acid position 254 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: S254R and S254K;

[0376] (iii) an amino acid substitution at a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: L309V, L309Y, and L309E;

[0377] (iv) an amino acid substitution at a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: Q311R, Q311V, Q311L, and Q311K;

[0378] (v) an amino acid substitution at a position corresponding to amino acid position 428 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428L, S428M, S428Y, S428H, and S428R;

[0379] (vi) amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG; and

[0380] (vii) an amino acid substitution at a position corresponding to amino acid position 434 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S434F, S434W, S434H, S434R, and S434Y;

[0381] wherein amino acid positions are based on EU numbering, wherein the two or more amino acid substitutions are at different positions, and wherein the polypeptide has increased binding affinity to feline FcRn compared to (a) an Fc domain of a wild-type feline IgG, and (b) a polypeptide comprising only one of the two or more amino acid substitutions.

[0382] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 286 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: T286E and T286D.

[0383] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 289 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: T289K and T289H.

[0384] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 301 of wild-type feline IgG, wherein the amino acid substitution is R301L.

[0385] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 334 of wild-type feline IgG, wherein the amino acid substitution is R334D.

[0386] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 426 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of: S426L and S426H.

[0387] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 437 of wild-type feline IgG, wherein the amino acid substitution is T437L.

[0388] In some embodiments, the two or more amino acid substitutions are selected from the group consisting of:

[0389] (i) a combination of S252Y and Q311R and / or Q311L;

[0390] (ii) a combination of S434Y and one or more of S254R, S254K, L262E, T286D, T286E, T289K, E293D, E293K, L309V, L309E, K326D, and Q347L;

[0391] (iii) S434F and E380D;

[0392] (iv) a combination of S428L and one or more of S252R, T286E, Q311V, Q311K, D312T, I377V, I383L, or N389cR;

[0393] (v) S428L, E380D and S434R;

[0394] (vi) S428L, E380T and S434R;

[0395] (vii) a combination of S252R and L262Q;

[0396] (viii) T260E, L309E and Q355L;

[0397] (ix) S290V and R344D; and

[0398] (x)R301L, E380V and T437L.

[0399] In some embodiments, the two or more amino acid substitutions are T286E, Q311V, and S428Y.

[0400] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

[0401] In some cases, the present disclosure provides a feline IgG CH2 region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 6. Also provided are feline IgG CH2 region variants comprising an amino acid sequence that has 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid differences from any one of SEQ ID NOs: 4 to 6.

[0402] In other cases, the disclosure features a feline IgG CH3 region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 7 to 9. Also featured are feline IgG CH3 region variants comprising an amino acid sequence that has 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid differences from any one of SEQ ID NOs: 7 to 9.

[0403] In certain cases, the disclosure features a feline IgG Fc region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3. Also disclosed are feline IgG Fc region variants comprising an amino acid sequence that has 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 acid differences from any one of SEQ ID NOs: 1 to 3.

[0404] In some embodiments, one or more polypeptides are provided comprising a feline IgG Fc CH2 region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 6.

[0405] In some embodiments, the polypeptides are characterized by comprising one or more feline IgG Fc CH3 region variants comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 7 to 9.

[0406] In some embodiments, the invention features one or more polypeptides comprising a feline IgG Fc region variant comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3.

[0407] As described elsewhere, in some embodiments, the polypeptide further comprises at least one additional amino acid substitution in the region corresponding to amino acid positions 250-256, amino acid positions 285-288, amino acid positions 307-315, amino acid positions 376-380, amino acid positions 383 to 392, or amino acid positions 428-437 of wild-type feline IgG, wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0408] In some embodiments, the polypeptide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) additional amino acid substitutions selected from those disclosed in Table 4 below.

[0409] Table 4. List of amino acid substitutions (Groups 1 and 2) that increase binding of feline IgG1a Fc variants to feline FcRN

[0410]

[0411] Amino acid substitutions can be made on one or both chains of the CH2 domain, CH3 domain, or Fc domain. In some cases, the substitutions on both chains of the CH2 domain, CH3 domain, or Fc domain are identical. In some cases, the substitutions on both chains of the CH2 domain, CH3 domain, or Fc domain are different. In some cases, the Fc region comprises one or more additional substitutions that increase or decrease effector function and / or improve product heterogeneity.

[0412] Other substitutions that can be combined with half-life extending substitutions

[0413] The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process that requires the coordination of a complex series of activities to produce the desired polypeptide / protein. These developments include optimizing specificity, affinity, functional activity, expression levels in engineered cell lines, long-term stability, eliminating or enhancing effector functions, and developing commercially viable manufacturing and purification methods. The present disclosure encompasses substitutions at one or more additional amino acid positions of Fc region variants that promote achievement of any one or more of the aforementioned objectives.

[0414] In some embodiments, the Fc region variant comprises amino acid substitutions at one or more additional amino acid positions that increase or decrease effector function and / or improve product heterogeneity.

[0415] In some embodiments, a substitution is introduced that reduces the effector function of the cat Fc region. Such substitutions will be familiar to those skilled in the art and may be at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) positions of cat IgG. Illustrative examples include WO 2019 / 035010 A1.

[0416] In some embodiments, substitutions that enhance binding to protein A are introduced into wild-type cat IgG Fc regions to facilitate purification by protein A chromatography. Such substitutions can be at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) positions of cat IgG. Illustrative examples include WO 2019 / 035010 A1.

[0417] In some embodiments, additional amino acid substitutions can be made to change the binding affinity to FcRn compared to the parent polypeptide or wild-type polypeptide (e.g., to increase or decrease the binding affinity to FcRn). In some embodiments, substitutions are made to change the binding affinity to FcRn compared to the parent polypeptide or wild-type polypeptide (e.g., to increase or decrease the binding affinity to FcRn). In some variants, the modification can be one, two, three, or four modifications selected from the group consisting of: 308F, 428L, 434M, and 434S, wherein the numbering is according to EU numbering. In some embodiments, the Fc variant 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, wherein the numbering is according to EU numbering. In some embodiments, Fc variants include one or more modifications selected from the group consisting of the following: 428L / 434S, 308F / 428L / 434S, wherein numbering is according to EU numbering. In some embodiments, Fc variants include one or more modifications selected from the group consisting of the following: 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S and 308F / 319L / 434S, wherein numbering is according to EU numbering. A detailed description of these modifications is described in, for example, US8883973B2, which is incorporated herein by reference in its entirety.

[0418] In some embodiments, the polypeptide comprises the hinge region of a feline antibody. In some embodiments, the hinge region of a feline antibody can be modified to extend half-life. In some embodiments, according to EU numbering, the modification is 228P.

[0419] In some embodiments, binding to FcRn is pH dependent. H310 and H435 (EU numbering) may be crucial 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.

[0420] In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations according to EU numbering), or a LALA-PG mutation (L234A, L235A, P329G mutations according to EU numbering). In some embodiments, the LALA mutation is P234A, M234A, or S234A. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residues at positions 234 and 235 (EU numbering) are Ala.

[0421] Peptides comprising feline IgG Fc variants

[0422] The present disclosure encompasses any polypeptide that may benefit from an increased half-life in cats. To extend half-life, these polypeptides are designed to comprise an Fc region variant disclosed above (eg, a CH2 region, a CH3 region, or a CH2+CH3 region).

[0423] In some embodiments, the polypeptides of the present disclosure comprise an antibody hinge region. The hinge region can be positioned between the antigen or ligand binding domain of the polypeptide and the Fc region variant. In some cases, the hinge region is attached to the C-terminus of a cytokine, growth factor, enzyme, or peptide, and the hinge region is attached to the N-terminus of the Fc region variant. Exemplary hinge region sequences are provided below.

[0424] IgG1a: KTDHPPGPKPCDCPKCP (SEQ ID NO: 10);

[0425] IgG1b: KTDHPPGPKPCDCPKCP (SEQ ID NO: 11); and

[0426] IgG2: KTASTIESKTGEGPKCP (SEQ ID NO: 12);

[0427] If a hinge region is used, the hinge region in the recombinant protein of the present disclosure may comprise zero to six (i.e., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12. In some cases, the hinge region used in the recombinant protein of the present disclosure is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.

[0428] In some embodiments, a linker sequence can be used instead of an antibody hinge sequence to connect a polypeptide (e.g., an antibody, a ligand binding domain of a receptor, an enzyme, a ligand, a peptide) to a cat Fc region variant disclosed herein. In certain embodiments, the linker is made up of 1 to 20 amino acids connected by a peptide bond, wherein the amino acid is selected from 20 naturally occurring amino acids. As is well known to those skilled in the art, some of these amino acids can be glycosylated. In other embodiments, 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is made up of most non-sterically hindered 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: 13); Ser Gly Gly Gly (SEQ ID NO: 14); Gly Gly Gly Gly Ser (SEQ ID NO: 15); Ser Gly GlyGly Gly (SEQ ID NO: 16); Gly Gly Gly Gly Gly Ser (SEQ ID NO:17); Ser Gly Gly GlyGly Gly (SEQ ID NO:18); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:19); Ser Gly GlyGly Gly Gly Gly (SEQ ID NO:20); (Gly Gly Gly Gly Ser) n (SEQ ID NO: 15), wherein n is an integer of one or greater (e.g., 1, 2, 3, 4, 5); and (Ser Gly Gly Gly Gly) n (SEQ ID NO: 16), wherein n is an integer of one or greater (e.g., 1, 2, 3, 4, 5).

[0429] Non-peptide linkers can also be used to link one or more polypeptides of interest to the Fc region variants disclosed herein. For example, an alkyl linker such as -NH(CH2) can be used. n C(O)-, wherein n = 2 to 20. These alkyl linking groups may also be substituted by any non-sterically hindering groups, such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0430] One or more polypeptides disclosed herein may include a binding domain. The binding domain may specifically bind to a protein, subunit, domain, motif, and / or epitope of a selected target as described herein. In some embodiments, the binding domain includes the ligand binding portion of an antibody, antibody fragment, or receptor. In some embodiments, the antibody or antibody fragment includes six complementary determining regions (CDRs) of an immunoglobulin molecule. In other embodiments, the antibody fragment is selected from the group consisting of: Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, nano antibodies, and diabodies. In other embodiments, the ligand binding portion of a receptor includes the ligand binding domain of a cat receptor protein or the extracellular domain of a cat receptor protein. In some embodiments, one or more polypeptides (e.g., fusion polypeptides) may include a protein, wherein the protein is a therapeutic protein as described herein. In some embodiments, the target (e.g., for a binding domain) or therapeutic protein (e.g., for a fusion polypeptide) is selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, IgE, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenic factor, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, β-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, tissue protein 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 / 10. CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD 33 (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 botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1,CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNAse, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, E MMPRIN, ENA, endothelin receptor, neprilysin, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrinogen, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, GLP1, GLP2, glucagon, Glut 4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, GnRH, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMVUL, 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, HIVIIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, cardiac myosin, cytomegalovirus (CMV), growth hormone (GH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptor (e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9 R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), interferon (INF)-α, INF-β, INF-γ, inhibin , iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratin Transforming growth factor (TGF), 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, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Mullerian inhibitor, Mug, MuSK, NAIP, NAP, NAV 1.7, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, neurotrophin-4 or neurotrophin-6, neurotrophin, neuron growth factor (NGF), NGFR, NGF-β, 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, PC NA, 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 α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific protein, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TGF-βRIII,TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5 Supplier Ck-1, Tie, TIMP, TIQ, TMEF F2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1Apo-2、DR4)、TNFRSF10B(TRAIL R2DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK RFN14)、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 RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40). ACT35, TXGP1R, TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DCTRAIL R2 TNFRH2), TNFRST23 (DCTRAIL R1TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL). Apo-2 division, TL2, TNFSF11 (TRANCE / RANK division ODF, OPG division), TNFSF12 (TWEAK Apo-3 division, DR3 division), TNFSF13 (APRIL TALL2).TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a catenin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, UPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral 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 receptors for hormones and growth factors.

[0431] In some embodiments, the antibody or antibody fragment comprises one or more complementary 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 below. For example, the antibody or antibody fragment may comprise all six CDRs of the antibody listed as binding to a specific target in Table 5. In some embodiments, the antibody or antibody fragment may be any antibody or antibody fragment disclosed in U.S. Patent Application Publication Nos. 2020 / 0062840, 2022 / 0119513, 2022 / 0106391, 2022 / 0177594, or 2022 / 0127351; U.S. Patent No. 9,328,164; and International Patent Application Publication Nos. WO 2020 / 056393 or WO 2023 / 097275.

[0432] Table 5. Exemplary CDR sequences of feline antibodies

[0433]

[0434]

[0435]

[0436]

[0437] In some embodiments, the binding domain specifically binds to one or more therapeutic targets or antigens in cats, such as, but not limited to, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA -4. EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (myostatin), GLP1, GLP2, GnRH, growth hormone releasing factor, IgE, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, I L-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL- 25, IL-31, IL-33, interleukin receptors (e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LFA-1, neuron growth factor (NGF) , NGFR, NGF-β, OSMR, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-β pan-specific protein, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, T GF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR and VEGFR-3 (flt-4).

[0438] In some embodiments, the one or more polypeptides may include a protein, wherein the protein is a therapeutic protein, e.g., EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, or a thrombopoietin binding peptide.In some embodiments, the therapeutic protein is ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA -4. EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (myostatin), GLP1, GLP2, GnRH, growth hormone releasing factor, IgE, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, I L-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL- 25, IL-31, IL-33, interleukin receptors (e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LFA-1, neuron growth factor (NGF) , NGFR, NGF-β, OSMR, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-β pan-specific protein, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, T GF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9(4-1BB CD137, ILA), VEFGR-1(fit-1), VEGF, VEGFR or VEGFR-3(flt-4).

[0439] For example, one or more 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: 156; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 157; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 158; a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 159; a CDR-L2 comprising the amino acid sequence of ATS; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 161.

[0440] 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: 101-150, 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: 100.

[0441] 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: 101, 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: 100.

[0442] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline 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: 102, 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: 100.

[0443] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Asp at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 103, 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: 100.

[0444] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 104, 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: 100.

[0445] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 105, 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: 100.

[0446] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 106, 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: 100.

[0447] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Tyr at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 107, 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: 100.

[0448] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Val at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 108, 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: 100.

[0449] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 109, 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: 100.

[0450] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Try at an amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 110, 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: 100.

[0451] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Leu at an amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 111, 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: 100.

[0452] In some embodiments, the polypeptide comprises a Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 112, 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: 100.

[0453] In some embodiments, the polypeptide comprises Tyr at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and Glu at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 113, 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: 100.

[0454] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline 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: 114, 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: 100.

[0455] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 115, 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: 100.

[0456] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Glu at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 116, 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: 100.

[0457] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Leu at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 117, 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: 100.

[0458] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 118, 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: 100.

[0459] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Tyr at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 119, 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: 100.

[0460] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 120, 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: 100.

[0461] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 121, 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: 100.

[0462] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 311 of the wild-type feline 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: 122, 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: 100.

[0463] In some embodiments, the polypeptide comprises a Met at the amino acid position corresponding to amino acid position 252 of the wild-type feline IgG and a Try at the amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 123, 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: 100.

[0464] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Leu at an amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 124, 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: 100.

[0465] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 125, 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: 100.

[0466] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 252 of the wild-type feline IgG, and a Glu at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 126, 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: 100.

[0467] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline 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: 127, 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: 100.

[0468] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 128, 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: 100.

[0469] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Glu at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 129, 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: 100.

[0470] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG and a Leu at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 130, 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: 100.

[0471] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 131, 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: 100.

[0472] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Tyr at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 132, 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: 100.

[0473] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 133, 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: 100.

[0474] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Glu at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 134, 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: 100.

[0475] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 135, 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: 100.

[0476] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 311 of the wild-type feline 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: 136, 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: 100.

[0477] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG and a Try at the amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 137, 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: 100.

[0478] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG and a Leu at an amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 138, 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: 100.

[0479] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 139, 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: 100.

[0480] In some embodiments, the polypeptide comprises a Met at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Glu at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 140, 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: 100.

[0481] In some embodiments, the polypeptide comprises a Leu at the amino acid position corresponding to amino acid position 428 of the wild-type feline 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: 141, 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: 100.

[0482] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 286 of the wild-type feline 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: 142, 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: 100.

[0483] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG and a Leu at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 143, 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: 100.

[0484] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Val at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 144, 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: 100.

[0485] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Tyr at an amino acid position corresponding to amino acid position 301 of the wild-type feline 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: 145, 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: 100.

[0486] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 309 of the wild-type feline 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: 146, 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: 100.

[0487] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Try at an amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 147, 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: 100.

[0488] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Leu at an amino acid position corresponding to amino acid position 377 of the wild-type feline 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: 148, 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: 100.

[0489] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and an Asp at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 149, 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: 100.

[0490] In some embodiments, the polypeptide comprises a Leu at an amino acid position corresponding to amino acid position 428 of the wild-type feline IgG, and a Glu at an amino acid position corresponding to amino acid position 392 of the wild-type feline 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: 150, 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: 100.

[0491] In some embodiments, the therapeutic protein is any protein described herein. In some embodiments, the one or more polypeptides further comprise a feline IgG CH2 domain, an IgG CH3 domain, or an IgG Fc region as described herein. The modified feline IgG CH2 domain, IgG CH3 domain, or IgG Fc region can extend the half-life of the therapeutic protein in vivo.

[0492] Pharmaceutical composition

[0493] In one aspect, the invention features a pharmaceutical composition comprising (i) any of the polypeptides disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0494] To prepare a pharmaceutical or sterile composition of one or more polypeptides described herein, the one or more polypeptides can be combined 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)).

[0495] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers, for example, in the form of a lyophilized powder, slurry, aqueous solution, or suspension (see, e.g., Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, one or more polypeptides of the invention are diluted to an appropriate concentration in sodium acetate solution at pH 5-6, and NaCl or sucrose is added to maintain tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to increase stability.

[0496] The toxicity and therapeutic efficacy of a polypeptide composition administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 In particular aspects, one or more polypeptides that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used to formulate a range of dosages for cats. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration employed.

[0497] Any suitable mode of administration can be used. Exemplary suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, directly intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial. In some embodiments, one or more polypeptides can be administered by an invasive route such as injection. In other embodiments, one or more polypeptides are administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery.

[0498] The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0499] Alternatively, one or more polypeptides can be administered in a local rather than systemic manner, for example, by direct injection of the antibody into an arthritic joint characterized by immunopathology or a pathogen-induced lesion, typically in a reservoir or sustained-release formulation. Furthermore, one or more polypeptides can be administered in a targeted drug delivery system, for example, in liposomes coated with tissue-specific antibodies, targeting, for example, an arthritic joint characterized by immunopathology or a pathogen-induced lesion. The liposomes will be targeted to the diseased tissue and selectively taken up by the diseased tissue.

[0500] The administration regimen depends on several factors, including but not limited to the age, weight and physical condition of the cat being treated, serum or tissue turnover of the therapeutic antibody, the level of symptoms, the immunogenicity of one or more therapeutic polypeptides, and the accessibility of target cells in the biomatrix. Preferably, the administration regimen delivers one or more polypeptides of sufficient therapeutic quality to achieve an improvement in the target disease state while minimizing undesirable side effects. Therefore, the amount of the biologic delivered depends, in part, on the severity of the disease being treated, and on the one or more specific therapeutic polypeptides. Guidance for selecting appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Milgrom et al. New Engl. J. Med. 341: 1966-1973 (1999); Slamon et al. New Engl. J. Med. 344: 783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342: 613-619 (2000); Ghosh et al. New Engl. J. Med. 348: 24-32 (2003); Lipsky et al. New Engl. J. Med. 343: 1594-1602 (2000)).

[0501] The determination of the appropriate dosage of one or more polypeptides is performed by one skilled in the art, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, the dosage is initially slightly less than the optimal dosage, and then the dosage is increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, symptomatic diagnostic measures of inflammation or the levels of inflammatory cytokines produced.

[0502] Nucleic acid, vector, host cell and preparation method

[0503] The present disclosure also encompasses one or more nucleic acids encoding one or more polypeptides described herein, one or more vectors comprising the one or more nucleic acids, and host cells comprising the one or more nucleic acids or the one or more vectors.

[0504] In one aspect, the invention features one or more nucleic acids encoding any of the polypeptides disclosed herein.

[0505] In another aspect, the invention features one or more expression vectors containing one or more nucleic acids encoding any of the polypeptides disclosed herein.

[0506] In another aspect, the invention features a host cell comprising one or more nucleic acids encoding any of the polypeptides disclosed herein, or one or more expression vectors comprising one or more nucleic acids encoding any of the polypeptides disclosed herein.

[0507] In another aspect, the present invention provides a method for preparing a polypeptide, the method comprising:

[0508] (i) providing one or more nucleic acids encoding any of the polypeptides disclosed herein;

[0509] (ii) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; and, optionally,

[0510] (iii) collecting the polypeptide produced in (ii) from the host cell culture.

[0511] One or more polypeptides described herein can be produced in bacteria or eukaryotic cells. Some polypeptides (e.g., Fab) can be produced in bacterial cells (e.g., E. coli cells). Polypeptides can also be produced in eukaryotic cells (such as transformed cell lines (e.g., CHO, 293E, COS, 293T, Hela)). In addition, polypeptides (e.g., scFv) can be expressed in yeast cells (such as Pichia pastoris (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hansenula or yeast). In order to produce the antibody of interest, one or more polynucleotides encoding one or more polypeptides are constructed, the one or more polynucleotides are introduced into one or more expression vectors, and then the one or more expression vectors are expressed in a suitable host cell. In order to improve expression, the nucleotide sequence of the gene can be recoded without changing (or changing to a minimum - for example, removing the C-terminal residues of the heavy or light chain) amino acid sequence. Potentially recoded regions include regions related to translation initiation, codon usage, and possible unintended mRNA splicing. One of ordinary skill can readily envision polynucleotides encoding the Fc region variants described herein.

[0512] Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the polypeptide (eg, antibody).

[0513] If one or more polypeptides are to be expressed in bacterial cells (for example, intestinal bacteria), expression vectors can have the feature of allowing carrier to amplify in bacterial cells. In addition, when using intestinal bacteria (such as JM109, DH5α, HB101 or XL1-Blue) as host, carriers can have promoters that can be allowed to effectively express in intestinal bacteria, such as lacZ promoter (Ward et al., 341:544-546 (1989)), araB promoter (Better et al., Science, 240:1041-1043 (1988)) or T7 promoter. The example of such carriers includes, for example, M13 series carriers, pUC series carriers, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), " QIAexpress system " (QIAGEN), pEGFP and pET (when using this expression vector, host is preferably the BL21 expressing T7 RNA polymerase). Expression vectors can contain the signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169: 4379 (1987)) can be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation.

[0514] If one or more polypeptides are to be expressed in animal cells (such as CHO, COS and NIH3T3 cells), the expression vector may contain a promoter for expression in these cells, for example, the SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)) (e.g., early simian virus 40 promoter), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)) or CMV promoter (e.g., human cytomegalovirus immediate early promoter). In addition to the nucleic acid sequence encoding the Fc region variant, the recombinant expression vector may also carry additional sequences (such as sequences that regulate replication of the vector in the host cell (e.g., an origin of replication)) and a selective marker gene. The selective marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, U.S. Pat. No. 4,399,216, U.S. Pat. No. 4,634,665 and U.S. Pat. No. 5,179,017). For example, typically the selectable marker gene confers resistance to drugs (such as G418, hygromycin or methotrexate) on a host cell into which the vector is introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

[0515] In some embodiments, one or more polypeptides are produced in mammalian cells. Exemplary mammalian host cells for expressing one or more polypeptides include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, as described in Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA, 77: 4216-4220, which are used together with a DHFR selective marker, for example, as described in Kaufman and Sharp (1982), Mol. Biol. 159: 601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte lines (e.g., NSO myeloma cells and SP2 cells), and cells from transgenic animals (e.g., transgenic mammals). For example, the cell is a mammary epithelial cell.

[0516] In an exemplary system for antibody expression, recombinant expression vectors encoding both the antibody heavy chain and the antibody light chain of the antibody are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy chain gene and the antibody light chain gene are each operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, adenovirus, etc., such as a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high-level transcription of the gene. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select CHO cells that have been transfected with the vector. The selected transformed host cells are cultured to allow expression of the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0517] Treatment

[0518] One or more polypeptides disclosed herein can be used to treat or prevent any disease or condition in a cat in need thereof. The present invention is particularly useful for treating chronic conditions that require repeated dosing. Due to the extended half-life of protein therapeutics, the frequency of dosing and / or the dosage level can be reduced.

[0519] In one aspect, the invention features a method of treating or preventing a feline disease or condition in a cat in need thereof, the method 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.

[0520] In another aspect, the invention features any of the polypeptides disclosed herein, or a pharmaceutical composition comprising (i) any of the polypeptides disclosed herein, and (ii) a pharmaceutically acceptable excipient, for use in treating or preventing a feline disease or disorder in a cat in need thereof.

[0521] Any suitable feline disease or condition can be treated. In some embodiments, the feline disease or condition is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, kidney disease, fertility-related disorder, infectious disease, or cancer.

[0522] In other embodiments, the feline disease or condition is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

[0523] In some embodiments, the disease, condition, illness or symptom treated or prevented is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, bone disease / musculoskeletal disease, cardiovascular disease, nervous system disease, kidney disease, metabolic disease, immune disease, genetic disease / hereditary disease, fertility-related disorder, infectious disease or cancer. In certain embodiments, the disease or condition treated or prevented is atopic dermatitis, allergic dermatitis, food allergy, osteoarthritis pain, perioperative pain, dental pain, cancer pain, arthritis, anemia, obesity or diabetes.

[0524] Antibodies can be used not only to treat or prevent disease, but also to regulate normal biological functions, such as managing fertility or behavior.

[0525] In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered in a bolus or by continuous infusion over a period of time. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered intramuscularly, intraperitoneally, intracerebrospinal fluid, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation.

[0526] In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered in an amount ranging from 0.01 mg / kg to 50 mg / kg body weight per dose. In some embodiments, one or more polypeptides disclosed herein, or a pharmaceutical composition comprising one or more polypeptides disclosed herein, are administered, for example, at 0.01 mg / kg to 55 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.01 mg / kg to 45 mg / kg, 0.01 mg / kg to 40 mg / kg, 0.01 mg / kg to 35 mg / kg, 0.01 mg / kg to 30 mg / kg, 0.01 mg / kg to 25 mg / kg, 0.01 mg / kg to 20 mg / kg, 0.01 mg / kg to 15 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 5 mg / kg, or 0.01 mg / kg 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 dosage of the antibody would be in the range of 0.01 mg / kg to 10 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 or 10 mg / kg (or any combination thereof) can be administered to an animal. In some embodiments, one or more polypeptides disclosed herein, or a pharmaceutical composition comprising one or more polypeptides disclosed herein, is administered at 2 mg / kg body weight per dose.

[0527] In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered within one month, two months, three months, four months, five months, or six months of each other, or within one week, two weeks, or three weeks of each other. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once a week. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once every two weeks. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once every three weeks. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once every month. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once every two months. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once every three months. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered once every four months. In some embodiments, one or more polypeptides disclosed herein, or a pharmaceutical composition comprising one or more polypeptides disclosed herein, are administered once every five months. In some embodiments, one or more polypeptides disclosed herein, or a pharmaceutical composition comprising one or more polypeptides disclosed herein, are administered once every six months. In some embodiments, one or more polypeptides disclosed herein, or a pharmaceutical composition comprising one or more polypeptides disclosed herein, are administered to cats once or over a series of treatments. In some embodiments, the dose is administered once a week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more weeks without treatment.

[0528] In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered in combination, simultaneously, sequentially, or in conjunction with one or more other therapeutic agents. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered in combination with one or more other therapeutic agents. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered synchronously with one or more other therapeutic agents. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered sequentially with one or more other therapeutic agents. In some embodiments, one or more polypeptides disclosed herein, or pharmaceutical compositions comprising one or more polypeptides disclosed herein, are administered in conjunction with one or more other therapeutic agents. Any suitable other therapeutic agent may be used.

[0529] diagnosis

[0530] One or more polypeptides disclosed herein can also be used for various diagnostic purposes, for example, to determine whether a cat has any particular disease or condition. In some embodiments, one or more polypeptides may comprise 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) as described herein. In some embodiments, one or more polypeptides further comprise a labeling group. Generally speaking, labeling groups can be divided into several categories, depending on the assay in which the labeling group is to be detected: a) isotopic labels, which can be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antibody via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art, and these methods can be used to practice the present invention.

[0531] 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).

[0532] Specific labels may also include optical dyes, including but not limited to chromophores, phosphors, and fluorophores, the latter of which are specific in many cases. Fluorophores may be "small molecule" fluorophores, or protein fluorophores.

[0533] Fluorescent labels can be any molecule that can be detected by intrinsic fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, 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 Detection Systems, Inc., New York, NY). Probes, Eugene, Oreg.), FITC, rhodamine and Texas Red (Pierce, Rockford, 111.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable optical dyes (including fluorophores) are described in the Molecular Probes Handbook, edited by Richard P. Haugland, which is incorporated by reference in its entirety.

[0534] Suitable protein fluorescent labels also include, but are not limited to, green fluorescent protein (including GFP from Renilla, Ptilosarcus, and Aequorea species (Chalfie et al., 1994, Science, 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H1J9; Stauber, 1998, Biotechniques, 24:462-471; Heim et al., 1996, Curr. Biol., 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol., 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA, 85:2603-2607), and Renilla fluorescent markers (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Patent 5,292,658, U.S. Patent 5,418,155, U.S. Patent 5,683,888, U.S. Patent 5,741,668, U.S. Patent 5,777,079, U.S. Patent 5,804,387, U.S. Patent 5,874,304, U.S. Patent 5,876,995, and U.S. Patent 5,925,558). All of the references cited above in this paragraph are expressly incorporated herein by reference in their entirety.

[0535] Determination

[0536] Fc γ RI and FcγRIII binding:

[0537] Binding to FcγRI and FcγRIII is an indicator 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.

[0538] C1q binding:

[0539] Binding to CIq, the first component of complement, is an indicator of the ability of an antibody to mediate CDC.To assess this property of an antibody, assays that measure antibody binding to CIq can be performed using methods known in the art.

[0540] half life:

[0541] 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). Exemplary animal models include non-human primate models and transgenic mouse models. The transgenic mouse model can be empty in the mouse FcRnα chain and expresses a cat FcRnα transgene (e.g., under the control of a constitutive promoter). The cat FcRnα chain can be paired with mouse β2-microglobulin in vivo to form a functional chimeric FcRn heterodimer. For example, the half-life of an antibody (e.g., a cat antibody) can be measured in the following manner: the antibody is injected into a cat model and the level of the antibody in the serum is measured over a certain period of time.

[0542] Example

[0543] Example 1: Using Biacore TM Surface plasmon resonance analysis at 8K

[0544] For using Biacore TMSurface plasmon resonance (SPR) analysis was performed at 8K, with bovine serum albumin (BSA) immobilized on a CM5 sensor chip. The sensor chip surfaces of flow cells 1 and 2 were activated for 420 seconds (10 μL / min) with a fresh mixture of 50 mmol / L N-hydroxysuccinimide and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. BSA diluted with 10 mM sodium acetate (pH 4.5) was then injected into flow cell 2 to achieve conjugation, while flow cell 1 was set as a blank. After the amine coupling reaction, the remaining active coupling sites on the chip surface were blocked by a 420-second injection of 1 mM ethanolamine hydrochloride. The running buffer for the binding experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 5.5), and the operation was performed at 25°C. The supernatant of the variant was injected onto the chip surface and captured onto immobilized BSA via a SASA (single domain antibody against serum albumin) tag (see, e.g., US2013 / 0129727A1) for 60 seconds. 200 nM feline FcRn (GenBank KF773786 (IgG receptor FcRn large subunit p51) and European Nucleotide Archive AY829266.1 (feline β-2-microglobulin)) was injected for 120 seconds, followed by dissociation with running buffer within 120 seconds. The flow rate during the BSA immobilization phase was 10 μL / min, and the flow rate during the association and dissociation phases was 30 μL / min. All data were obtained using Biacore TM Processed with 8K Evaluation Software Version 1.1.

[0545] Example 2: Binding kinetics of feline IgG1a variants to feline FcRn measured using the C1 biosensor

[0546] For feline IgG1a variants (S252Y, S252M, T286D, T286E, R301L, R301V, R301Y, L309V, L309E, Q311V, I377Y, I377L, R392D, R392E, S428M, S428L, S252Y+T286D, S252Y+T286E, S252Y+R301L, S252Y+R301V, S252Y+R301 1Y, S252Y+L309V, S252Y+L309D, S252Y+I377Y, S252Y+I377L, S252Y+R392D, S252Y+R392E, S252M+ T286D, S252M+T286E, S252M+R301L, S252M+R301V, S252M+R301Y, S252M+L309V, S252M+L309D, S252 M+Q311V, S252M+I377Y, S252M+I377L, S252M+R392D, S252M+R392E, S428M+T286D, S428M+T286E, S 428M+R301L, S428M+R301V, S428M+R301Y, S428M+L309V, S428M+L309E, S428M+L309D, S428M+Q311V The binding kinetics of the wild-type (S428L+I377Y, S428M+I377L, S428M+R392D, S428M+R392E, S428L+T286D, S428L+R301L, S428L+R301V, S428L+R301Y, S428L+L309D, S428L+I377Y, S428L+I377L, S428L+R392D, S428L+R392E, and wild-type) to feline FcRn (GenBank KF773786 (feline FcRn large subunit p51) and European Nucleotide Archive AY829266.1 (feline β-2-microglobulin)) at pH 6.0 were evaluated. EU numbering is used to identify positions. In this study, cat Fc variants carrying a single amino acid replacement or a combination of amino acid replacements were synthesized as cat IgG1a (Kanai et al., 2000, Vet. Immunol. Immunopathol. 73:53) using the variable domains described by Gearing DP et al. (2016, J Vet Intern Med, 30:1129). Synthesized cat IgGa variant DNA was subcloned into a mammalian expression vector and transiently transfected into CHO cells. Conditioned culture medium was purified using protein A chromatography.

[0547] For feline FcRn binding experiments, all assays were performed on Biacore TM The PCR reaction was performed on an 8K+ system at 25°C. For this set of experiments, antibodies were immobilized onto S-Series C1 sensor chips using standard amine coupling reagents. A mixture of 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mmol / L N-hydroxysuccinimide (NHS) was injected over 420 seconds to activate the surface. Antibodies were then injected at concentrations ranging from 0.5 μg / ml to 2 μg / ml in 10 mM sodium acetate (pH 5.0) over 120 seconds. Finally, 1 M ethanolamine was injected over 420 seconds. The running buffer was 1× phosphate-buffered saline (PBS)-P+ (Cytiva, catalog #28995084) (pH adjusted to 6.0).

[0548] To evaluate the binding affinity of feline IgG1a variants to feline FcRn at pH 6.0, feline FcRn concentrations ranging from 1.56 nM to 2000 nM were selected and injected in single-cycle mode.

[0549] Each antibody was injected at 5 μL / min for 90 seconds at four concentrations, followed by a 180-second dissociation period. Each concentration series was injected three times in this format, with at least three buffer-only cycles performed to allow for appropriate reference subtraction. The surface was regenerated by injecting 1×PBS-P+ (pH 7.4) twice within 30 seconds, followed by a 60-second wait. Three priming cycles were performed to stabilize the surface prior to analysis.

[0550] Data were evaluated using Insight Evaluation software by fitting to a 1:1 kinetic interaction model or fitting to steady-state affinity. Acceptable parameters were selected using quality metrics including U and T values. For kinetic rate constants, U values ​​less than 15 were considered acceptable, while for kinetic rate constants, T values ​​greater than 100 were considered acceptable. Steady-state affinity parameters were considered acceptable when these values ​​were outside the range.

[0551] All variants as well as the wild type are expected to not bind to feline FcRn at pH 7.4, and the tested variants show increased affinity for feline FcRn at acidic pH (e.g., pH 5.5 or pH 6.0) compared to wild type Fc.

[0552] Example 3: Pharmacokinetic Study of Feline IgG1a Fc Variants with Enhanced FcRn Binding and Wild-Type Feline IgG1a

[0553] Pharmacokinetic (PK) studies were performed in male and female cats. Feline IgG1a Fc variants, including antibodies carrying the wild-type feline IgG1a Fc domain (SEQ ID NO: 1), were prepared using anti-NGF variable domains as described in Gearing et al. (2016, J Vet Intern Med, 30: 1129), supra.

[0554] Animals were randomly divided into groups so that each group contained the same number of males and females. Each animal was administered a single intravenous dose of 2 mg / kg antibody. Approximately 0.5 ml of whole blood was collected at the following time points: 0 hour (pre-dose), 4 hours and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days after injection. Serum was separated from the whole blood and the presence of antibodies was determined by ELISA specific for anti-NGF antibodies. The serum concentrations of the six anti-NGF monoclonal antibody (mAb) variants were described by a two-compartment pharmacokinetic (PK) model with linear clearance using a nonlinear mixed effect (NLME) model. Population parameters were estimated using a stochastic approximation of the 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 lognormal distribution. Pharmacokinetic parameters (β) were determined based on body weight (BW) using mAb typical coefficients. BW,Cl =0.75,β BW,V1 =β BW,V2 =1,β BW,Q =2 / 3). Equation for a single parameter (Dong et al., 2011. Clin Pharmacokinet, 50:131) yes:

[0555]

[0556] in is a typical parameter of the population, η is a random variable with mean 0 and standard deviation ω, BW i is the animal's weight i, and BW ref The reference weight is 2kg.

[0557] Categorical covariates of clearance, intercompartmental exchange coefficient, and peripheral volume were used to discriminate antibody variants according to the following equation:

[0558]

[0559] Among them, if the individual variable covariate belongs to this category, Ω i=1, otherwise, Ω i = 0. Wild-type (WT) mAb variants were used as reference. Antibodies containing feline IgG1a Fc variants are expected to have increased terminal half-lives compared to antibodies containing wild-type feline IgG1a Fc.

[0560] Example 4: Pharmacokinetic Study of Feline IgG1a Fc Variants with Two or Three Fc Substitutions and Wild-Type Feline IgG1a

[0561] Pharmacokinetic (PK) studies were performed in male and female cats. Feline IgG1a Fc variants (including antibodies carrying a wild-type feline IgG1a Fc domain) were prepared using anti-NGF variable domains as described by Gearing et al. (2016, J Vet Intern Med, 30:1129). The feline IgG1a variants tested in this study included: S252Y+T286D, S252Y+T286E, S252Y+R301L, S252Y+R301V, S252Y+R301Y, S252Y+L309V, S252Y+L309D, S252Y+I377Y, S252Y+I377L, S252Y+R392D, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E, S252Y+I392E 52Y+R392E, S252M+T286D, S252M+T286E, S252M+R301L, S252M+R301V, S252M+R301Y, S252M+L309V, S252M+L309D, S252M+Q311V, S252M+I377Y, S252M+I377L, S252M+R392D , S252M+R392E, S428M+T286D, S428M+T286E, S428M+R301L, S428M+R301V, S428M+R30 1Y, S428M+L309V, S428M+L309E, S428M+L309D, S428M+Q311V, S428M+I377Y, S428M+I 377L, S428M+R392D, S428M+R392E, S428L+T286D, S428L+R301L, S428L+R301V, S428L+R301Y, S428L+L309D, S428L+I377Y, S428L+I377L, S428L+R392D, S428L+R392E and wild type.

[0562] Animals were randomly divided into groups with equal numbers of males and females in each group. Each animal was administered a single intravenous dose of 2 mg / kg antibody. Approximately 0.5 ml of whole blood was collected at the following time points: 0 hour (pre-dose), 4 hours and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days after injection. Serum was separated from the whole blood and the presence of antibodies was determined by ELISA specific for feline anti-NGF antibodies. The serum concentrations of seven anti-NGF monoclonal antibody (mAb) variants were described by a two-compartment pharmacokinetic (PK) model with linear clearance using a nonlinear mixed effects (NLME) model (population parameters were estimated using a stochastic approximation of the 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 lognormal distribution. Pharmacokinetic parameters (β) were determined based on body weight (BW) using mAb typical coefficients. BW,Cl =0.75,β BW,V1 =β BW,V2 =1,β BW,Q =2 / 3). Equation for a single parameter (Dong et al., 2011. Clin Pharmacokinet, 50:131) yes:

[0563]

[0564] in is a typical parameter of the population, η is a random variable with mean 0 and standard deviation ω, BW i is the animal's weight i, and BW ref The reference weight is 2kg.

[0565] Categorical covariates of clearance, intercompartmental exchange coefficient, and peripheral volume were used to discriminate antibody variants according to the following equation:

[0566]

[0567] Among them, if the individual variable covariate belongs to this category, Ω i =1, otherwise, Ω i = 0. Wild-type (WT) mAb was used as a reference.

[0568] It is expected that the combination of amino acid substitutions in the IgG Fc region will significantly improve the terminal half-life of anti-NGF IgG1a antibodies in cats compared to anti-NGF IgG1a antibodies carrying (i) wild-type feline IgG1a Fc region or (ii) a feline IgG1a Fc variant with only a single amino acid substitution.

[0569] Example 5: Binding kinetics of feline IgG Fc variants to feline FcRn

[0570] A group of cat Fc variants were expressed and purified in IgG format. IgG comprises a light chain containing the amino acid sequence of SEQ ID NO: 100, and a heavy chain containing the amino acid sequence of any one of SEQ ID NO: 101-150. The variable domains of the heavy and light chains of IgG are described in International Patent Application Publication WO 2023 / 97275, which is incorporated herein by reference in its entirety. The cat IgG1a constant domain contains a MALA mutation (according to EU numbering, M234A and L244A), which reduces potential effector activity (ADCC and CDC). The heavy and light chains were synthesized and subcloned into PC DNA TM 3.4 vector (Thermo Fisher Scientific) with a signal sequence at the N-terminus of the chain. The heavy chain construct and the light chain construct were co-transfected into E XPI CHO TM The cells were incubated for 7 days and then M AB S ELECT TM S U R E TM Conditioned medium was purified using protein A resin. The purified antibodies were buffer exchanged in PBS (pH 7.4). The FcRn complex consists of a large subunit (p51) and a small subunit (β2-microglobulin, p14), and feline FcRn protein was produced by co-expressing these two proteins in CHO cells. The soluble portion of the feline FcRn large subunit p51 isoform X1 (NCBI reference sequence number XP_044901959.1) with a 6×His tag (HHHHHH, SEQ ID NO: 153) at the C-terminus and a signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 154) at the N-terminus is shown in SEQ ID NO: 151. The soluble portion of the feline FcRn large subunit p51 isoform X1 (NCBI reference sequence number XP_044901959.1) with a signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 154) at the N-terminus is shown in SEQ ID NO: 151. The feline β2-microglobulin II (WSHPQFEK, SEQ ID NO: 155) (NCBI Reference No. NP_001009876.1) is shown in SEQ ID NO: 152. Conditioned medium from transfected CHO cells was conditioned using H IS T RAP TM Purify by FF chromatography and prepare in PBS (pH 7.2). Analytical size exclusion chromatography using a G3000SWxi column showed that the purity of feline FcRn was >95%.

[0571] Feline FcRn binding assays at pH 5.9 were performed in B IACORE TM This was done on a T200 instrument. An S-series Protein L sensor chip (Cytiva, catalog number BR29205137) was used for capture of antibody variants via kappa (κ) light chain. Feline variants were captured onto the Protein L chip in 60 seconds at a flow rate of 10 μL / min. 1× PBX-P+ (Cytiva, catalog number 28995084) adjusted to pH 5.9 was used as the running buffer. Feline FcRn was flowed through the sensor chip at 30 μL / min with a contact time of 120 seconds and a dissociation time of 600 seconds. Regeneration of the flow cell was accomplished by flowing 10 mM glycine (pH 1.7) at 30 μL / min in 30 seconds. Data were obtained by B IACORE TM Evaluation was performed using T200 evaluation software v3.2.1 by fitting to a 1:1 kinetic interaction model. Kinetic binding data for feline IgG variants at pH 5.9 are shown in Table 6 below.

[0572] Table 6. Binding data of feline Fc variants to feline FcRn-bound IgG at pH 5.9

[0573]

[0574]

[0575] Taken together, these data demonstrate that the feline Fc variants tested have superior FcRn binding properties compared to wild-type feline Fc. For example, feline Fc variants having the substitutions S252Y / T286D, S252Y / T286E, S252Y / L309V, S252M / T286D, S252M / T286E, S252M / L309V, S252M / Q311V, S252M / R392E, S428M / T286D, S428M / T286E, S428M / L309V, S428M / L309E, S428M / Q311V, and S428L / T286D have improved affinity for feline FcRn compared to wild-type Fc or feline Fc variants having a single amino acid substitution (e.g., S252Y, S252M, S428M, or S428L).

[0576] Other implementation plans

[0577] Although the present invention has been described in conjunction with specific embodiments, the foregoing specific embodiments are intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (a) a Tyr at a position corresponding to amino acid position 252 of wild-type feline IgG, and (b) at least one amino acid substitution at a position selected from the group consisting of: (i) a position corresponding to amino acid position 286 of wild-type feline IgG; (ii) a position corresponding to amino acid position 301 of wild-type feline IgG; (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp or Val; (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and (v) a position corresponding to amino acid position 392 of wild-type feline IgG; wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

2. The polypeptide of claim 1 , wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

3. The polypeptide of claim 1 or claim 2, wherein the polypeptide comprises Leu, Tyr or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

5. The polypeptide of any one of claims 1 to 4, wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises: (i) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (ii) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (iii) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (iv) Tyr at the amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (v) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (vi) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (vii) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (viii) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (ix) Tyr at the amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Tyr at the amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (x) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG; or (xi) Tyr at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG.

7. A polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (a) a Met at a position corresponding to amino acid position 252 of wild-type feline IgG, and (b) at least one amino acid substitution at a position selected from the group consisting of: (i) a position corresponding to amino acid position 286 of wild-type feline IgG; (ii) a position corresponding to amino acid position 301 of wild-type feline IgG; (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp or Val; (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Val; (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and (vi) a position corresponding to amino acid position 392 of wild-type feline IgG; wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

8. The polypeptide of claim 7, wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

9. The polypeptide of claim 7 or claim 8, wherein the polypeptide comprises Leu, Tyr or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

10. The polypeptide of any one of claims 7 to 9, wherein the polypeptide comprises Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG.

11. The polypeptide of any one of claims 7 to 10, wherein the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

12. The polypeptide of any one of claims 7 to 11, wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

13. The polypeptide of any one of claims 7 to 12, wherein the polypeptide comprises: (i) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (ii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (iii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (iv) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Tyr at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (v) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (vi) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (vii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (viii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 311 of said wild-type feline IgG; (ix) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (x) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Tyr at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (xi) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG; or (xii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG.

14. A polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (a) a Met at a position corresponding to amino acid position 428 of wild-type feline IgG, and (b) at least one amino acid substitution at a position selected from the group consisting of: (i) a position corresponding to amino acid position 286 of wild-type feline IgG; (ii) a position corresponding to amino acid position 301 of wild-type feline IgG; (iii) a position corresponding to amino acid position 309 of wild-type feline IgG; (iv) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Val; (v) a position corresponding to amino acid position 377 of wild-type feline IgG; and (vi) a position corresponding to amino acid position 392 of wild-type feline IgG; wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

15. The polypeptide of claim 14, wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

16. The polypeptide of claim 14 or claim 15, wherein the polypeptide comprises Leu, Tyr or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

17. The polypeptide of any one of claims 14 to 16, wherein the polypeptide comprises Asp, Glu or Val at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG.

18. The polypeptide of any one of claims 14 to 17, wherein the polypeptide comprises Val at the amino acid position corresponding to amino acid position 311 of the wild-type feline IgG.

19. The polypeptide of any one of claims 14 to 18, wherein the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

20. The polypeptide of any one of claims 14 to 19, wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

21. The polypeptide of any one of claims 14 to 20, wherein the polypeptide comprises: (i) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (ii) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (iii) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (iv) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Tyr at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (v) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (vi) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (vii) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (viii) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (ix) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 311 of said wild-type feline IgG; (x) Met at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (xi) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Tyr at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (xii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG; or (xiii) Met at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG.

22. A polypeptide comprising a feline IgG Fc region variant, wherein the feline IgG Fc region variant comprises (a) Leu at a position corresponding to amino acid position 428 of wild-type feline IgG, and (b) at least one amino acid substitution at a position selected from the group consisting of: (i) a position corresponding to amino acid position 286 of wild-type feline IgG; (ii) a position corresponding to amino acid position 301 of wild-type feline IgG; (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is Asp; (iv) a position corresponding to amino acid position 377 of wild-type feline IgG; and (v) a position corresponding to amino acid position 392 of wild-type feline IgG; wherein amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

23. The polypeptide of claim 22, wherein the polypeptide comprises an Asp at the amino acid position corresponding to amino acid position 286 of the wild-type feline IgG.

24. The polypeptide of claim 22 or claim 23, wherein the polypeptide comprises Leu, Tyr or Val at the amino acid position corresponding to amino acid position 301 of the wild-type feline IgG.

25. The polypeptide of any one of claims 22 to 24, wherein the polypeptide comprises an Asp at the amino acid position corresponding to amino acid position 309 of the wild-type feline IgG.

26. The polypeptide of any one of claims 22 to 25, wherein the polypeptide comprises Leu or Tyr at the amino acid position corresponding to amino acid position 377 of the wild-type feline IgG.

27. The polypeptide of any one of claims 22 to 26, wherein the polypeptide comprises Asp or Glu at the amino acid position corresponding to amino acid position 392 of the wild-type feline IgG.

28. The polypeptide of any one of claims 22 to 27, wherein the polypeptide comprises: (i) Leu at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 286 of said wild-type feline IgG; (ii) Leu at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (iii) Leu at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Tyr at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (iv) Leu at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Val at said amino acid position corresponding to amino acid position 301 of said wild-type feline IgG; (v) Leu at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 309 of said wild-type feline IgG; (vi) Leu at said amino acid position corresponding to amino acid position 428 of said wild-type feline IgG, and Leu at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (vii) Leu at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Tyr at said amino acid position corresponding to amino acid position 377 of said wild-type feline IgG; (viii) Leu at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Asp at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG; or (ix) Leu at said amino acid position corresponding to amino acid position 252 of said wild-type feline IgG, and Glu at said amino acid position corresponding to amino acid position 392 of said wild-type feline IgG.

29. The polypeptide of any one of claims 1 to 28, wherein the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO: 1, a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO: 2, or a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO:

3.

30. The polypeptide of any one of claims 1 to 29, wherein the polypeptide binds to the feline FcRn at a higher level at acidic pH than at neutral pH.

31. The polypeptide of claim 30, wherein the polypeptide binds to the feline FcRn at a higher level at pH 5.5 to pH 6.0 than at pH 7.

4.

32. The polypeptide of any one of claims 1 to 31, further comprising 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.

33. The polypeptide of any one of claims 1 to 32, wherein the polypeptide further comprises a binding domain.

34. The polypeptide of claim 33, wherein the binding domain comprises an antibody, an antibody fragment, or a ligand binding portion of a receptor.

35. The polypeptide of claim 34, wherein the antibody or the antibody fragment comprises six complementarity determining regions (CDRs) of an immunoglobulin molecule.

36. The polypeptide of claim 35, wherein the antibody fragment is selected from the group consisting of: Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, nanobody, and diabody.

37. The polypeptide of claim 35, wherein the ligand binding portion of the receptor comprises a ligand binding domain of a feline receptor protein or an extracellular domain of a feline receptor protein.

38. A polypeptide as described in claim 35, wherein the binding domain specifically binds to an antigen selected from the group consisting of: NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52 and complement system complexes.

39. A pharmaceutical composition comprising (i) the polypeptide of any one of claims 1 to 38, and (ii) a pharmaceutically acceptable excipient.

40. One or more nucleic acids encoding a polypeptide according to any one of claims 1 to 38.

41. One or more expression vectors comprising one or more nucleic acids of claim 40.

42. A host cell comprising one or more nucleic acids of claim 40 or one or more expression vectors of claim 41.

43. A method for preparing a polypeptide, the method comprising: (i) providing one or more nucleic acids according to claim 40; (ii) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; and, optionally, (iii) collecting the polypeptide produced in (ii) from the host cell culture.

44. A method of treating or preventing a feline disease or condition in a cat in need thereof, the method comprising administering to the cat an effective amount of a composition comprising the polypeptide of any one of claims 1 to 38 or the pharmaceutical composition of claim 39.

45. The method of claim 44, wherein the feline disease or condition is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, kidney disease, fertility-related condition, infectious disease, or cancer.

46. ​​The method of claim 44, wherein the feline disease or condition is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

47. A polypeptide according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 36 for use in treating or preventing a feline disease or disorder in a cat in need thereof.

48. A polypeptide or pharmaceutical composition for use as claimed in claim 47, wherein the feline disease or condition is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, kidney disease, fertility-related condition, infectious disease or cancer.

49. The polypeptide or pharmaceutical composition for use according to claim 47, wherein the feline disease or condition is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia or obesity.

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