Mutations in the constant regions of canine antibodies

Abstract

The present invention relates generally to canine antibody variants and their uses. In particular, the present invention relates to mutations in the constant regions of canine antibodies to improve various characteristics.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 142,774, filed January 28, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates generally to canine antibody variants and their uses. In particular, the invention relates to one or more mutations in the Fc constant region of canine antibodies to improve various characteristics. [Background technology]

[0003] Canine IgG monoclonal antibodies (mAbs) have been developed as effective therapeutic agents in veterinary medicine. Several years ago, four canine IgG subclasses were identified and characterized (Bergeron et al., 2014, Vet Immunol Immunopathol., vol. 157(1-2), pages 31-41). However, the extension of the half-life of canine IgG has not been well studied.

[0004] The neonatal Fc receptor (FcRn) extends the half-life of IgG in a pH-dependent interaction with its fragment crystallizable (Fc) region through a recycling mechanism. Specifically, the Fc region, which spans the interface of the CH2 and CH3 domains, interacts with FcRn on the surface of cells to regulate IgG homeostasis. This interaction favors acidic interactions after IgG pinocytosis, thus protecting IgG from degradation. Endocytosed IgG is then recycled to the cell surface and released into the bloodstream at alkaline pH, thereby maintaining sufficient serum IgG for proper function. Thus, the pharmacokinetic profile of IgG depends on the structural and functional properties of their Fc region.

[0005] Three canine IgG subclasses have been shown to bind canine FcRn and compared to human IgG analogs. The half-lives of canine IgGs need to be thoroughly studied, as it is not possible to expect or predict without any experimental support whether they will closely align with human IgG.

[0006] Extending the half-life of IgG can allow for less frequent administration of antibody drugs and / or lower doses, thus reducing veterinary visits, improving patient compliance, and reducing concentration-dependent cytotoxicity / adverse events.

[0007] Thus, there is a need to identify mutations in the Fc constant region to improve half-life. Summary of the Invention

[0008] The present invention relates to mutant canine IgGs that provide higher affinity for FcRn compared to wild-type canine IgG. Specifically, the inventors of the present application have surprisingly and unexpectedly found that substituting one or more amino acid residues improves affinity for FcRn.

[0009] In one aspect, the invention provides a modified IgG comprising a canine IgG constant domain comprising at least one amino acid substitution compared to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 247, 252, 254, 256, 311, 312, 314, 431, 434, or 439, as numbered according to the Eu index in Kabat.

[0010] In some embodiments the constant domain comprises the substitutions P247V, L252Y, L252P, L252W, L252A, L252D, L252G, L252H, L252I, L252K, L252M, L252N, L252Q, L252S, L252T, L252V, L252F, L252R, A 254F, A254G, A254H, A254N, A254Q, A254R, A254W, A254C, A254D, A254I, A254K, A 254L, A254M, A254P, A254S, A254T, A254V, A254Y, T256H, T256I, T256K, T256L, T 256Q, T256Y, T256A, T256C, T256D, T256F, T256G, T256M, T256N, T256P, T256R, T 256S, T256V, T256W, T256E, Q311H, Q311R, Q311Y, Q311W, D312P, L314K, A431K, N 434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, and one or more of E439K.

[0011] In another aspect, the invention provides a polypeptide comprising a canine IgG constant domain comprising at least one amino acid substitution compared to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 247, 252, 254, 256, 311, 312, 314, 431, 434, or 439, as numbered according to the Eu index in Kabat.

[0012] In yet another aspect, the invention provides an antibody comprising a canine IgG constant domain comprising at least one amino acid substitution compared to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 247, 252, 254, 256, 311, 312, 314, 431, 434, or 439, as numbered according to the Eu index in Kabat.

[0013] In a further aspect, the invention provides a method for generating or producing an antibody or molecule, the method comprising providing a vector or host cell comprising an antibody comprising a canine IgG constant domain, the canine IgG constant domain comprising one or more amino acid substitutions compared to a wild-type canine IgG constant domain, the one or more substitutions being at amino acid residue 247, 252, 254, 256, 311, 312, 314, 431, 434, or 439, or a combination thereof.

[0014] In another aspect, the invention provides a fusion molecule comprising a canine IgG constant domain comprising at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 247, 252, 254, 256, 311, 312, 314, 431, 434, or 439, as numbered according to the Eu index in Kabat.

[0015] In another aspect, the invention provides a method for increasing antibody serum half-life in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody comprising a canine IgG constant domain, the canine IgG constant domain comprising at least one amino acid substitution compared to a wild-type canine IgG constant domain, the substitution being at amino acid residue 252, 254, 256, 311, 434, or 439, as numbered according to the EU index in Kabat. In one exemplary embodiment, the canine IgG constant domain comprises one or more of the following mutations: L252F, L252R, L252Y, L252M, A254T, A254S, T256E, Q311W, N434H, N434Y, and E439K. In another exemplary embodiment, the canine IgG constant domain comprises one or more mutations selected from the group of: (1) L252F; (2) L252R, N434H, and Q311W; (3) L252R; (4) Q311W; (5) L252R, A254T, T256E, and N434H; (6) L252Y and A254T; or (7) L252M, A254S, and E439K.

[0016] Other features and advantages of the present invention will become apparent from the following detailed description examples and drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0017] This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] [Figure 1] The domain structure of IgG is shown. [Figure 2A] FIG. 1 shows an alignment of the amino acid sequences of wild-type (WT) human IgG1, WT canine IgGGA, WT canine IgGB, WT canine IgGC, and WT canine IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are colored red, purple, blue, and green, respectively. [Figure 2B] Figure 2 continued. [Figure 2C] Figure 2 continued. [Figure 2D] Figure 2 continued. [Figure 2E] Figure 2 continued. [Figure 2F] Figure 2 continued. [Diagram 3] 1 shows the canine Fc IgGB WT nucleotide sequence. [Figure 4]It has been shown that certain canine IgG point mutations increase half-life in dogs. Mut1 refers to L252F, Mut2 refers to the combination of mutations L252R, N434H, and Q311W, Mut3 refers to L252R, Mut4 refers to the combination of mutations L252Y, N434Y, and Q311W, Mut5 refers to Q311W, Mut6 refers to the combination of mutations L252R, A254T, T256E, and N434H, Mut7 refers to the combination of mutations L252Y and A254T, and Mut8 refers to the combination of mutations L252M, A254S, and E439K.

[0019] Brief Description of the Sequence Listing SEQ ID NO:1 refers to the amino acid sequence of the wild-type canine IgGA constant region.

[0020] SEQ ID NO:2 refers to the amino acid sequence of the wild-type canine IgGB(65) constant region.

[0021] SEQ ID NO:3 refers to the amino acid sequence of the wild-type canine IgGC constant region.

[0022] SEQ ID NO: 4 refers to the amino acid sequence of the wild-type canine IgGD constant region.

[0023] SEQ ID NO:5 refers to the nucleic acid sequence of the wild-type canine IgGB(65) codon optimized, according to one embodiment.

[0024] SEQ ID NO:6 refers to the nucleic acid sequence of the wild-type canine IgGB(65) constant domain, according to one embodiment.

[0025] SEQ ID NO:7 refers to the amino acid sequence of a wild-type canine IgGB CH1 domain.

[0026] SEQ ID NO:8 refers to the amino acid sequence of a wild-type canine IgGB hinge domain.

[0027] SEQ ID NO: 9 refers to the amino acid sequence of a wild-type canine IgGB CH2 domain.

[0028] SEQ ID NO: 10 refers to the amino acid sequence of a wild-type canine IgGB CH3 domain.

[0029] SEQ ID NO:11 refers to the nucleic acid sequence of a wild-type canine IgGB CH1 domain.

[0030] SEQ ID NO: 12 refers to the nucleic acid sequence of a wild-type canine IgGB hinge domain.

[0031] SEQ ID NO: 13 refers to the nucleic acid sequence of a wild-type canine IgGB CH2 domain.

[0032] SEQ ID NO: 14 refers to the nucleic acid sequence of a wild-type canine IgGB CH3 domain.

[0033] SEQ ID NO: 15 refers to the amino acid sequence of the wild-type human IgG1 constant region.

[0034] SEQ ID NO: 16 refers to the nucleic acid sequence of the wild-type canine IgGA constant region.

[0035] SEQ ID NO: 17 refers to the nucleic acid sequence of the wild-type canine IgGC constant region.

[0036] SEQ ID NO: 18 refers to the nucleic acid sequence of the wild-type canine IgGD constant region.

[0037] SEQ ID NO: 19 refers to the nucleic acid sequence of the wild-type human IgG1 constant region.

[0038] SEQ ID NO: 20 refers to the nucleic acid sequence of the variable heavy chain CDR1 of the anti-IL31 antibody designated herein as ZTS-8183.

[0039] SEQ ID NO:21 refers to the amino acid sequence of the variable heavy chain CDR1 of the anti-IL31 antibody designated herein as ZTS-8183.

[0040] SEQ ID NO:22 refers to the nucleic acid sequence of the variable heavy chain CDR2 of the anti-IL31 antibody designated herein as ZTS-8183.

[0041] SEQ ID NO:23 refers to the amino acid sequence of the variable heavy chain CDR2 of the anti-IL31 antibody designated herein as ZTS-8183.

[0042] SEQ ID NO:24 refers to the nucleic acid sequence of the variable heavy chain CDR3 of the anti-IL31 antibody designated herein as ZTS-8183.

[0043] SEQ ID NO:25 refers to the amino acid sequence of the variable heavy chain CDR3 of the anti-IL31 antibody designated herein as ZTS-8183.

[0044] SEQ ID NO:26 refers to the nucleic acid sequence of the variable light chain CDR1 of the anti-IL31 antibody designated herein as ZTS-8183.

[0045] SEQ ID NO:27 refers to the amino acid sequence of the variable light chain CDR1 of the anti-IL31 antibody designated herein as ZTS-8183.

[0046] SEQ ID NO:28 refers to the nucleic acid sequence of the variable light chain CDR2 of the anti-IL31 antibody designated herein as ZTS-8183.

[0047] SEQ ID NO:29 refers to the amino acid sequence of the variable light chain CDR2 of the anti-IL31 antibody designated herein as ZTS-8183.

[0048] SEQ ID NO:30 refers to the nucleic acid sequence of the variable light chain CDR3 of the anti-IL31 antibody designated herein as ZTS-8183.

[0049] SEQ ID NO:31 refers to the amino acid sequence of the variable light chain CDR3 of the anti-IL31 antibody designated herein as ZTS-8183.

[0050] SEQ ID NO: 32 refers to the nucleic acid sequence of the variable light chain of the anti-IL31 antibody designated herein as ZTS-8183.

[0051] SEQ ID NO: 33 refers to the amino acid sequence of the variable light chain of the anti-IL31 antibody designated herein as ZTS-8183.

[0052] SEQ ID NO:34 refers to the nucleic acid sequence of the variable heavy chain of the anti-IL31 antibody designated herein as ZTS-8183.

[0053] SEQ ID NO:35 refers to the amino acid sequence of the variable heavy chain of the anti-IL31 antibody designated herein as ZTS-8183. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054]

[0057] The subject matter of the present invention may be more readily understood by reference to the following detailed description, which forms a part of this disclosure: It is to be understood that the present invention is not limited to the specific products, methods, conditions, or parameters described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to limit the invention as claimed.

[0055]

[0058] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0056]

[0059] As used above, and throughout this disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings:

[0057] definition

[0060] In this disclosure, the singular forms "a," "an," and "the" include plural referents, and reference to a particular numerical value includes at least that particular value unless the context clearly indicates otherwise. Thus, for example, a reference to a "molecule" or "compound" is a reference to one or more of such molecules or compounds and equivalents thereof known to those of skill in the art. The term "plurality," as used herein, means more than one. When a range of values ​​is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that the particular value forms another embodiment by using the antecedent "about." All ranges are inclusive and combinable.

[0058]

[0061] In this specification and claims, the numbering of amino acid residues in immunoglobulin heavy chains is that of the Eu index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "Eu index in Kabat" refers to the residue numbering of an IgG antibody, as reflected herein in Figure 2.

[0059]

[0062] The term "isolated" when used in reference to a nucleic acid is a nucleic acid that is identified and separated from at least one contaminant nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid is in a form or setting different from that in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid molecule present in a natural cell. An isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses an encoded polypeptide, for example, the nucleic acid molecule is in a plasmid or chromosomal location different from that of the natural cell. An isolated nucleic acid can be in single-stranded or double-stranded form. When an isolated nucleic acid molecule is used to express a protein, an oligonucleotide or polynucleotide will minimally contain a sense or coding strand, but may contain both a sense strand and an antisense strand (i.e., may be double-stranded).

[0060]

[0063] A nucleic acid molecule is "operably linked" or "operably associated" when it is placed in a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a coding sequence of a nucleic acid if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence of a nucleic acid if it is positioned to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof that does not include an Fc region as it occurs in nature) when the expressed fusion protein is positioned to include a heterologous protein or functional fragment thereof adjacent either upstream or downstream of the variant Fc region polypeptide, and the heterologous protein may be immediately adjacent to the variant Fc region polypeptide or separated from it by a linker sequence of any length and composition. Similarly, a polypeptide (used interchangeably herein with "protein") molecule is "operably linked" or "operably associated" when it is placed in a functional relationship with another polypeptide.

[0061]

[0064] As used herein, the term "functional fragment," when referring to a polypeptide or protein (e.g., a variant Fc region, or a monoclonal antibody), refers to a fragment of that protein that retains at least one function of the full-length polypeptide. Fragments can range in size from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. A functional fragment of a variant Fc region polypeptide of the invention retains at least one "amino acid substitution" as defined herein. A functional fragment of a variant Fc region polypeptide retains at least one function known in the art to be associated with an Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, downregulation of a cell surface receptor, or may, for example, increase the in vivo or in vitro half-life of a polypeptide to which it is operatively associated).

[0062]

[0065] The term "purified" or "to purify" refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody can be purified by complete or substantial (at least 90%, 91%, 92%, 93%, 94%, 95%, or more preferably at least 96%, 97%, 98%, or 99%) removal of at least one contaminating non-immunoglobulin protein, and also by removal of immunoglobulin proteins that do not bind to the same antigen. Removal of non-immunoglobulin proteins and / or removal of immunoglobulins that do not bind to a particular antigen results in an increase in the percentage of antigen-specific immunoglobulins in a sample. In another example, a polypeptide (e.g., an immunoglobulin) expressed in a bacterial host cell is purified by complete or substantial removal of host cell proteins, thereby increasing the percentage of the polypeptide in a sample.

[0063]

[0066] The term "native" referring to a polypeptide (e.g., an Fc region) is used herein to indicate that the polypeptide has an amino acid sequence that consists of the amino acid sequence of a polypeptide that commonly occurs in nature, or a naturally occurring polymorphism thereof. A native polypeptide (e.g., a native Fc region) can be produced by recombinant means or can be isolated from a natural source.

[0064]

[0067] As used herein, the term "expression vector" refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of an operably linked coding sequence in a particular host organism.

[0065]

[0068] As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro, in situ, or in vivo.

[0066]

[0069] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of an immunoglobulin heavy chain Fc region may vary, a canine IgG heavy chain Fc region is usually defined to extend, for example, from amino acid residue 231 to the carboxyl terminus. In some embodiments, variants include only a portion of the Fc region, which may or may not include the carboxy terminus. An immunoglobulin Fc region generally includes two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants having no such constant domains (or only a portion of such constant domains) are contemplated.

[0067]

[0070] The "CH2 domain" of a canine IgG Fc region typically extends, for example, from about 231 amino acids to about 340 amino acids (see FIG. 2). The CH2 domain is unique in that it is not tightly paired with another domain. Two N-linked branched carbohydrate chains are interposed between the two CH2 domains in an intact native IgG molecule.

[0068]

[0071] The "CH3 domain" of a canine IgG Fc region generally spans residues from the C-terminus of the Fc region through the CH2 domain, e.g., extending from about amino acid residue 341 to about amino acid residue 447 (see Figure 2).

[0069]

[0072] A "functional Fc region" has an "effector function" of a native sequence Fc region. At least one effector function of a polypeptide comprising a variant Fc region of the invention may be enhanced or decreased compared to a polypeptide comprising a native Fc region or the parent Fc region of the variant. Examples of effector functions include, but are not limited to, Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions may require that the Fc region be operably linked to a binding domain (e.g., an antibody variable domain) and may be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, target cell depletion from whole blood samples or fractionated blood samples, and the like).

[0070]

[0073] A "native sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence identical to the amino acid sequence of an Fc region typically found in nature. An exemplary native sequence canine Fc region is shown in Figure 2 and comprises the native sequence of a canine IgG Fc region.

[0071]

[0074] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region (or a fragment thereof) by reason of at least one "amino acid substitution" as defined herein. In preferred embodiments, the variant Fc region has at least one amino acid substitution, preferably 1, 2, 3, 4, or 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, compared to the native sequence Fc region or the Fc region of a parent polypeptide. In alternative embodiments, the variant Fc region may be generated according to the methods disclosed herein, and the variant Fc region may be fused to a selected heterologous polypeptide, such as an antibody variable domain or a non-antibody polypeptide, e.g., a binding domain of a receptor or ligand.

[0072]

[0075] As used herein, the term "derivative" in the context of a polypeptide refers to a polypeptide comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions. As used herein, the term "derivative" also refers to a polypeptide that has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, an antibody may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Derivative polypeptides may be generated by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, a derivative polypeptide has similar or identical functions as the polypeptide from which it was derived. It is understood that a polypeptide comprising a variant Fc region of the invention may be a derivative as defined herein, and preferably, the derivatization occurs within the Fc region.

[0073]

[0076] As used herein with respect to a polypeptide (e.g., an Fc region or a monoclonal antibody), "substantially of canine origin" indicates that the polypeptide has an amino acid sequence that is at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, or even more preferably at least 95%, 96%, 97%, 98%, or 99% homologous to that of a naturally occurring canine amino polypeptide.

[0074]

[0077] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). A preferred FcR is a native sequence FcR. Further preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the Fc gamma RI, Fc gamma RII, Fc gamma RIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. Another preferred FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0075]

[0078] The phrases "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells (e.g., nonspecific) that express FcR (e.g., natural killer ("NK") cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

[0076]

[0079] As used herein, the phrase "effector cells" refers to white blood cells (preferably canines) that express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc gamma RIII and perform ADCC effector functions. Examples of white blood cells that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from a native source (e.g., blood or PBMCs).

[0077]

[0080] A variant polypeptide having an "altered" FcRn binding affinity is one that has either an improved (i.e., increased, greater, or higher) or decreased (i.e., reduced, decreased, or lower) FcRn binding affinity when measured at pH 6.0 compared to the variant's parent polypeptide or a polypeptide comprising a native Fc region. A variant polypeptide that exhibits increased binding or increased binding affinity to FcRn binds to FcRn with a higher affinity than the parent polypeptide. A variant polypeptide that exhibits decreased binding or decreased binding affinity to FcRn binds to FcRn with a lower affinity than its parent polypeptide. Such variants that exhibit decreased binding to FcRn may have little or no binding to FcRn, e.g., 0-20% binding to FcRn compared to the parent polypeptide. A variant polypeptide that binds to FcRn with "improved affinity" compared to its parent polypeptide is one that binds to FcRn with a higher binding affinity than the parent polypeptide, when the amounts of variant and parent polypeptides in a binding assay are essentially the same and all other conditions are the same. For example, a variant polypeptide with improved FcRn-binding affinity may exhibit about a 1.10-fold to about a 100-fold (more typically, about a 1.2-fold to about a 50-fold) increase in FcRn-binding affinity compared to the parent polypeptide, when FcRn-binding affinity is determined, e.g., in an ELISA assay or other method available to one of skill in the art.

[0078]

[0081] As used herein, an "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with another, different, "replacement" amino acid residue. The replacement residue or residues may be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and are selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile): leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of amino acid substitution herein. "Non-naturally occurring amino acid residue" refers to a residue other than the naturally occurring amino acid residues listed above, which is capable of being covalently linked to an adjacent amino acid residue in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991).

[0079]

[0082] The term "assay signal" refers to the output from any method of detecting protein-protein interactions, including but not limited to colorimetric assays, fluorescence intensity, or absorbance measurements from decays per minute. Assay formats can include ELISA, facs, or other methods. A change in the "assay signal" can reflect a change in cell viability and / or a change in kinetic off-rate, kinetic on-rate, or both. A "higher assay signal" refers to a measured output number that is greater than another number (e.g., a variant can have a higher (larger) measured number in an ELISA assay compared to the parent polypeptide). A "lower" assay signal refers to a measured output number that is less than another number (e.g., a variant can have a lower (smaller) measured number in an ELISA assay compared to the parent polypeptide).

[0080]

[0083] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. Binding affinity can be measured by the kinetic off-rate (generally expressed in units of the reciprocal of time, e.g., seconds). -1 The equilibrium dissociation constant is directly related to the ratio of the kinetic on-rate (generally reported in units of concentration per unit time, e.g., moles / second) to the kinetic on-rate (generally reported in units of concentration per unit time, e.g., moles / second). Generally, unless each of these parameters is experimentally determined (e.g., by BIACORE or SAPIDYNE measurements), it is not possible to state definitively whether a change in the equilibrium dissociation constant is due to differences in the on-rate, the off-rate, or both.

[0081]

[0084] As used herein, the term "hinge region" refers to the range of amino acids spanning canine IgG, e.g., from canine IgG positions 216 to 230. Hinge regions of other IgG isotypes can be aligned with the IgG sequence by placing the cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions.

[0082]

[0085] "Clq" is a polypeptide that contains the binding site for the Fc region of immunoglobulin. Clq, together with two serine proteases, Clr and Cls, forms the complex Cl, the first component of the CDC pathway.

[0083]

[0086] As used herein, the term "antibody" is used synonymously with "immunoglobulin" or "Ig" and is used in the broadest sense to specifically cover monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological or functional activity. Single chain antibodies, and chimeric, canine, or caninized antibodies, including portions derived from different species, as well as chimeric or CDR-grafted single chain antibodies, and the like, are also encompassed by the present invention and the term "antibody." The various portions of these antibodies can be synthetically linked together chemically by conventional techniques or prepared as a contiguous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric or caninized chain can be expressed to produce a contiguous protein. See, for example, U.S. Patent No. 4,816,567, U.S. Patent No. 4,816,397, WO86 / 01533, U.S. Patent No. 5,225,539, and U.S. Patent No. 5,585,089, and U.S. Patent No. 5,698,762. See also, for primatized antibodies, Newman, R. et al. BioTechnology, 10:1455-1460, 1993, and for single chain antibodies, Ladner et al., U.S. Patent No. 4,946,778 and Bird, RE et al., Science, 242:423-426, 1988. It is understood that all forms of antibodies that contain an Fc region (or a portion thereof) are encompassed within the term "antibody" herein. Furthermore, the antibodies can be labeled with a detectable label, immobilized on a solid phase, and / or conjugated to a heterologous compound (e.g., an enzyme or a toxin) according to methods known in the art.

[0084]

[0087] As used herein, the term "antibody fragment" refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. Antibody fragments preferably retain at least a portion of the hinge and, optionally, the CH1 region of the IgG heavy chain. In other preferred embodiments, the antibody fragment includes at least a portion of the CH2 region or the entire CH2 region.

[0085]

[0088] As used herein, the term "functional fragment", when used in reference to a monoclonal antibody, is intended to refer to a portion of a monoclonal antibody that still retains functional activity. Functional activity can be, for example, antigen binding activity or specificity, receptor binding activity or specificity, effector function activity, etc. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single chain Fv (scFv); and Fc fragments. Such terms are described, for example, in Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989), Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.), Huston et al., Cell Biophysics, 22:189-224 (1993), Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The term functional fragment is intended to include fragments produced, for example, by protease digestion or reduction of monoclonal antibodies and by recombinant DNA methods known to those skilled in the art.

[0086]

[0089] As used herein, the term "fragment" refers to a polypeptide that contains an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more contiguous amino acid residues of the amino acid sequence of another polypeptide. In preferred embodiments, a fragment of a polypeptide retains at least one function of the full-length polypeptide.

[0087]

[0090] As used herein, the term "chimeric antibody" includes monovalent, divalent, or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through a disulfide bridge with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy-light chain dimers associated through at least one disulfide bridge. A chimeric heavy chain of an antibody for use in a canine comprises an antigen-binding region derived from a heavy chain of a non-canine antibody linked to at least a portion of a canine heavy chain constant region, such as CH1 or CH2. A chimeric light chain of an antibody for use in a canine comprises an antigen-binding region derived from a light chain of a non-canine antibody linked to at least a portion of a canine light chain constant region (CL). Antibodies, fragments, or derivatives with chimeric heavy and light chains of the same or different variable region binding specificities can also be prepared by appropriate association of the individual polypeptide chains according to known method steps. Using this approach, a host expressing a chimeric light chain and a host expressing a chimeric heavy chain are cultured separately, and the immunoglobulin chains are harvested separately and then assembled. Alternatively, the hosts can be co-cultured, the chains spontaneously assembled in the culture medium, and the assembled immunoglobulin or fragments subsequently harvested, or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, for example, U.S. Patent Nos. 6,284,471, 5,807,715, 4,816,567, and 4,816,397).

[0088]

[0091] As used herein, a "caninized" form of a non-canine (e.g., murine) antibody (i.e., a caninized antibody) is an antibody that contains minimal or no sequences derived from non-canine immunoglobulin. In most cases, caninized antibodies are canine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-canine species (donor antibody) such as mouse, rat, rabbit, human, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, caninized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further improve antibody performance. Generally, a caninized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-canine immunoglobulin and all or substantially all of the FR residues are those of a canine immunoglobulin sequence. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin.

[0089]

[0092] As used herein, the term "immunoadhesin" denotes an antibody-like molecule which combines the binding domain of a heterologous "adhesin" protein (e.g., receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of an adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (antigen binding site) (i.e., is "heterologous"), with an immunoglobulin constant domain sequence.

[0090]

[0093] As used herein, the term "ligand binding domain" refers to any naturally occurring receptor, or any region or derivative thereof, that retains at least a qualitative ligand binding ability of the corresponding naturally occurring receptor. In certain embodiments, the receptor is derived from a cell surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergene family. Other receptors that are not members of the immunoglobulin supergene family but are nevertheless specifically covered by this definition are receptors for cytokines, in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L-, and P-selectin).

[0091]

[0094] As used herein, the term "receptor binding domain" refers to any naturally occurring ligand of a receptor, including, for example, a cell adhesion molecule, or any region or derivative of such a naturally occurring ligand that retains at least a qualitative receptor binding ability of the corresponding naturally occurring ligand.

[0092]

[0095] As used herein, an "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the polypeptide, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, an isolated polypeptide is purified (1) to greater than 95% by weight, preferably greater than 99% by weight, of the polypeptide as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a rolling cup sequenator, or (3) to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated polypeptide includes the polypeptide in situ in a recombinant cell, since at least one component of the polypeptide's natural environment is absent. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0093]

[0096] As used herein, the terms "disorder" and "disease" are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder).

[0094]

[0097] As used herein, the term "receptor" refers to a polypeptide capable of binding at least one ligand. Preferred receptors are cell surface or soluble receptors having an extracellular ligand-binding domain and, optionally, other domains (e.g., a transmembrane domain, an intracellular domain, and / or a membrane anchor). The receptors evaluated in the assays described herein can be intact receptors, or fragments or derivatives thereof (e.g., fusion proteins comprising the binding domain of the receptor fused to one or more heterologous polypeptides). Furthermore, the receptors evaluated for their binding properties can be present intracellularly or isolated, and can optionally be coated onto an assay plate or some other solid phase, or can be directly labeled and used as probes.

[0095] Canine wild-type IgG

[0098] Canine IgG is well known in the art and is fully described, for example, in Bergeron et al., 2014, Vet Immunol Immunopathol., vol. 157(1-2), pages 31-41. In one embodiment, the canine IgG is A In another embodiment, the canine IgG is IgG B In yet another embodiment, the canine IgG is IgG C In a further embodiment, the canine IgG is D In certain embodiments, the canine IgG is B It is.

[0096]

[0099] IgG A , IgG B , IgG C , and IgG D The amino acid and nucleic acid sequences of 、 It is well known in the art.

[0097] [000100] In one example, an IgG of the invention comprises a constant domain, e.g., a CH1, CH2, or CH3 domain, or a combination thereof. In another example, a constant domain of the invention comprises an Fc region, e.g., a CH2 or CH3 domain, or a combination thereof.

[0098] [000101] In particular examples, the wild-type constant domain comprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4. In some embodiments, the wild-type IgG constant domain is a homolog, variant, isomer, or functional fragment of SEQ ID NO: 1, 2, 3, or 4, but does not have any of the mutations described herein. Each possibility represents a separate embodiment of the present invention.

[0099] [000102] IgG constant domains also include polypeptides having an amino acid sequence substantially similar to that of the heavy and / or light chain, where substantially the same amino acid sequence is defined herein as a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the compared amino acid sequence, as determined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988).

[0100] [000103] The present invention also includes nucleic acid molecules encoding IgG or portions thereof as described herein. In one embodiment, the nucleic acid may encode an antibody heavy chain, for example, including a CH1, CH2, CH3 region, or a combination thereof. In another embodiment, the nucleic acid may encode an antibody heavy chain, for example, including any one of the VH region or portions thereof, or any one of the VH CDRs, including any variants thereof. The present invention also includes nucleic acid molecules encoding an antibody light chain, for example, including any one of the CL region or portions thereof, any one of the VL region or portions thereof, or any one of the VL CDRs, including any variants thereof. In certain embodiments, the nucleic acid encodes both the heavy and light chains, or portions thereof.

[0101] [000104] The amino acid sequence of the wild-type constant domain set forth in SEQ ID NO: 1, 2, 3, or 4 is encoded by its corresponding nucleic acid sequence. For example, the amino acid sequence of the wild-type constant domain set forth in SEQ ID NO: 2 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 6. In some embodiments, the amino acid sequence of the wild-type constant domain set forth in SEQ ID NO: 1, 2, 3, or 4 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 16, 5, 17, or 18, respectively.

[0102] Modified Canine IgG [000105] The inventors of the present application have surprisingly and unexpectedly found that substituting one or more amino acid residues improves affinity to FcRn. As used herein, amino acid position numbers refer to positions numbered according to the Eu index in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0103] [000106] Thus, in one embodiment, the invention provides a modified IgG comprising a canine IgG constant domain comprising at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 247, 252, 254, 256, 311, 312, 314, 431, 434, or 439, numbered according to the Eu index in Kabat.

[0104] [000107] In some embodiments, the constant domain is selected from the group consisting of the substitutions P247V, L252Y, L252P, L252W, L252A, L252D, L252G, L252H, L252I, L252K, L252M, L252N, L252Q, L252S, L252T, L252V, L252F ... 252R, A254F, A254G, A254H, A254N, A254Q, A254R, A254W, A254C, A254D, A254I, A2 54K, A254L, A254M, A254P, A254S, A254T, A254V, A254Y, T256H, T256I, T256K, T25 6L, T256Q, T256Y, T256A, T256C, T256D, T256F, T256G, T256M, T256N, T256P, T256 R, T256S, T256V, T256W, T256E, Q311H, Q311R, Q311Y, Q311W, D312P, L314K, A431K , N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, and E439K.

[0105] [000108] In certain examples, the invention comprises one or more mutations as described herein in the wild-type amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4. In some embodiments, the mutant IgG constant domain is a homolog, variant, isomer, or functional fragment having one or more mutations as described herein. Each possibility represents a separate embodiment of the invention.

[0106] [000109] The amino acid sequence of a variant constant domain is encoded by its corresponding variant nucleic acid sequence.

[0107] Methods for producing the antibody molecules of the invention [000110] Methods for producing antibody molecules are well known in the art and are fully described in U.S. Patent Nos. 8,394,925, 8,088,376, 8,546,543, 10,336,818, and 9,803,023, and U.S. Patent Application Publication No. 2006 / 0067930, which are incorporated herein by reference in their entirety. Any suitable method, process, or technique known to those skilled in the art can be used. The antibody molecules with variant Fc regions of the present invention can be produced according to methods well known in the art. In some embodiments, the variant Fc region can be fused to a selected heterologous polypeptide, such as an antibody variable domain or binding domain of a receptor or ligand.

[0108] [000111] With the advent of molecular biology methods and recombinant technology, one skilled in the art can produce antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences that code for the specific amino acid sequences found in the polypeptide structure of an antibody. Such antibodies can be produced either by cloning the gene sequences that code for the polypeptide chains of the antibody, or by directly synthesizing the polypeptide chains and assembling the synthesized chains to form active tetrameric (H2L2) structures that have affinity for specific epitopes and antigenic determinants. This has allowed the ready generation of antibodies with sequences that are characterized by neutralizing antibodies from different species and sources.

[0109] [000112] Regardless of the source of the antibodies or how they are recombinantly constructed or synthesized, in vitro or in vivo, using transgenic animals, laboratory or commercial sized large cell cultures, using transgenic plants, or by direct chemical synthesis without the use of living organisms at any stage of the process, all antibodies have a similar overall three-dimensional structure. This structure is often provided as H2L2, referring to the fact that antibodies generally contain two light (L) chain amino acids and two heavy (H) chain amino acids. Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting regions are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence from antibodies of different antigen specificities. The variable region of either the H or L chain contains amino acid sequences capable of specifically binding to antigen targets.

[0110] [000113] As used herein, the term "antigen-binding region" refers to a portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer to the antibody its specificity and affinity for the antigen. The antibody-binding region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the H or L chains that provide the antigen-binding region, there are smaller sequences called "hypervariable" that are the reason for the extreme variability between antibodies of different specificities. Such hypervariable regions are also called "complementarity determining regions" or "CDR regions". These CDR regions are responsible for the basic specificity of the antibody for a particular antigenic determinant structure.

[0111] [000114] Although CDRs represent non-contiguous stretches of amino acids within the variable region, it has been found that, regardless of species, the positions where these important amino acid sequences are located within the variable heavy and light chain regions have similar positions within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e. highly conserved) regions and variable regions, within the latter of which there are CDRs and so-called "framework regions", which are composed of amino acid sequences within the variable region of the heavy or light chain, but outside the CDRs.

[0112] [000115] The present invention further provides a vector comprising at least one of the above-mentioned nucleic acids. Since the genetic code is degenerate, more than one codon may be used to code for a particular amino acid. Using the genetic code, one or more different nucleotide sequences may be identified, each of which may be capable of coding for the amino acid. The probability that a particular oligonucleotide will in fact constitute an actual coding sequence can be estimated by considering unusual base pairing relationships and the frequency with which a particular codon is actually used (to code for a particular amino acid) in the eukaryotic or prokaryotic cell expressing the antibody or moiety. Such "codon usage rules" are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe's "codon usage rules," a single nucleotide sequence or set of nucleotide sequences containing a theoretical "most likely" nucleotide sequence capable of coding for a canine IgG sequence can also be identified. It is also contemplated that antibody coding regions for use in the present invention may also be provided by modifying existing antibody genes using standard molecular biology techniques to generate variants of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequences of the antibody or peptide.

[0113] [000116] For example, one class of substitutions is conservative amino acid substitutions. Such substitutions substitute a given amino acid in a canine antibody peptide with another amino acid with similar characteristics. Conservative substitutions are typically found in the substitution of aliphatic amino acids Ala, Val, Leu, and lie for each other; exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, exchange of amide residues Asn and Gin, exchange of basic residues Lys and Arg, exchange of aromatic residues Phe and Tyr, etc. Guidance on which amino acid changes are likely to be phenotypically silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0114] [000117] A variant canine antibody or peptide may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative mutations or mutations in non-critical residues or non-critical regions. Functional variants may also contain substitutions of similar amino acids that do not change or only slightly change function. Alternatively, such substitutions may have a positive or negative effect to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions in critical residues or critical regions.

[0115] [000118] Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites important for ligand-receptor binding can also be determined by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904 (1992), de Vos et al., 255 Science 306-12 (1992).

[0116] [000119] In addition, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Moreover, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques which are well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill in the art and are well described in the scientific literature. Some of the most common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in most basic textbooks, such as Proteins-Structure and Molecular Properties (2nd ed., TECreighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews are available on this subject, such as by Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. 182 Meth. Enzymol. 626-46 (1990), and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0117] [000120] In another aspect, the present invention provides antibody derivatives. An antibody "derivative" contains additional chemical moieties that are not normally part of the protein. Covalent modifications of proteins are included within the scope of the present invention. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to water-insoluble support matrices or other macromolecular carriers.

[0118] [000121] Derivatives also include radiolabeled monoclonal antibodies that are labeled with, for example, radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, tritium (H 3 ); conjugates of monoclonal antibodies containing biotin or avidin with enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters), or fluorescent agents (such as phycobyl proteins) are used.

[0119] [000122] Another derivative bifunctional antibody of the invention is a bispecific antibody, generated by combining portions of two separate antibodies that recognize two different antigenic groups. This can be accomplished by crosslinking or recombinant techniques. In addition, moieties can be added to the antibody or portions thereof to increase half-life in vivo (e.g., by extending the time to clearance from the bloodstream). Such techniques include, for example, the addition of PEG moieties (also called pegylation), and are well known in the art. See U.S. Patent Application Publication No. 2003 / 0031671.

[0120] [000123] In some embodiments, nucleic acids encoding the subject antibodies are directly introduced into host cells and the cells are incubated under conditions sufficient to induce expression of the encoded antibodies. After the subject nucleic acids are introduced into the cells, the cells are typically incubated for a period of about 1-24 hours, usually at 37°C, sometimes under selection, to allow expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the medium in which the cells are growing. Traditionally, monoclonal antibodies are produced as native molecules in mouse hybridoma lines. In addition to that technology, the present invention provides recombinant DNA expression of antibodies. This allows for the production of antibodies in a selected host species, as well as the production of a spectrum of antibody derivatives and fusion proteins.

[0121] [000124] A nucleic acid sequence encoding at least one antibody, portion, or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques, including restriction enzyme digestion to provide blunt or sticky ends for ligation, suitable ends, appropriate filling of sticky ends, alkaline phosphatase treatment to avoid undesired ligation, and ligation with a suitable ligase. Techniques for such manipulations are disclosed, for example, by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 (supra), and can be used to construct nucleic acid sequences encoding antibody molecules or antigen-binding regions thereof.

[0122] [000125] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide when it contains a nucleotide sequence containing transcriptional and translational regulatory information and such a sequence is "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the DNA sequence to be expressed are connected in such a way as to permit gene expression as a peptide or antibody moiety in recoverable amounts. The exact nature of the regulatory regions necessary for gene expression may vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 2001 (supra); Ausubel et al., 1993 (supra).

[0123] [000126] Thus, the present invention encompasses the expression of antibodies or peptides in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacterial, yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or in host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin. Any other suitable mammalian cells known in the art can also be used.

[0124] [000127] In one embodiment, the nucleotide sequence of the present invention will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993 (supra). Important factors in choosing a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the number of copies of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0125] [000128] Examples of prokaryotic vectors known in the art include plasmids such as those capable of replicating in E. coli (e.g., pBR322, CoIE1, pSC101, pACYC184, .pi.vX, etc.). Such plasmids are disclosed, for example, by Maniatis et al., 1989 (supra); Ausubel et al., 1993 (supra). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed in Gryczan, THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include Streptomyces bacteriophages such as p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987) and phLC31 (Chater et al., SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986), Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978), and Ausubel et al., 1993 (supra).

[0126] [000129] Alternatively, gene expression elements useful for expressing antibody or peptide encoding cDNA include, but are not limited to, (a) the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites, such as those derived from the SV40 late region (Okayarea et al., 1983); and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983).

[0127] [000130] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987) using the SV40 early promoter and enhancer, mouse immunoglobulin heavy chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements as expression elements. For immunoglobulin genes composed of cDNA portions, genomic DNA portions (Whittle et al., 1 Protein Engin. 499 (1987)), the transcription promoter can be human cytomegalovirus, the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be native chromosomal immunoglobulin sequences.

[0128] [000131] In one embodiment, for expression of cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron of more than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements listed above to achieve expression of the proteins in mammalian cells.

[0129] [000132] Each fusion gene can be assembled or inserted into an expression vector. Recipient cells capable of expressing immunoglobulin chain gene products are then transfected with the peptide or H or L chain coding genes alone or co-transfected with H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the incorporated genes, and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.

[0130] [000133] In one embodiment, the fusion genes encoding the peptide or H and L chains, or portions thereof, are assembled into separate expression vectors that are then used to co-transfect recipient cells. Alternatively, the fusion genes encoding the H and L chains can be assembled on the same expression vector. The recipient cell line for transfection of the expression vector and production of the antibody can be a myeloma cell. The myeloma cell can synthesize, assemble, and secrete the immunoglobulins encoded by the transfected immunoglobulin genes, and has the machinery for immunoglobulin glycosylation. The myeloma cell can be grown in culture or in the peritoneal cavity of a mouse, where the secreted immunoglobulin can be obtained from ascites. Other suitable recipient cells include lymphocytes, such as B lymphocytes of canine or non-canine origin, hybridoma cells of canine or non-canine origin, or interspecies heterohybridoma cells.

[0131] [000134] Expression vectors carrying the antibody constructs or polypeptides of the present invention can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[0132] [000135] Yeast may offer substantial advantages over bacteria in the production of immunoglobulin heavy and light chains. Yeast carries out post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies now exist that utilize strong promoter sequences and high copy number plasmids that can be used for the production of desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0133] [000136] Yeast gene expression systems can be routinely evaluated for levels of production, secretion, and stability of peptides, antibodies, fragments, and regions thereof. Any of a range of yeast gene expression systems incorporating promoter and terminator elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium can be utilized. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Several approaches can be selected to evaluate the optimal expression plasmid for expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985).

[0134] [000137] Bacterial strains can also be utilized as hosts for the production of antibody molecules or peptides described by the present invention. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vector carries a replication site as well as specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be selected to evaluate expression plasmids for the production of antibodies, fragments, and regions, or antibody chains, encoded by cloned immunoglobulin cDNAs in bacteria (Glover, 1985 (supra); Ausubel, 1993 (supra); Sambrook, 2001 (supra); Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0135] [000138] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, folding and assembly of hand L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. Mammalian cells that may be useful as hosts for the production of antibody proteins include cells of fibroblast origin, such as Vero (ATCC CRL81) or CHO-K1 (ATCC CRL61) cells, in addition to the cells of lymphoid origin mentioned above. Many vector systems are available for the expression of cloned peptide hand L chain genes in mammalian cells (see Glover, 1985, supra). Different approaches can be followed to obtain complete H2L2 antibodies. It is possible to co-express hand L chains in the same cell to achieve intracellular association and linkage of hand L chains to complete tetrameric H2L2 antibodies and / or peptides. Co-expression can be achieved by using either the same or different plasmids in the same host. Genes for both hand L chain and / or peptide can be placed on the same plasmid, which can then be transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the L chain, and then the resulting cell line can be transfected with a H chain plasmid containing a second selectable marker. To generate cell lines with improved properties, such as higher production of assembled H2L2 antibody molecules or improved stability of transfected cell lines, cell lines that produce peptide and / or H2L2 molecules via either route can be transfected with plasmids encoding additional copies of peptide, H, L, or H plus L chains, along with additional selectable markers.

[0136] [000139] For long-term, high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines can be engineered that stably express antibody molecules. Rather than using expression vectors that contain a viral origin of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. Following introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful in screening and evaluation of compounds / components that interact directly or indirectly with the antibody molecule.

[0137] [000140] Once an antibody of the invention is produced, it may be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. In many embodiments, the antibody is secreted from the cells into the culture medium and harvested from the culture medium.

[0138] Pharmaceutical and veterinary uses [000141] The present invention also provides pharmaceutical compositions comprising a molecule of the invention and one or more pharma- ceutically acceptable carriers. More specifically, the present invention provides pharmaceutical compositions comprising a pharma- ceutically acceptable carrier or diluent and, as an active ingredient, an antibody or peptide according to the invention.

[0139] [000142] A "pharmaceutical acceptable carrier" includes any excipient that is nontoxic to cells or animals exposed at the dosages and concentrations employed. The pharmaceutical composition may include one or additional therapeutic agents.

[0140] [000143] "Pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissue of an animal without undue toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit / risk ratio.

[0141] [000144] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents, and absorption delaying agents.

[0142] [000145] Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphates, citrates, and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; and other carbohydrates including monosaccharides, disaccharides, and glucose, mannose, or dextrins; EDTA; salt forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®; isotonicity agents such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride; and combinations thereof.

[0143] [000146] The pharmaceutical compositions of the present invention may be formulated in a variety of ways, including, for example, liquid, semi-solid, or solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the compositions are in the form of injectable or infusible solutions. The compositions may be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The compositions may be formulated as immediate release, controlled release, sustained release, or delayed release compositions.

[0144] [000147] The compositions of the invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein may be performed by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of the antibody (typically performed in a pharmaceutical formulation) to airway surfaces. Topical administration to airway surfaces may be performed by intranasal administration (e.g., by use of a dropper, swab, or inhaler). Topical administration of the antibody to airway surfaces may also be performed by inhalation administration, such as by making respirable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibody as an aerosol suspension and then having the subject inhale the respirable particles. Methods and devices for administering respirable particles of a pharmaceutical formulation are well known, and any conventional technique may be used.

[0145] [000148] In some desired embodiments, the antibody is administered by parenteral injection. For parenteral administration, the antibody or molecule may be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharma- ceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, and the like. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting agents and buffers, toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the antibody in these formulations can vary widely, for example, less than about 0.5% by weight, usually from about 1% or more up to 15% or 20% by weight, and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular form of administration selected. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA.SCI. (15th ed., Mack Pub.Co., Easton, Pa., 1980).

[0146] [000149] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution technique can be used. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of antibody activity loss, and that usage levels may need to be adjusted to compensate. Compositions containing the present antibodies or a cocktail thereof can be administered for the prevention of recurrence and / or therapeutic treatment of existing disease. Suitable pharmaceutical carriers are described in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference textbook in the art. In therapeutic applications, the compositions are administered to subjects already suffering from a disease in an amount sufficient to cure or at least partially arrest or alleviate the disease and its complications.

[0147] [000150] Effective doses of the compositions of the invention for the treatment of the conditions or diseases described herein will vary depending on many different factors, including, for example and without limitation, the pharmacodynamic characteristics of the particular agent and its form and route of administration; the target site; the physiological condition of the animal; other medicinal agents being administered; whether the treatment is prophylactic or therapeutic; the age, health, and weight of the recipient; the nature and extent of the type of symptoms; types of concurrent treatments, frequency of treatments, and the desired effect.

[0148] [000151] Single or multiple administrations of the compositions can be carried out with dose levels and pattern selected by the treating veterinarian. In any event, the pharmaceutical formulation should provide a sufficient quantity of the antibody of the invention to effectively treat the subject.

[0149] [000152] Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0150] [000153] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount." A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary depending on factors such as the disease state, age, sex, and weight in an individual, and the ability of the molecule to elicit a desired response in an individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

[0151] [000154] In another aspect, the compositions of the present invention may be used to treat various diseases and disorders, for example, in dogs. As used herein, the terms "treat" and "treatment" refer to therapeutic procedures, including prophylactic or preventative measures, where the subject is one in which undesired physiological changes associated with a disease or condition are prevented or slowed down (alleviated). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of a disease or condition, stabilization of a disease or condition (i.e., the disease or condition does not get worse), delay or slowing of the progression of a disease or condition, improvement or alleviation of a disease or condition, and remission (partial or complete) of a disease or condition. Those in need of treatment include those already with the disease or condition, and those prone to having the disease or condition, or those in which the disease or condition is to be prevented.

[0152] [000155] All patent and literature references cited herein are incorporated by reference in their entirety.

[0153] [000156] The following examples are provided to supplement the preceding disclosure and provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the subject matter described herein. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in view thereof are obvious to those skilled in the art and fall within the true scope of the present invention and can be made without departing from the true scope of the present invention. EXAMPLES

[0154] Example 1 Construction of canine IgG Fc variants [000157] Construction of all canine IgGs (Figure 1) was performed as described by Bergeron et al. (Bergeron et al., 2014, Vet Immunol Immunopathol., vol. 157(1-2), pages 31-41), utilizing plasmids containing sequences encoding canine constant regions of the IgGB(65) subclass, and inserting the VH / VL sequences of each mAb investigated herein upstream and in frame with nucleotides encoding the constant domain. Mutations were incorporated at each respective position in the CH1, CH2, or CH3 domains (Figure 2) of each plasmid by direct DNA synthesis of the constant regions as gene fragments, which were then subcloned into each variable region of interest.

[0155] Expression and purification [000158] Monoclonal antibody (mAbs) variants were expressed in EXPICHO-S (Chinese Hamster Ovary) cells, a mammalian suspension cell line obtained from Thermo Fisher. Suspension EXPICHO-S cells were maintained at 0.14-8.0 x 10e6 cells / ml in EXPICHO Expression Medium (Gibco). Cells were diluted according to the ExpiCHO protocol user manual on day -1 and on the day of transfection. Diluted cells were transfected as described in the protocol using reagents supplied by the ExpiFectamine CHO Transfection Kit (Gibco) according to Max titer conditions. After 12-14 days of incubation, cultures were harvested and clarified. Antibodies were purified from the clarified supernatant via Protein A chromatography by a MabSelect Sure LX (GE Healthcare) pre-equilibrated with PBS. After sample loading, the resin was washed with PBS and then with 20 mM sodium acetate, pH 5.5. Samples were eluted from the column with 20 mM acetic acid, pH 3.5. After elution, pools were made and neutralized by adding 1 M sodium acetate to 4%. Samples were exchanged into final buffer (e.g., PBS, etc.) at intervals depending on available volume and intended use. Concentration was measured by absorbance at 280 nm.

[0156] SDS-PAGE [000159] Non-reduced (nr) and reduced sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) were performed using 4-12% Bis-Tris NuPAGE gels in MES-SDS running buffer and SeeBlue Plus 2 standards (all from Invitrogen). For non-reduced samples, 1 mM of the alkylating agent N-ethylmaleimide (NEM) was added, and for reduced samples, the reducing agent dithiothreitol (DTT) was added. Gels were stained with Coomassie blue to detect protein bands.

[0157] analysis SEC [000160] Analytical SEC was performed using a TSK gel SuperSW3000, 4.6 mm, 10 x 30 cm, 4 μm column from TOSOH BioScience at 0.25 ml / min in 200 mM sodium phosphate pH 7.2 running buffer.

[0158] NR-CGE [000161] Non-reducing capillary gel electrophoresis (nrCGE) was performed using a Beckman Coulter PA800 plus analyzer using A55625 capillary cartridges according to the manufacturer's instructions.

[0159] SMAC [000162] Stand-up monolayer adsorption chromatography (SMAC) was performed using a Sepax Zenix SEC-300, 4.6 x 300 mm column in 200 mM sodium phosphate pH 7.2 running buffer at 0.35 ml / min.

[0160] HIC [000163] Hydrophobic interaction chromatography (HIC) was performed using a Sepax Proteomix HIC Butyl-NP5, 4.6 x 100 mm column. A linear gradient from 100% 1.8 M ammonium sulfate in 0.1 M sodium phosphate pH 6.5 to 100% 0.1 M sodium phosphate pH 6.5 was applied at 0.75 ml / min for 20 min.

[0161] Octet-BLI [000164] The assay was performed using Forte Bio's Octet QKe with an amine-reactive second generation biosensor. The sample is exchanged into 1x Gibco PBS without calcium and magnesium and diluted to a concentration of 0.5mg / mL. After establishing a biosensor baseline, the biosensor is immersed in 100uL of sample for 600 seconds.

[0162] [000165] Antibodies were screened for binding to Protein A or Protein G sensors via Octet QKe quantification (Pall ForteBio Corp, Menlo Park, CA, USA). Protein A-bound constructs were purified and protein quality quantified as described by Bergeron et al.

[0163] Example 2 FcRn binding assay [000166] Canine FcRn was isolated and prepared according to Bergeron et al. discussed above, and mutant Fc IgG was assayed against canine FcRn. Standard PCR was used to amplify canine FcRn-α subunit and β-microglobulin. FcRn-α subunit and β-microglobulin were co-transfected into HEK293 cells, and FcRn complexes were purified by IMAC affinity purification via the c-terminal His tag. FcRn complexes were biotinylated through a BirA enzyme biotinylation reaction. KD's were measured by Biacore 3000 or Biacore T200 (GE Healthcare, Pittsburgh, PA, USA) using SA sensor chips.

[0164] [000167] FcRn was captured on the sensor surface using a modified SA capture method. The following capture running buffer and titration method was used: 10 mM MES, 150 mM NaCl, 0.005% Tween® 20, 0.5 mg / mL BSA, pH 6. The running buffer and titration were 1×HBS-P, 0.5 mg / mL BSA, pH 7.4. Fc variant IgG was flowed over the receptor surface and affinity was determined using Scrubber2 software analysis (BioLogic Software Pty, Ltd., Campbell, Australia) or T200 evaluation software (Table 1). A blank experiment containing only buffer was subtracted from all experiments. The flow cells were regenerated using 50 mM Tris pH 8. Experiments were performed at 15° C.

[0165] [000168] The mutations made at each position have a significant effect on the affinity of IgG for FcRn at pH 6. The increase in FcRn affinity for IgG is not dependent on the VHVL domain and is universal for all canine IgGs.

[0166] [000169] Binding of wild-type (WT) and mutant IgG to canine FcRn was measured by surface plasmon resonance (Biacore). Biophysical characterization was also performed.

[0167] [000170] As shown in Table 2, data on biophysical characterization indicate that multiple mutations exhibited improved biophysical properties, particularly with respect to polyreactivity. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0168] [000171] The results clearly show that mutations made at various positions have profound effects on the affinity of IgG for FcRn.

[0169] Example 3 Fc variant IgG PK study in dogs [000172] Pharmacokinetic (PK) studies were performed to demonstrate the impact of extending the half-life of various canine IgG point mutations: (1) L252F, (2) L252R, N434H, Q311W, (3) L252R, (4) L252Y, N434Y, Q311W, (5) Q311W, (6) L252R, A254T, T256E, N434H, (7) L252Y, A254T, and (8) L252M, A254S, E439K.

[0170] [000173] Eight Fc-modified caninized monoclonal antibodies listed in Table 5 were used in the experiment, as well as wild-type caninized monoclonal antibodies. The eight Fc-modified caninized monoclonal antibodies were administered subcutaneously at a single 1 mg / kg dose to approximately 4-year-old beagle dogs (n=2). Serum concentrations of these molecules were analyzed using ligand binding methods.

[0171] [000174] The pharmacokinetic properties of eight Fc-modified canine monoclonal antibodies were evaluated in dogs using a non-compartmental approach (linear trapezoidal rule for AUC calculation) with Watson™. Additional calculations, including correction of AUC for overlap of concentration-time profiles after the second and third injection of drug, were performed in Excel™. Concentration-time and pharmacokinetic data summaries, including simple statistics (mean, standard deviation, coefficient of variation), were calculated using Excel™ or Watson™. No other statistical analyses were performed.

[0172] [000175] The results are summarized in Table 5 below. [Table 5]

[0173] [000176] The results, as shown in the table above, indicate that certain modifications of the Fc region have a significant effect on their half-life times. Specifically, the canine IgG point mutations (1) L252F, (2) L252R, N434H, Q311W, (3) L252R, (5) Q311W, (6) L252R, A254T, T256E, N434H, (7) L252Y, A254T, and (8) L252M, A254S, E439K are effective in enhancing half-life compared to wild type.

[0174] [000177] The half-life (T1 / 2) of wild-type mAb ZTS-8183 was approximately 11 days. However, the half-lives (T1 / 2) of the Fc-modified canine monoclonal antibodies ZTS-00530712, ZTS-00530713, ZTS-00530714, ZTS-00530716, ZTS-00530717, ZTS-00530718, and ZTS-00530719 were 23.0, 4.23, 13.9, 22.2, 31.4, 27.8, and 25.3, respectively.

[0175] [000178] As shown in Table 5 above and Figure 4, the half-life (T1 / 2) of the Fc-modified canine monoclonal antibody, ZTS-00530715 (i.e., the combination of mutations L252Y, N434Y, and Q311W) is not significant.

[0176] [000179] In summary, the results clearly show that canine IgG point mutations (1) L252F, (2) L252R, N434H, Q311W, (3) L252R, (5) Q311W, (6) L252R, A254T, T256E, N434H, (7) L252Y, A254T, and (8) L252M, A254S, E439K are highly effective in enhancing half-life in dogs.

[0177] [000180] Although preferred embodiments of the present invention have been described, it should be understood that the present invention is not limited to the precise embodiments, and various changes and modifications may be made by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

Claims

1. 1. A polypeptide comprising a canine IgG constant domain, comprising at least one amino acid substitution compared to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 252, 254, 256, 311, 312, 314, 431, 434, 439, or 247, as numbered according to the EU index in Kabat.

2. The constant domain is L252Y, L252P, L252W, L252A, L252D, L252G, L252H, L252I, L252K, L252M, L252N, L252Q, L252S, L252T, L252V, L252F, L252R, A254F, A254G, A254H, A254I, A254K, A254M, A254N, A254Q, A254S, A254T, A254V, A254F, A254G, A254H, A254I, A254K, A254R ...R, A254R, A254R, A254R, A254R, A254R, A254R, A254R, A25 4N, A254Q, A254R, A254W, A254C, A254D, A254I, A254K, A254L, A254M, A254P, A254 S, A254T, A254V, A254Y, T256H, T256I, T256K, T256L, T256Q, T256Y, T256A, T256C , T256D, T256F, T256G, T256M, T256N, T256P, T256R, T256S, T256V, T256W, T256E, Q311H, Q311R, Q311Y, Q311W, D312P, L314K, A431K, N434A, N434C, N434D, N434E, N 2. The polypeptide of claim 1, comprising one or more of the following substitutions: 434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, E439K, and P247V.

3. 2. The polypeptide of claim 1, wherein the polypeptide has a higher affinity for FcRn than an IgG polypeptide having the wild-type canine IgG constant domain.

4. The polypeptide of claim 1 , wherein the polypeptide is a canine or caninized IgG polypeptide.

5. The polypeptide of claim 1 , wherein the IgG constant domain comprises an Fc constant region having a CH2 domain, a CH3 domain, or a combination thereof.

6. 2. The polypeptide of claim 1, wherein the wild-type canine IgG constant domain comprises the amino acid sequence set forth in SEQ ID NO:

1.

7. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharma- ceutically acceptable carrier.

8. A kit comprising, in a container, the polypeptide of claim 1 and instructions for use.

9. A modified IgG comprising the polypeptide described in claim 1.

10. An antibody comprising the polypeptide described in claim 1.

11. A fusion molecule comprising the polypeptide described in claim 1.

12. 2. A vector comprising a nucleic acid sequence encoding the amino acid sequence of the polypeptide of claim 1, wherein the wild-type canine IgG constant domain comprises the amino acid sequence set forth in SEQ ID NO:

1.

13. An isolated cell comprising the vector of claim 12.

14. A method for producing a molecule, the method comprising providing a cell as described in claim 13 and culturing the cell.

15. 1. A method for increasing the serum half-life of an antibody in a dog, said method comprising modifying an antibody to contain a canine IgG constant domain, said canine IgG constant domain comprising at least one amino acid substitution compared to a wild-type canine IgG constant domain, said substitution being at amino acid residue 252, 254, 256, 311, 434, or 439, as numbered according to the Eu index in Kabat.

16. 16. The method of claim 15, wherein the canine IgG constant domain comprises one or more of the mutations L252F, L252R, L252Y, L252M, A254T, A254S, T256E, Q311W, N434H, N434Y, and E439K.

17. 16. The method of claim 15, wherein the canine IgG constant domain comprises a mutation selected from the group of: (1) L252F; (2) L252R, N434H, and Q311W; (3) L252R; (4) Q311W; (5) L252R, A254T, T256E, and N434H; (6) L252Y and A254T; or (7) L252M, A254S, and E439K.

18. The method of claim 15, wherein the antibody is an anti-IL31 antibody.

19. The antibody described in claim 10, wherein the antibody is an anti-IL31 antibody.

20. The fusion molecule of claim 11, wherein the molecule is an anti-IL31 molecule.

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