Canine antibody variant

Abstract

The present invention generally relates to canine antibodies variants and their use. Specifically, the present invention relates to mutations in the constant regions of canine antibodies for improving various characteristics.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 354,039, filed on June 21, 2022, the entire content of which is incorporated herein by reference.

[0002] The present invention generally relates to canine antibody variants and their use. Specifically, the present invention relates to one or more mutations in the Fc constant region of canine antibodies for improving various characteristics.

Background Art

[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).

[0004] The neonatal Fc receptor (FcRn) extends the half - life of IgG in its pH - dependent interaction with the fragment crystallizable (Fc) region through a recycling mechanism. Specifically, the Fc region that spreads across the interface of the CH2 and CH3 domains interacts with FcRn on the cell surface to regulate IgG constancy. This interaction is promoted by the acidic interaction after IgG pinocytosis, and thus IgG is protected from degradation. Then, the endocytosed IgG is recycled to the cell surface and released into the bloodstream at alkaline pH, thereby maintaining sufficient serum IgG for proper function. Therefore, the pharmacokinetic profiles of IgG depend on the structural and functional properties of their Fc regions.

[0005] Three canine IgG subclasses have been bound to canine FcRn and compared to human IgG analogs. The half-life of canine IgG needs to be studied extensively because without some experimental verification, one cannot anticipate or predict whether they closely match human IgG.

[0006] Prolonging the half-life of IgG can enable a reduction in the dosing frequency and / or a decrease in the dosage of an antibody drug, which in turn reduces veterinary visits, improves patient compliance, and decreases concentration-dependent cytotoxicity / adverse events.

[0007] Therefore, there is a need to identify mutations in the Fc constant region to improve the half-life. SUMMARY OF THE INVENTION

[0008] The present invention relates to a mutant canine IgG that provides a higher FcRn affinity 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 the affinity for FcRn.

[0009] In one aspect, the present invention provides a modified IgG comprising a canine IgG constant domain that contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, and the substitution is at amino acid residues 286, 311, 312, 426, or 436 numbered according to the Eu index in Kabat.

[0010] In some embodiments, the constant domain comprises one or more of the substitutions T286C, T286D, T286E, T286G, T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

[0011] In another aspect, the invention provides a polypeptide comprising a canine IgG constant domain that comprises at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 286, 311, 312, 426, or 436 numbered according to the Eu index in Kabat.

[0012] In yet another aspect, the invention provides an antibody comprising a canine IgG constant domain that comprises at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 286, 311, 312, 426, or 436 numbered according to the Eu index in Kabat.

[0013] In a further aspect, the present invention provides a method for generating or manufacturing an antibody or molecule, the method comprising providing a vector or host cell having an antibody comprising a canine IgG constant domain, wherein the canine IgG constant domain comprises one or more amino acid substitutions compared to the wild-type canine IgG constant domain, and the one or more substitutions are at amino acid residues 286, 311, 312, 426, or 436, or combinations thereof.

[0014] In another aspect, the present 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 residues 286, 311, 312, 426, or 436 numbered according to the Eu index in Kabat.

[0015] In another aspect, the present invention provides a method for increasing the antibody serum half-life in dogs, the method comprising administering to the dog a therapeutically effective amount of an antibody comprising a canine IgG constant domain, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to the wild-type canine IgG constant domain, and the substitution is at amino acid residues amino acid residues 286, 311, 312, 426, or 436 numbered according to the Eu index in Kabat.

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

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

[0018]

Figure 1

Figure 2-1

Figure 2-2

Figure 2-3

Figure 2-4

Figure 2-5

Figure 2-6

Figure 3

Figure 4

Mode for Carrying Out the Invention

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

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

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

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

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

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

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

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

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

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

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

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

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

[0032] SEQ ID NO: 14 refers to the nucleic acid sequence of the 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] The subject matter of the present invention can be more readily understood by reference to the following detailed description, which forms a part of this disclosure. The present invention is not limited to the specific products, methods, conditions, or parameters described and / or shown herein, and it should be understood that the terms used herein are for the purpose of describing particular embodiments by way of example only and are not intended to limit the claimed invention.

[0039] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular.

[0040] As used above and throughout this disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise indicated.

[0041] Definitions

[0044] In the present disclosure, the singular forms "a", "an", and "the" include plural referents, and references to a particular numerical value include at least that particular value unless the context clearly indicates otherwise. Thus, for example, reference to a "molecule" or "compound" is a reference to one or more such molecules or compounds known to those skilled in the art and their equivalents. The term "plural" as used herein means more than one. When a range of values is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, it is understood that the use of the preceding "about" forms another embodiment with the particular value. All ranges are inclusive and combinable.

[0042]

[0045] In this specification and the claims, the numbering of amino acid residues in the immunoglobulin heavy chain 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). The "Eu index in Kabat" refers to the residue numbering of IgG antibodies and is reflected in Figure 2 herein.

[0043]

[0046] The term "isolated", when used in reference to a nucleic acid, is a nucleic acid that has been identified and separated from at least one contaminating substance 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 the nucleic acid molecules present in natural cells. An isolated nucleic acid molecule includes a nucleic acid molecule contained within a cell that normally expresses the encoded polypeptide. For example, the nucleic acid molecule is at a plasmid or chromosomal location different from that of the natural cell. An isolated nucleic acid may exist in single-stranded form or double-stranded form. When using an isolated nucleic acid molecule to express a protein, the oligonucleotide or polynucleotide includes at least either the sense strand or the coding strand, but may also include both the sense strand and the antisense strand (i.e., it may be double-stranded).

[0044]

[0047] A nucleic acid molecule is "operably linked" or "operably bound" 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 when it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence of a nucleic acid when 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 a functional fragment thereof that does not include an Fc region when present in nature) when the expressed fusion protein includes a heterologous protein or a functional fragment thereof adjacent to either upstream or downstream of the variant Fc region polypeptide. The heterologous protein may be adjacent to the immediate vicinity of the variant Fc region polypeptide or may be separated therefrom by a linker sequence of any length and composition. Similarly, a polypeptide (used interchangeably with "protein" herein) molecule is "operably linked" or "operably bound" when it is placed in a functional relationship with another polypeptide.

[0045]

[0048] 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. The fragment can range in size from six amino acids to one amino acid less than the entire amino acid sequence of the full-length polypeptide. Functional fragments of the variant Fc region polypeptides of the invention retain at least one "amino acid substitution" as defined herein. Functional fragments of the variant Fc region polypeptides retain at least one function known in the art to be associated with the Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, downregulation of cell surface receptors, or can increase the in vivo or in vitro half-life of a polypeptide, for example, when operably linked).

[0046]

[0049] The term "purified" or "purify" refers to substantially removing at least one contaminating substance from a sample. For example, an antigen-specific antibody may 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 non-immunoglobulin proteins, which are at least one contaminating substance, and also by removal of immunoglobulin proteins that do not bind the same antigen. Removal of non-immunoglobulin proteins and / or removal of immunoglobulins that do not bind a particular antigen results in an increase in the percentage of antigen-specific immunoglobulin in the 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 the sample.

[0047]

[0050] As used herein, the term "native" when referring to a polypeptide (e.g., the Fc region) is used to indicate that the polypeptide has the amino acid sequence of a naturally occurring and commonly occurring polypeptide or an amino acid sequence consisting of its naturally occurring polymorphism. Native polypeptides (e.g., native Fc regions) can be produced by recombinant means or isolated from natural sources.

[0048]

[0051] As used herein, the term "expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the coding sequence operably linked in a particular host organism.

[0049]

[0052] 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 placed in vitro, in situ, or in vivo.

[0050]

[0053] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. The generally accepted boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the Fc region of a canine IgG heavy chain is typically defined as extending, for example, from amino acid residue 231 to its carboxyl terminus. In some embodiments, the variant includes only a portion of the Fc region and may or may not include the carboxyl terminus. The Fc region of an immunoglobulin generally includes CH2 and CH3, which are two constant domains. 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 portions of such constant domains) are contemplated.

[0051]

[0054] The "CH2 domain" of the canine IgG Fc region generally extends, for example, from about amino acid 231 to about amino acid 340 (see Figure 2). The CH2 domain is unique in that it does not pair closely with another domain. Two N-linked branched carbohydrate chains intervene between the two CH2 domains of the native IgG molecule as such.

[0052]

[0055] The "CH3 domain" of the canine IgG Fc region generally extends, for example, from about amino acid residue 341 to about amino acid residue 447, covering the residues from the C-terminus of the Fc region to the CH2 domain (see Figure 2).

[0053]

[0056] The "functional Fc region" has the "effector function" of the native sequence Fc region. At least one effector function of the polypeptide containing the variant Fc region of the present invention can be enhanced or decreased as compared with the polypeptide containing the native Fc region or the parental 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 evaluated using various assays (e.g., Fc binding assay, ADCC assay, CDC assay, depletion of target cells from whole blood samples or fractionated blood samples, etc.).

[0054]

[0057] The "native sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of the Fc region naturally and commonly found. An exemplary native sequence canine Fc region is shown in Figure 2 and includes the native sequence of the canine IgG Fc region.

[0055]

[0058] The "variant Fc region" includes an amino acid sequence that is different from that of the native sequence Fc region (or a fragment thereof) due to at least one "amino acid substitution" as defined herein. In a preferred embodiment, 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 the parent polypeptide, compared to the native sequence Fc region or the Fc region of the parent polypeptide. In an alternative embodiment, the variant Fc region may be generated according to the methods disclosed herein, and this 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 a ligand.

[0056]

[0059] As used herein, the term "derivative" in the context of a polypeptide refers to a polypeptide comprising an amino acid sequence that has been modified 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, without limitation, an antibody may be modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cell ligand or another protein, etc. Derivative polypeptides may be generated by chemical modification using techniques known to those of skill in the art, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicaamycin, etc. Further, the derivative polypeptide has the same or identical function as the polypeptide from which it is derived. A polypeptide comprising the variant Fc region of the present invention may be a derivative as defined herein, and preferably, it is understood that derivatization occurs within the Fc region.

[0057]

[0060] 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% identical to that of a native canine amino polypeptide.

[0058]

[0061] The terms "Fc receptor" or "FcR" are used to describe receptors that bind to the Fc region (e.g., the Fc region of an antibody). Preferred FcRs are native sequence FcRs. Further, preferred FcRs are those that bind to 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 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 to be identified in the future, are encompassed by the term "FcR" herein.

[0059]

[0062] The phrases "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which non-specific cytotoxic cells (e.g., non-specific) (e.g., natural killer ("NK") cells, neutrophils, and macrophages) that express FcRs recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. NK cells, which are primary cells for mediating ADCC, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

[0060]

[0063] As used herein, the term "effector cell" refers to leukocytes (preferably of canine origin) that express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc gamma RIII and perform an ADCC effector function. Examples of leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources (e.g., blood or PBMC).

[0061]

[0064] A variant polypeptide having an "altered" FcRn binding affinity is one in which the FcRn binding affinity is either improved (i.e., increased, greater, or higher) or decreased (i.e., reduced, decreased, or lower) as measured at pH 6.0 compared to the variant's parent polypeptide or a polypeptide comprising the native Fc region. A variant polypeptide that exhibits increased binding or increased binding affinity for FcRn binds to FcRn with a higher affinity than the parent polypeptide. A variant polypeptide that exhibits decreased binding or decreased binding affinity for FcRn binds to FcRn with a lower affinity than its parent polypeptide. Such a variant that exhibits decreased binding to FcRn has little or no binding to FcRn, e.g., it can have 0 to 20% binding to FcRn compared to the parent polypeptide. When the amounts of the variant polypeptide and the parent polypeptide in the binding assay are essentially the same and all other conditions are identical, a variant polypeptide that binds to FcRn with an "improved affinity" compared to its parent polypeptide is one that binds to FcRn with a higher binding affinity than the parent polypeptide. For example, in an ELISA assay or other methods available to those skilled in the art where FcRn binding affinity is determined, a variant polypeptide having an improved FcRn binding affinity can exhibit an increase in FcRn binding affinity of about 1.10-fold to about 100-fold (more typically, about 1.2-fold to about 50-fold) compared to the parent polypeptide.

[0062]

[0065] As used herein, "amino acid substitution" refers to replacing at least one existing amino acid residue in a given amino acid sequence with a different "replacement" amino acid residue. The replacement residue or residues can be "naturally occurring amino acid residues" (i.e., those 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). Substitutions with one or more non-naturally occurring amino acid residues are also encompassed by the definition of amino acid substitution herein. "Non-naturally occurring amino acid residues" refer to residues other than the naturally occurring amino acid residues listed above and are capable of covalently bonding to adjacent amino acid residues within the 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).

[0063]

[0066] 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 decay per unit time. Assay formats can include ELISA, FACS, or other methods. Changes in the "assay signal" can reflect changes in cell viability and / or changes in the kinetic off-rate, kinetic on-rate, or both. "Higher assay signal" refers to the measured output number being greater than another number (e.g., a variant may have a higher (greater) measured number in an ELISA assay compared to the parental polypeptide). "Lower" assay signal refers to the measured output number being less than another number (e.g., a variant may have a lower (smaller) measured number in an ELISA assay compared to the parental polypeptide).

[0064]

[0067] 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 is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g., seconds -1 reported) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g., moles / second). Generally, it is not possible to clearly state whether changes in the equilibrium dissociation constant are due to differences in the on-rate, off-rate, or both, unless each of these parameters is experimentally determined (e.g., by BIACORE or SAPIDYNE measurements).

[0065]

[0068] As used herein, the term "hinge region" refers to the range of amino acids spanning the canine IgG from, for example, positions 216 to 230 of canine IgG. The hinge regions of other IgG isotypes can be aligned with the IgG sequence by placing cysteine residues that form inter-heavy chain disulfide (S-S) bonds at the same positions.

[0066]

[0069] "Clq" is a polypeptide that includes the binding site of the Fc region of immunoglobulins. Clq, together with two serine proteases, Clr and Cls, forms the complex Cl, which is the first component of the CDC pathway.

[0067]

[0070] As used herein, the term "antibody" is used synonymously with "immunoglobulin" or "Ig" and is used in the broadest sense, encompassing monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological or functional activity. Also included in the present invention and the term "antibody" are single-chain antibodies, and chimeric, canine, or caninized antibodies, and chimeric or CDR-grafted single-chain antibodies, which contain portions from different species. The various portions of these antibodies can be joined together chemically or synthetically by conventional techniques, or prepared as a continuous protein using genetic engineering techniques. For example, nucleic acids encoding chimeric or caninized chains can be expressed to produce a continuous 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, U.S. Patent No. 5,585,089, and U.S. Patent No. 5,698,762. Also see Newman, R. et al. BioTechnology, 10:1455-1460, 1993 for primatized antibodies, and Ladner et al. U.S. Patent No. 4,946,778 and Bird, R.E. et al., Science, 242:423-426, 1988 for single-chain antibodies. It is understood that all forms of antibodies that include the Fc region (or a portion thereof) are encompassed within the term "antibody". Further, 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.

[0068]

[0071] As used herein, the term "antibody fragment" refers to a portion of a whole 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 comprises at least a portion or the whole of the CH2 region.

[0069]

[0072] As used herein, the term "functional fragment," when used with respect to a monoclonal antibody, is intended to refer to a portion of the monoclonal antibody that still retains functional activity. The functional activity can be, for example, antigen-binding activity or specificity, receptor-binding activity or specificity, effector function activity, etc. Examples of monoclonal antibody functional fragments include individual heavy or light chains such as VL, VH, and Fd and fragments thereof, monovalent fragments such as Fv, Fab, and Fab’, divalent 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, R.A. (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, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The term "functional fragment" is intended to include fragments generated, for example, by protease digestion or reduction of a monoclonal antibody and by recombinant DNA methods known to those skilled in the art.

[0070]

[0073] As used herein, the term "fragment" refers to a polypeptide that includes the 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 a preferred embodiment, the fragment of the polypeptide retains at least one function of the full-length polypeptide.

[0071]

[0074] As used herein, the term "chimeric antibody" includes monovalent, divalent, or multivalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain that is associated through a disulfide bridge with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy chain-light chain dimers that are associated through at least one disulfide bridge. The chimeric heavy chain of an antibody for use in canines includes an antigen-binding region derived from the heavy chain of a non-canine antibody that is linked to at least a portion of a canine heavy chain constant region, such as CH1 or CH2. The chimeric light chain of an antibody for use in canines includes an antigen-binding region derived from the light chain of a non-canine antibody that is linked to at least a portion of a canine heavy chain constant region (CL). Antibodies, fragments, or derivatives having 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, the immunoglobulin chains are recovered separately, and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to spontaneously associate in the culture medium, followed by recovery of the assembled immunoglobulin, or fragment, or both the heavy and light chains can be expressed in the same host cell. Methods for generating chimeric antibodies are well known in the art (see, e.g., U.S. Patent Nos. 6,284,471, 5,807,715, 4,816,567, and 4,816,397).

[0072]

[0075] As used herein, the "caninized" form of a non-canine animal (e.g., mouse) antibody (i.e., a caninized antibody) is an antibody that minimally contains or does not contain any sequences derived from non-canine animal immunoglobulins. In most cases, a caninized antibody is a canine animal immunoglobulin (recipient antibody) in which residues from the hypervariable regions of the recipient have been replaced by residues from the hypervariable regions of a non-canine animal species (donor antibody) such as a mouse, rat, rabbit, human, or non-human primate that have the desired specificity, affinity, and capacity. In some cases, the framework region (FR) residues of the canine animal immunoglobulin are replaced by the corresponding non-canine animal residues. Additionally, a caninized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are generally made to further improve antibody performance. Generally, a caninized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-canine animal immunoglobulin, and all or substantially all of the FR residues are of a canine animal immunoglobulin sequence. A caninized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically that of a canine animal immunoglobulin.

[0073]

[0076] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding domain of a heterologous "adhesin" protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, an immunoadhesin contains a fusion of an adhesin amino acid sequence having the desired binding specificity that is outside of (i.e., "heterologous" to) the antigen recognition and binding site (antigen-binding site) of an antibody and an immunoglobulin constant domain sequence.

[0074]

[0077] As used herein, the term "ligand binding domain" refers to any native receptor, or any region or derivative thereof, that retains at least the qualitative ligand binding ability of the corresponding native 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 nonetheless specifically encompassed by this definition are cytokine receptors, and in particular, receptors having 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).

[0075]

[0078] As used herein, the term "receptor binding domain" refers to any native ligand of a receptor, such as a cell adhesion molecule, or any region or derivative of such a native ligand that retains at least the qualitative receptor binding ability of the corresponding native ligand.

[0076]

[0079] As used herein, an "isolated" polypeptide is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, an isolated polypeptide is (1) greater than 95% by weight, preferably greater than 99% by weight, of the polypeptide as determined by the Lowry method, (2) purified to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a rotary cup sequenator, or (3). Because at least one component of the polypeptide's natural environment is absent, an isolated polypeptide is contained in recombinant cells in situ. However, usually an isolated polypeptide will be prepared by at least one purification step.

[0077]

[0080] As used herein, the terms "disorder" and "disease" are used synonymously 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., a pathological condition that predisposes a patient to a particular disorder).

[0078]

[0081] As used herein, the term "receptor" refers to a polypeptide capable of binding to at least one ligand. Preferred receptors are cell surface or soluble receptors having an extracellular ligand-binding domain and optionally other domains (e.g., transmembrane domains, intracellular domains, and / or membrane anchors). The receptors evaluated in the assays described herein can be the native receptor, or a fragment or derivative thereof (e.g., a fusion protein comprising the binding domain of the receptor fused to one or more heterologous polypeptides). Further, the receptors whose binding properties are evaluated can be present intracellularly, or can be isolated and optionally coated onto an assay plate or some other solid phase, or directly labeled and used as a probe.

[0079] Canine wild-type IgG

[0082] 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 IgG 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 IgG D . In a particular embodiment, the canine IgG is IgG B .

[0080]

[0083] IgG A , IgG B , IgG C , and IgG D amino acid and nucleic acid sequences are also 、 well known in the art.

[0081]

[0084] In one example, the IgG of the present invention includes a constant domain, such as a CH1, CH2, or CH3 domain, or a combination thereof. In another example, the constant domain of the present invention includes an Fc region that includes, for example, a CH2 or CH3 domain, or a combination thereof.

[0082]

[0085] In certain examples, the wild-type constant domain includes 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 mutations described herein. Each possibility represents a separate embodiment of the present invention.

[0083]

[0086] The IgG constant domain also includes a polypeptide having an amino acid sequence substantially similar to the amino acid sequence of the heavy and / or light chain. The substantially identical 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).

[0084]

[0087] The present invention also includes a nucleic acid molecule encoding the IgG or a portion thereof described herein. In one embodiment, the nucleic acid can encode an antibody heavy chain that includes, for example, a CH1, CH2, CH3 region, or a combination thereof. In another embodiment, the nucleic acid can encode an antibody heavy chain that includes any one of, for example, a VH region or a portion thereof, including any variant thereof, or any one of the VH CDRs. The present invention also includes a nucleic acid molecule encoding an antibody light chain that includes any one of, for example, a CL region or a portion thereof, including any variant thereof, a VL region or a portion thereof, including any variant thereof, or any one of the VL CDRs. In certain embodiments, the nucleic acid encodes both the heavy and light chains, or a portion thereof.

[0085]

[0088] The amino acid sequences of the wild-type constant domains described in SEQ ID NOs: 1, 2, 3, or 4 are encoded by their corresponding nucleic acid sequences. For example, the amino acid sequence of the wild-type constant domain described in SEQ ID NO: 2 is encoded by the nucleic acid sequence described in SEQ ID NO: 5 or 6. In some embodiments, the amino acid sequences of the wild-type constant domains described in SEQ ID NOs: 1, 2, 3, or 4 are encoded by the nucleic acid sequences described in SEQ ID NOs: 16, 5, 17, or 18, respectively.

[0086] Modified canine IgG

[0089] The inventors of the present application have surprisingly and unexpectedly found that substituting one or more amino acid residues improved the affinity for FcRn. As used herein, the position numbers of amino acids refer to the 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)).

[0087]

[0090] Accordingly, in one embodiment, the present invention provides a modified IgG comprising a canine IgG constant domain that contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residues 286, 311, 312, 426, or 436 numbered according to the Eu index in Kabat.

[0088]

[0091] In some embodiments, the constant domain comprises one or more of substitution T286C, T286D, T286E, T286G, T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

[0089]

[0092] In certain instances, the invention comprises one or more of the mutations described herein in the wild-type amino acid sequences set forth in SEQ ID NOs: 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 of the mutations described herein. Each possibility represents a separate embodiment of the invention.

[0090]

[0093] The amino acid sequence of the mutant constant domain is encoded by its corresponding mutant nucleic acid sequence.

[0091] Methods for making the antibody molecules of the invention

[0094] Methods for making 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 hereby incorporated by reference in their entirety. Any suitable method, process, or technique known to those of skill in the art can be used. Antibody molecules having the variant Fc regions of the 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.

[0092]

[0095] With the advent of methods in molecular biology and recombinant techniques, those of skill in the art can generate antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences that encode specific amino acid sequences found in the polypeptide structure of the antibody. Such antibodies can be generated either by cloning the gene sequence that encodes the polypeptide chain of the antibody or by directly synthesizing the polypeptide chain and assembling the synthesized chains to form an active tetramer (H2L2) structure having an affinity for specific epitopes and antigenic determinants. This has enabled the easy generation of antibodies having sequences that are characterized by neutralizing antibodies from different species and sources.

[0093]

[0096] Regardless of how they are recombinantly constructed or synthesized, whether from an antibody source, using transgenic animals which are a source of antibodies, or in vitro or in vivo, in laboratory-sized or commercially sized large cell cultures, using transgenic plants, or by direct chemical synthesis without the use of a living body at any stage of the process, all antibodies have an overall similar three-dimensional structure. This structure is often presented as H2L2, referring to the fact that an antibody generally contains two light chain (L) amino acids and two heavy chain (H) amino acids. Both chains have regions capable of interacting with an antigen target that is structurally complementary. The region that interacts with the target is called the "variable" or "V" region and is characterized by differences in the amino acid sequence between antibodies of different antigen specificities. The variable region of either the H or L chain contains an amino acid sequence capable of specifically binding to the antigen target.

[0094]

[0097] As used herein, the term "antigen-binding region" refers to a portion of an antibody molecule that contains the amino acid residues that interact with an antigen and confer upon 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 region of the H or L chain that provides the antigen-binding region, there are smaller sequences referred to as "hypervariable" which are responsible for the extreme variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity-determining regions" or "CDR regions". These CDR regions are responsible for the basic specificity of the antibody for a particular antigen determinant structure.

[0095]

[0098] CDRs represent non - contiguous ranges of amino acids within the variable regions, and regardless of the species, the positions where these important amino acid sequences are located within the variable heavy and light chain regions have been found to have similar positions within the amino acid sequence of the variable chain. All variable heavy and light chains of antibodies have three CDR regions each, which are non - contiguous with each other. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) regions and variable regions, and within the latter, there are CDRs and so - called "framework regions" composed of amino acid sequences that are within the variable regions of the heavy or light chains but outside the CDRs.

[0096]

[0099] The present invention further provides a vector comprising at least one of the above - mentioned nucleic acids. Since the genetic code deteriorates, two or more codons can be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which may be capable of encoding an amino acid. The probability that a particular oligonucleotide will actually constitute the actual coding sequence can be estimated by considering abnormal base - pairing relationships in eukaryotic or prokaryotic cells expressing the antibody or portion, and the frequency with which a particular codon is actually used (to encode a particular amino acid). 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 a set of nucleotide sequences can be identified that includes the theoretical "most likely" nucleotide sequence capable of encoding the Canidae IgG sequence. Also, it is contemplated that antibody - coding regions for use in the present invention can also be provided by modifying existing antibody genes using standard molecular biological techniques that give rise to variants of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequence of the antibody or peptide.

[0097] [000100]For example, one class of substitutions is conservative amino acid substitutions. Such substitutions replace a given amino acid in a canid antibody peptide with another amino acid having similar characteristics. Typically seen as conservative substitutions are the replacements of each other among the aliphatic amino acids Ala, Val, Leu, and Ile, the exchange of the hydroxyl residues Ser and Thr, the exchange of the acidic residues Asp and Glu, the substitution between the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg, the replacement between the aromatic residues Phe and Tyr, and the like. Guidance regarding which amino acid changes are likely to be phenotypically silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0098] [000101]Variant canid antibodies or peptides may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative mutations, or mutations in residues or regions that are not important. Functional variants may also include similar amino acid substitutions that do not change or only slightly change the function. Alternatively, such substitutions may have a positive or negative impact to some extent. 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 important residues or important regions.

[0099] [000102]Amino acids essential for the 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 a single alanine mutation into all residues in the molecule. The resulting mutant molecules are then tested for biological activities 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).

[0100] [000103]Furthermore, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. In addition, 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 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 phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, 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 an amino acid to a protein such as arginylation, and ubiquitination. Such modifications are well known to those skilled in the art and are described in detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamate residues, hydroxylation, and ADP ribosylation are described, for example, in most basic textbooks such as Proteins-Structure and Molecular Properties (2nd ed., T.E. Creighton, W.H. Freeman & Co., N.Y., 1993). For this topic, many detailed reviews are available, such as by Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, N.Y., 1983), Seifter et al. 182 Meth. Enzymol. 626-46 (1990), and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0101] [000104]In another aspect, the present invention provides antibody derivatives. An "derivative" of an antibody includes additional chemical moieties that are not normally part of the protein. Covalent modifications of the protein are included within the scope of the present invention. Such modifications can be introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization using bifunctional agents well known in the art is useful for cross-linking antibodies or fragments to water-insoluble support matrices or other macromolecular carriers.

[0102] [000105]Derivatives also include radiolabeled monoclonal antibodies that are labeled. For example, radioactive iodine (251, 131I), carbon (14C), sulfur (35S), indium, tritium (3H 3 ), etc.; 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 acridin esters), or fluorescent agents (such as phycobiliproteins) are used.

[0103] [000106]Another derivative bispecific antibody of the present invention is a bispecific antibody generated by combining two separate antibody moieties that recognize two different antigenic groups. This can be achieved by cross-linking or recombinant techniques. In addition, moieties can be added to the antibody or a portion thereof to increase the in vivo half-life (e.g., by extending the time to clearance from the bloodstream). Such techniques include, for example, adding a PEG moiety (also referred to as pegylation), which is well known in the art. See U.S. Patent Application Publication No. 2003 / 0031671.

[0104] [000107]In some embodiments, the nucleic acid encoding the antibody of the subject is introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. After the nucleic acid of the subject has been introduced into the cell, the cell is typically incubated at 37° C., sometimes under selection, for a period of about 1 to 24 hours in order 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. Conventionally, monoclonal antibodies have been produced as natural molecules in mouse hybridoma strains. In addition to that technology, the present invention provides for recombinant DNA expression of antibodies. This allows for the production of antibodies in selected host species, as well as various derivatives and fusion proteins of the antibody.

[0105] [000108]A nucleic acid sequence encoding at least one antibody, portion, or polypeptide of the invention can be recombined with vector DNA according to conventional techniques including blunt or sticky ends for ligation, restriction enzyme digestion to provide appropriate ends, filling in of sticky ends as appropriate, alkaline phosphatase treatment to avoid unwanted ligation, and ligation with an appropriate 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 these can be used to construct nucleic acid sequences encoding antibody molecules or antigen-binding regions thereof.

[0106] [000109]Nucleic acid molecules, such as DNA, contain nucleotide sequences that include transcriptional and translational regulatory information, and such sequences are said to be "capable of expressing" a polypeptide when they are "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is a linkage in which a regulatory DNA sequence and a DNA sequence to be expressed are connected in such a way as to permit gene expression in recoverable amounts as a peptide or antibody moiety. The exact nature of the regulatory regions required for gene expression can vary depending on the organism, as is well known in similar arts. See, for example, Sambrook et al., 2001 (supra), Ausubel et al., 1993 (supra).

[0107] [000110]Accordingly, the present invention encompasses the expression of antibodies or peptides in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds, 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.

[0108] [000111]In one embodiment, the nucleotide sequences of the present invention will be incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993 (supra). Important factors in the selection of a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells not containing 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 different species of host cells.

[0109] [000112]Examples of prokaryotic vectors known in the art include plasmids such as those that can replicate in E. coli (e.g., pBR322, CoIE1, pSC101, pACYC184, πvX, etc.). Such plasmids are disclosed, for example, by Maniatis et al., 1989 (supra), Ausubel et al, 1993 (supra). Examples of Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed in THE MOLEC.BIO.OF THE BACILLI 307-329 (Academic Press, NY, 1982) by Gryczan. 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).

[0110] [000113]Alternatively, gene expression elements useful for the expression of antibodies or peptides encoding cDNA include, but are not limited to, (a) the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), the Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982)), and the 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).

[0111] [000114]Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987) using the SV40 early promoter and its enhancer, the murine immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, the rabbit S-globin intervening sequence, immunoglobulin, and the rabbit S-globin polyadenylation site, and the SV40 polyadenylation element as expression elements. In immunoglobulin genes composed of partial cDNA, partial genomic DNA (Whittle et al, 1 Protein Engin. 499 (1987)), the transcription promoter can be the human cytomegalovirus, and the promoter enhancer can be the cytomegalovirus and murine / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be the native chromosomal immunoglobulin sequences.

[0112] [000115]In one embodiment, for the 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 greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain to be synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements listed above to achieve protein expression in mammalian cells.

[0113] [000116]Each fusion gene can be assembled into an expression vector or inserted into an expression vector. Subsequently, recipient cells capable of expressing the immunoglobulin chain gene product are transfected singly with the peptide or H or L chain coding gene, or co-transfected with the H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the integrated gene, and the expressed immunoglobulin chain or intact antibody or fragment is recovered from the culture.

[0114] [000117]In one embodiment, the peptide or the H and L chains, or a fusion gene encoding them or portions thereof, is then assembled into separate expression vectors 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 generation of the antibody can be myeloma cells. Myeloma cells can synthesize, assemble, and secrete the immunoglobulin encoded by the transfected immunoglobulin gene and have the mechanism of immunoglobulin glycosylation. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse from which 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.

[0115] [000118]The expression vector carrying the antibody construct or polypeptide of the present invention can be introduced into a suitable host cell by any of a variety of suitable means including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with a polycation such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[0116] [000119]Yeast can provide substantial advantages over bacteria in the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications including glycosylation. There are several recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used for the production of desired proteins in yeast. Yeast recognizes the leader sequences of cloned mammalian gene products and secretes peptides carrying the leader sequences (i.e., prepeptides). Hitzman et al., 11th Int’l Conference on Yeast, Genetics & Molec. Biol. (Montpellier, France, 1982).

[0117] [000120]Yeast gene expression systems can be routinely evaluated for levels of production, secretion, and stability of peptides, antibodies, fragments, and regions thereof. One of a series of yeast gene expression systems incorporating promoters and termination elements from constitutively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in a medium rich in glucose can be utilized. Known glycolytic genes can also provide very 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 for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985).

[0118] [000121]The bacterial strain can also be used as a host for the production of the antibody molecules or peptides described by the present invention. A plasmid vector containing a replicon and control sequences derived from a species compatible with the host cell is used in connection with these bacterial hosts. The vector carries a replication site and certain genes that make it possible to provide phenotypic selection in the transformed cells. Several approaches can be selected to evaluate the expression plasmid for the production of antibodies, fragments, and regions, or antibody chains encoded by the cloned immunoglobulin cDNA in bacteria (Glover, 1985 (supra), Ausubel, 1993 (supra), Sambrook, 2001 (supra), Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, N.Y. (1994 - 2001), Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997 - 2001).

[0119] [000122]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 the heavy and light chains, glycosylation of the antibody molecule, and secretion of the functional antibody protein. Mammalian cells that may be useful as hosts for the production of antibody proteins include fibroblast-derived cells such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells in addition to the above-mentioned lymphoid-derived cells. Many vector systems are available for the expression of cloned peptide heavy and light chain genes in mammalian cells (see Glover, 1985 (supra)). To obtain a complete H2L2 antibody, different approaches can be followed. It is possible to co-express the heavy and light chains in the same cell to achieve intracellular association and ligation of the heavy and light chains into the complete tetrameric H2L2 antibody and / or peptide. Co-expression can be carried out by using either the same or different plasmids in the same host. The genes for both the heavy and light chains and / or the peptide can be placed on the same plasmid, which is then transfected into the cells, thereby directly selecting cells that express both chains. Alternatively, first, cells can be transfected with a plasmid encoding one chain, for example, the light chain, and subsequently, the resulting cell line can be transfected with a heavy 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 the transfected cell line, cell lines that produce peptides and / or H2L2 molecules via either pathway can be transfected with a plasmid encoding additional copies of the peptide, H, L, or H plus L chains in combination with an additional selectable marker.

[0120] [000123]For the long-term high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines that stably express antibody molecules can be engineered. Instead of using an expression vector containing a viral origin of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. Following the introduction of foreign DNA, the 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, enabling cells to stably integrate the plasmid into the chromosome and grow to form a population (focus) that can be cloned and expanded into a cell line. Such engineered cell lines can be particularly useful in screening and evaluating compounds / components that interact directly or indirectly with the antibody molecule.

[0121] [000124]Once the antibodies of the invention are produced, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after Protein A, and size exclusion column chromatography), centrifugation, differential absorption solubility, or any other standard technique for protein purification. In many embodiments, the antibodies are secreted from the cells into the culture medium and harvested from the culture medium.

[0122] Pharmaceutical and veterinary uses [000125]The invention also provides a pharmaceutical composition comprising a molecule of the invention and one or more pharmaceutically acceptable carriers. More specifically, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, an antibody or peptide according to the invention.

[0123] [000126]A "pharmaceutically acceptable carrier" includes any excipient that is non-toxic to the cells or animals exposed at the dosages and concentrations used. The pharmaceutical composition can include one or additional therapeutic agents.

[0124] [000127] "Pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with animal tissues without undue toxicity, irritation, allergic response, or other problems commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0125] [000128] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coating agents, antibacterial and antifungal agents, wetting agents, preservatives, bagger, chelating agents, antioxidants, isotonic agents, and absorption delaying agents.

[0126] [000129] Pharmaceutically acceptable carriers include water; physiological 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 monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; EDTA; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®; isotonic agents such as saccharides, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride; and combinations thereof.

[0127] [000130] The pharmaceutical compositions of the present invention can be formulated in a variety of ways, including, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions, or suspensions, liposomes, suppositories, tablets, pills, or powders, etc. In some embodiments, the composition is in the form of an injectable or infusible solution. The composition can be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The composition can be formulated as an immediate release, controlled release, sustained release, or delayed release composition.

[0128] [000131]The compositions of the present invention can 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 chosen route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein can be carried out orally, by any suitable means including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), or by topical administration of the antibody (typically included in a pharmaceutical formulation) to the airway surface. Topical administration to the airway surface can be carried out by intranasal administration (e.g., by use of a dropper, swab, or inhaler). Topical administration of the antibody to the airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibody as an aerosol suspension and then causing the respirable particles to be inhaled by the subject. Methods and devices for administering respirable particles of a pharmaceutical formulation are well known and any conventional technique can be used.

[0129] [000132]In some desired embodiments, the antibody is administered by parenteral injection. In parenteral administration, the antibody or molecule can be formulated as a solution, suspension, emulsion, or lyophilized powder associated with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle can be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier such as an aqueous carrier, and such vehicles can be water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, etc. Liposomes and non-aqueous vehicles such as non-volatile oils can also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, buffers, toxicity adjusters, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations can vary widely, for example, less than about 0.5% by weight, usually about 1% by weight or more up to a maximum of 15% or 20% by weight, and will be selected mainly based on the specific dosage form chosen, based on factors such as fluid volume, viscosity, etc. The vehicle or lyophilized powder can contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulations are sterilized by generally used techniques. The actual methods for preparing compositions for parenteral administration are known or apparent to those skilled in the art and are described in more detail, for example, by REMINGTON’S PHARMA.SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[0130] [000133] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques can be used. Those skilled in the art will understand that lyophilization and reconstitution can result in varying degrees of antibody activity loss and that it may be necessary to adjust the usage levels to compensate. Compositions containing the present antibodies or cocktails thereof can be administered for the prevention of recurrence and / or therapeutic treatment of existing diseases. 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 composition is administered to a subject already suffering from a disease in an amount sufficient to cure, or at least partially arrest or mitigate, the disease and its complications.

[0131] [000134] The effective dosage of the composition of the present invention for the treatment of the conditions or diseases described herein will vary depending upon, for example, but not limited to, the pharmacodynamic characteristics of the particular agent and its dosage form and route of administration; the target site; the physiological state of the animal; other pharmaceuticals being administered; whether the treatment is prophylactic or therapeutic; the age, health, and weight of the recipient; the nature and degree of the symptoms; and the type of treatment being concurrently carried out, the frequency of the treatment, as well as many other different factors including the desired effect.

[0132] [000135] Single or multiple administrations of the composition can be carried out at dosage levels and patterns selected by the treating veterinarian. In any event, the pharmaceutical formulation should provide an amount of the antibody(ies) of the present invention sufficient to effectively treat the subject.

[0133] [000136] Therapeutic dosages can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0134] [000137]The pharmaceutical composition of the present invention may include a "therapeutically effective amount". A "therapeutically effective amount" refers to an effective amount in terms of dosage and duration that is necessary to achieve a desired therapeutic result. The therapeutically effective amount of a molecule can vary depending on factors such as the disease state, age, gender, and weight of an individual, as well as the ability of the molecule to induce a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effect exceeds any toxic or adverse effect of the molecule.

[0135] [000138]In another aspect, the compositions of the present invention can be used, for example, in the treatment of various diseases and disorders in dogs. As used herein, the terms "treating" and "treatment" refer to a therapeutic treatment that includes preventive or prophylactic measures, where the subject is one in which an undesired physiological change associated with a disease or condition is prevented or decelerated (mitigated). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, a decrease in the degree of a disease or condition, stabilization of a disease or condition (i.e., the disease or condition does not worsen), a delay or deceleration in the progression of a disease or condition, an 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 who already have a disease or condition, those who tend to have a disease or condition, or those who should prevent a disease or condition.

[0136] [000139]All patents and reference documents cited herein are hereby incorporated by reference in their entirety.

[0137] [000140]The following examples are provided to supplement the foregoing disclosure and to provide a better understanding of the subject matter described herein. These examples should not be regarded as limiting the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes from those perspectives will be apparent to those skilled in the art and are included within the true scope of the present invention and can be made without departing from the true scope of the present invention.

Examples

[0138] Example 1 Construction of Canine IgG Fc variants [000141]As described by Bergeron et al. (Bergeron et al., 2014, Vet Immunol Immunopathol., vol. 157(1-2), pages 31-41), the construction of all canine IgG (Figure 1) was performed. Therein, a plasmid containing the sequence encoding the canine constant region of the IgGB(65) subclass was utilized, and the VH / VL sequences of each mAb investigated herein were inserted upstream and in-frame with the nucleotides encoding the constant domain. By direct DNA synthesis of the constant region as a gene fragment, mutations were incorporated at each respective position of the CH1, CH2, or CH3 domain (Figure 2) of each plasmid, and then subcloned into each respective variable region of interest.

[0139] Expression and purification [000142] Monoclonal antibody (mAbs) variants were expressed in a mammalian suspension cell line, EXPICHO-S (Chinese hamster ovary) cells obtained from Thermo Fisher. Suspended EXPICHO-S cells were maintained at 0.14 - 8.0×10e6 cells / ml in EXPICHO expression medium (Gibco). The cells were diluted on day - 1 and the transfection day according to the ExpiCHO protocol user manual. According to the maximum titer conditions, the diluted cells were transfected using the reagents supplied from the ExpiFectamine CHO transfection kit (Gibco) as described in the protocol. After 12 - 14 days of incubation, the cultures were harvested and clarified. The antibody was purified from the clarified supernatant via protein A chromatography using 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. The sample was eluted from the column using 20 mM acetic acid, pH 3.5. After elution, a pool was made and neutralized by adding 1M sodium acetate to 4%. The sample was occasionally exchanged into the final buffer (e.g., PBS, others) according to the available volume and purpose of use. The concentration was measured by absorbance at 280 nm.

[0140] SDS - PAGE [000143] Non - reducing (nr) and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS - PAGE) was performed using a 4 - 12% Bis - Tris NuPAGE gel in MES - SDS running buffer and SeeBlue Plus 2 standards (all from Invitrogen). For non - reducing samples, 1 mM alkylating agent N - ethylmaleimide (NEM) was added, and for reducing samples, reducing agent dithiothreitol (DTT) was added. The gels were stained with Coomassie Blue and protein bands were detected.

[0141] Example 2 FcRn Binding Assay [000144] Canine FcRn was isolated and prepared according to Bergeron et al., discussed above, and mutant Fc IgGs were assayed against canine FcRn. Standard PCR was used to amplify canine FcRn-α subunit and β-microglobulin. FcRn-α subunit and β-microglobulin were cotransfected into HEK293 cells, and the FcRn complex was purified by IMAC affinity purification via the c-terminal His tag. The FcRn complex was biotinylated using the BirA enzyme biotinylation reaction. KD was measured using a Biacore T200 or Biacore 8K (Cytiva Life Science).

[0142] [000145] FcRn was captured onto the sensor surface using a modified SA capture method. The capture buffer and titration method used were 10 mM MES, 150 mM NaCl, 0.005% Tween® 20, 0.5 mg / mL BSA, pH 6. The running buffer and titration method used 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 T200 evaluation software or Bicaore 8K evaluation software (Table 1). A blank experiment containing buffer alone was run from all experiments. The flow cell was regenerated using 50 mM Tris, pH 8. Experiments were performed at 15°C.

[0143] [000146] Mutations made at each position have a significant effect on the affinity of IgG for FcRn at pH 6.

[0144] [000147] Binding of wild-type (WT) and mutant IgGs to canine FcRn was measured by surface plasmon resonance (Biacore). [Table 1-1] [Table 1-2]

Table 1-3

[0145] [000148]The results clearly show that the mutations created at various positions have a significant effect on the affinity of IgG to FcRn.

[0146] Example 3 FcRn Binding Verification Method [000149]The Fc regions of canine main-chain subclasses IgGA and IgGB were first designed using their respective CH2 and CH3 regions. The protein modeling function of AlphaFold 2.2 developed by DeepMind was implemented to model the 3D structures of each of the wild-type (WT) and mutant constructs of each canine skeletal subclass IgGA and IgGB.

[0147] [000150]Molecular Operating Environment (MOE) (MOE2019.0102) developed by Chemical Computing Group provides a flexible and automated graphical user interface for protein modeling. To analyze the structural differences, the sequence-to-profile alignment algorithm ranks the sequence templates using a scoring algorithm, and a score exceeding 85% ensures the selection of protein templates with physically realistic structures. The model was then optimized using the same pipeline, and the structural stability of the model was verified using a Ramachandran plot that checks the stereochemical quality of the protein structure.

[0148] Results [000151]The above method was performed on the wild-type (WT) construct and the following mutants: T286C, T286D, T286E, T286G, T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W. They were superimposed, and the mutated residues were shown in ball-and-stick form (Figure 4A). An RMSD plot was generated to calculate the root mean square deviation between two WT structures, Canidae-IgGA WT and Canidae-IgGB WT, which are relative to each other (Figure 4B). RMSD values of 2.0 Å or less are considered the criterion for regarding two structures as similar. The results showed that the Canidae IgGA construct was identical to the Canidae IgGB subclass with an average RMSD value of the structure of 1.21 Å. The RMSD at the five positions where the mutation scan was performed was also recorded in Table 3 with values in the range of 0.226 to 0.997 Å.

[0149] [000152]Following the same procedure, the following mutations in both Canidae subclasses were investigated: T286C, T286D, T286E, T286G, T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y (Table 3). Again, as with the WT, no dramatic fluctuations in the model were observed at each of the mutation positions between the two subclasses, as indicated by the RMSD value range of 0.226 - 0.997 Å (Table 3).

Table 2

Table 3-1

Table 3-2

Table 3-3

[0150] Conclusion [000153]Molecular modeling of all mutations across the two subclasses IgGA and IgGB of the canine skeleton was performed and verified using MOE2019.0102. The Fc folding and residue conformations between subclasses were shown to have an RMSD of less than 2 Å, indicating that the canine IgG1 and IgG2 protein structures have very high identity at all of the mutation positions described herein and can therefore function in a similar manner.

[0151] [000154]Although the preferred embodiments of the present invention have been described, it should be understood that the present invention is not limited to the exact embodiments and that various changes and modifications can be made by those skilled in the art without departing from the scope or spirit of the invention as defined by the appended claims.

Claims

1. A modified IgG comprising a canid IgG constant domain comprising at least one amino acid substitution compared to the wild-type canid IgG constant domain, wherein the substitution is located at amino acid residues 426, 286, 311, 312, or 436, numbered according to the Eu index in Kabat. The aforementioned steady-state domains are substituted with A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, T286C, T286D, T286E, T286G , T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311C , Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P , Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312 H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312 Modified IgG, including one or more of W, D312Y, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

2. The modified IgG according to claim 1, wherein the modified IgG has a higher affinity for FcRn than the IgG having the wild-type canid IgG constant domain.

3. The modified IgG according to claim 1, wherein the modified IgG is a canid or caninized IgG.

4. The modified IgG according to claim 1, wherein the IgG steady-state domain includes an Fc steady-state region having a CH3 domain.

5. The modified IgG according to claim 1, wherein the IgG steady-state domain includes an Fc steady-state region having CH2 and CH3 domains.

6. The modified IgG according to claim 1, wherein the wild-type canid IgG constant domain comprises the amino acid sequence shown in SEQ ID NO:

2.

7. A pharmaceutical composition comprising the modified IgG described in claim 1 and a pharmaceutically acceptable carrier.

8. A kit comprising a modified IgG as described in claim 1 in a container, and instructions for use.

9. A method for increasing the binding affinity of canine IgG to a neonatal Fc receptor (FcRn), the method comprising providing a modified IgG containing a canine IgG constant domain, wherein the canine IgG constant domain contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, and the substitution is located at amino acid residues 426, 286, 311, 312, or 436, numbered according to the Eu index in Kabat. The canid IgG constant domain is mutant A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, T286C, T286D, T286 E, T286G, T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286 V, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311 N, Q311P, Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312 G, D312H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312 A method comprising one or more of V, D312W, D312Y, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

10. The method according to claim 9, wherein the canid IgG constant domain has a serum half-life that is higher than that of IgG having the wild-type canid IgG constant domain.

11. The method according to claim 9, wherein the IgG steady domain includes an Fc steady region having a CH3 domain.

12. The method according to claim 9, wherein the IgG steady domain includes an Fc steady region having CH2 and CH3 domains.

13. The method according to claim 9, wherein the wild-type canid IgG constant domain comprises the amino acid sequence shown in SEQ ID NO:

2.

14. A polypeptide comprising a canid IgG constant domain, wherein the substitution is located at amino acid residues 426, 286, 311, 312, or 436, numbered according to the Eu index in Kabat, The aforementioned steady-state domains are substituted with A426C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, T286C, T286D, T286E, T286G , T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311 C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311 P, Q311R, Q311S, Q311T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D31 2H, D312I, D312K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D31 A polypeptide comprising one or more of the following: 2W, D312Y, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

15. A vector comprising a nucleic acid sequence encoding the amino acid sequence of the polypeptide described in Claim 14, wherein the wild-type canid IgG constant domain comprises the amino acid sequence shown in Sequence ID No.

2.

16. An isolated cell comprising the vector according to claim 15.

17. A method for producing a molecule, the method comprising providing the cells described in claim 16 and culturing the cells.

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