Canine antibody mutants

Abstract

The present invention relates generally to canine antibody variants and uses thereof. In particular, the present invention relates to mutations in the constant region of canine antibodies for improving various characteristics.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 354,039, filed on June 21, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to canine antibody mutants and uses thereof. In particular, the present invention relates to one or more mutations in the Fc constant region of canine antibodies for improving various characteristics. Background Art

[0004] Canine IgG monoclonal antibodies (mAbs) are being developed as effective therapeutic agents in veterinary medicine. A few years ago, four canine IgG subclasses were identified and characterized (Bergeron et al., 2014, Vet Immunol Immunopathol., Vol. 157 (1-2), pp. 31-41).

[0005] Through a cyclic mechanism, the neonatal Fc receptor (FcRn) prolongs the half-life of IgG in a pH-dependent interaction with the crystallizable (Fc) region of the IgG fragment. Specifically, the Fc region across the interface of the CH2 and CH3 domains interacts with FcRn on the surface of the cell to regulate IgG homeostasis. The acidic interaction after IgG pinocytosis is conducive to this interaction, and thus protects IgG from degradation. Then, the endocytosed IgG is recycled back to the cell surface and released into the bloodstream at an alkaline pH, thereby maintaining enough serum IgG to achieve normal function. Therefore, the pharmacokinetic profile of IgG depends on the structural and functional characteristics of its Fc region.

[0006] The three canine IgG subclasses bind to canine FcRn and have been compared to human IgG analogs. The half-life of canine IgG remains to be fully investigated, as it cannot be expected or predicted without any experimental support whether it will align closely with human IgG.

[0007] The extended half-life of IgG may allow for less frequent dosing and / or lower doses of antibody drugs, which in turn may reduce veterinary visits, improve patient compliance, and reduce concentration-dependent cytotoxicity / adverse events.

[0008] Therefore, there is a need to identify mutations in the Fc constant region to improve half-life. Summary of the invention

[0009] The present invention relates to mutant canine IgGs that provide higher affinity for FcRn relative to wild-type canine IgG. In particular, the inventors of the present application have discovered that substitution of one or more amino acid residues surprisingly and unexpectedly enhances affinity for FcRn.

[0010] In one aspect, the invention provides a modified IgG comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat.

[0011] In some embodiments, the constant domain comprises one or more of the following 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, D312 K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A42 6C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A4 26R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

[0012] In another aspect, the invention provides a polypeptide comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat.

[0013] In yet another aspect, the invention provides an antibody comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat.

[0014] In another aspect, the present invention provides a method for producing or manufacturing an antibody or molecule, the method comprising: providing a vector or host cell having an antibody comprising a canine IgG constant domain, the canine IgG constant domain comprising one or more amino acid substitutions relative to a wild-type canine IgG constant domain, wherein the one or more substitutions are at amino acid residues 286, 311, 312, 426 or 436, or a combination thereof.

[0015] In another aspect, the invention provides a fusion molecule comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat.

[0016] In another aspect, the invention provides a method for enhancing the serum half-life of an antibody in a dog, the method comprising: administering to the dog a therapeutically effective amount of an antibody comprising a canine IgG constant domain, the canine IgG constant domain comprising at least one amino acid substitution relative to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 286, 311, 312, 426 or 436, as numbered according to the EU index as in Kabat.

[0017] According to the following detailed description examples and drawings, other features and advantages of the present invention will become apparent. However, it should be understood that although the detailed description and specific examples indicate the preferred embodiments of the present invention, they are only given by way of illustration, because according to the detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] Figure 1 The domain structure of IgG is shown.

[0020] Figure 2An alignment of the amino acid sequences of wild-type (WT) human IgG1, canine IgGA, WT canine IgGB, WT canine IgGC, and WT canine IgGD is shown. The amino acid residues are numbered according to the Eu index as in Kabat. The CH1, hinge, CH2, and CH3 amino acid residues are colored red, purple, blue, and green, respectively.

[0021] Figure 3 The canine Fc IgGB WT nucleotide sequence is shown.

[0022] Figure 4 Protein modeling of canine IgGA and IgGB Fc regions is shown. Figure 4 A shows an overlay of the protein model, with the enlarged sub-image showing that five amino acid residue positions are displayed in ball-and-stick form. Figure 4 B shows the Root Mean Square Deviation (RMSD) comparison of wild-type constructs of canine IgGA and IgGB.

[0023] Brief Description of Sequence Listing

[0024] SEQ ID NO.: 1 refers to the amino acid sequence of the wild-type canine IgGA constant region.

[0025] SEQ ID NO.: 2 refers to the amino acid sequence of the constant region of wild-type canine IgGB (65).

[0026] SEQ ID NO.: 3 refers to the amino acid sequence of the wild-type canine IgGC constant region.

[0027] SEQ ID NO.: 4 refers to the amino acid sequence of the wild-type canine IgGD constant region.

[0028] SEQ ID NO.: 5 refers to the nucleic acid sequence of the optimized codons of wild-type canine IgGB (65) according to one embodiment.

[0029] SEQ ID NO.: 6 refers to the nucleic acid sequence of the wild-type canine IgGB (65) constant domain according to one embodiment.

[0030] SEQ ID NO.: 7 refers to the amino acid sequence of the wild-type canine IgGB CH1 domain.

[0031] SEQ ID NO.: 8 refers to the amino acid sequence of the wild-type canine IgGB hinge domain.

[0032] SEQ ID NO.: 9 refers to the amino acid sequence of a wild-type canine IgGB CH2 domain.

[0033] SEQ ID NO.: 10 refers to the amino acid sequence of a wild-type canine IgGB CH3 domain.

[0034] SEQ ID NO.: 11 refers to the nucleic acid sequence of the wild-type canine IgGB CH1 domain.

[0035] SEQ ID NO.: 12 refers to the nucleic acid sequence of the wild-type canine IgGB hinge domain.

[0036] SEQ ID NO.: 13 refers to the nucleic acid sequence of a wild-type canine IgGB CH2 domain.

[0037] SEQ ID NO.: 14 refers to the nucleic acid sequence of a wild-type canine IgGB CH3 domain.

[0038] SEQ ID NO.: 15 refers to the amino acid sequence of the wild-type human IgG1 constant region.

[0039] SEQ ID NO.: 16 refers to the nucleic acid sequence of the wild-type canine IgGA constant region.

[0040] SEQ ID NO.: 17 refers to the nucleic acid sequence of the wild-type canine IgGC constant region.

[0041] SEQ ID NO.: 18 refers to the nucleic acid sequence of the wild-type canine IgGD constant region.

[0042] SEQ ID NO.: 19 refers to the nucleic acid sequence of the wild-type human IgG1 constant region. DETAILED DESCRIPTION

[0043] The subject matter of the present invention may be more readily understood by reference to the following detailed description which forms a part of the present disclosure. It should be understood that the present invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is only for the purpose of describing specific embodiments by way of example and is not intended to limit the claimed invention.

[0044] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural meanings, and plural terms should include singular meanings.

[0045] As employed above and throughout the disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise indicated.

[0046] definition

[0047] In this disclosure, the singular forms "a", "an", and "the" include plural references, and unless the context clearly indicates otherwise, reference to a particular value includes at least that particular value. Thus, for example, reference to a "molecule" or "compound" is a reference to one or more of such molecules or compounds and equivalents thereof known to those skilled in the art, and so on. As used herein, the term "plurality" 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 by using the antecedent "about", it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0048] In the specification and claims, the numbering of amino acid residues in the immunoglobulin heavy chain is as in the Eu index as 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 as in Kabat" refers to the residue numbering of IgG antibodies and is used herein in Figure 2 reflected in.

[0049] When used with respect to nucleic acids, the term "isolated" refers to a nucleic acid that is identified and separated from at least one contaminant nucleic acid that is usually associated with it in its natural source. The isolated nucleic acid is in a form or environment different from the form or environment in which it exists in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acid molecule present in natural cells. The isolated nucleic acid molecule includes nucleic acid molecules contained in cells that are usually expressed in polypeptides encoded therein, wherein, for example, the nucleic acid molecule is located in a plasmid or chromosomal position different from the position of natural cells. The isolated nucleic acid can exist in single-stranded or double-stranded form. When the isolated nucleic acid molecule is used to express a protein, the oligonucleotide or polynucleotide will contain at least a sense strand or a coding strand, but can contain both a sense strand and an antisense strand (that is, can be double-stranded).

[0050] A nucleic acid molecule is "operably linked" or "operably attached" when it is in a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a coding sequence of a nucleic acid if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence of a nucleic acid if it is positioned so as to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof that does not comprise an Fc region when present in nature) if the nucleic acid molecule is positioned so that the expressed fusion protein comprises a heterologous protein or functional fragment thereof abutting a variant Fc region polypeptide upstream or downstream; the heterologous protein may be directly adjacent to the variant Fc region polypeptide or may be separated from the polypeptide by a linker sequence of any length and composition. Similarly, a polypeptide (used synonymously with "protein" herein) molecule is "operably linked" or "operably attached" when it is in a functional relationship with another polypeptide.

[0051] As used herein, when referring to a polypeptide or protein (e.g., a variant Fc region or a monoclonal antibody), the term "functional fragment" refers to a fragment of the protein that retains at least one function of the full-length polypeptide. The size of the fragment can range from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. The functional fragment of the variant Fc region polypeptide of the present invention retains at least one "amino acid substitution" as defined herein. The functional fragment of the variant Fc region polypeptide retains at least one function associated with the Fc region known in the art (e.g., ADCC, CDC, Fc receptor binding, Clq binding, cell surface receptor downregulation, or can, for example, increase the in vivo or in vitro half-life of the polypeptide to which it is operably attached).

[0052] The term "purified" or "purification" refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody can be purified by complete or substantial removal (at least 90%, 91%, 92%, 93%, 94%, 95% or more preferably, at least 96%, 97%, 98% or 99%) of at least one contaminating non-immunoglobulin; it can also be purified by removing immunoglobulins that do not bind to the same antigen. Removal of non-immunoglobulins and / or removal of immunoglobulins that do not bind to a specific antigen increases the percentage of antigen-specific immunoglobulins in the sample. In another example, a polypeptide (e.g., immunoglobulin) expressed in a bacterial host cell is purified by complete or substantial removal of host cell proteins; the percentage of the polypeptide in the sample is thereby increased.

[0053] The term "native" when it refers to a polypeptide (e.g., an Fc region) is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of the polypeptide (in the case where the polypeptide normally occurs in nature) or a naturally occurring polymorph thereof. A native polypeptide (e.g., a native Fc region) can be produced by recombinant means or can be isolated from a naturally occurring source.

[0054] As used herein, the term "expression vector" refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences required for the expression of the operably linked coding sequence in a particular host organism.

[0055] As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells, such as E. coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro, in situ, or in vivo.

[0056] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of the Fc region of an immunoglobulin heavy chain may vary, the canine IgG heavy chain Fc region is generally defined as extending from, for example, the amino acid residue at position 231 to its carboxyl end. In certain embodiments, the variant comprises only a portion of the Fc region and may or may not include the carboxyl end. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In certain embodiments, variants having one or more constant domains are contemplated. In other embodiments, variants not having such constant domains (or only having a portion of such constant domains) are contemplated.

[0057] The "CH2 domain" of a canine IgG Fc region generally extends from, e.g., about amino acid 231 to about amino acid 340 (see Figure 2 ). The CH2 domain is unique in that it is not tightly paired with another domain. Two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the complete native IgG molecule.

[0058] The "CH3 domain" of a canine IgG Fc region is generally the C-terminal end of the CH2 domain in the Fc region, for example, the residue stretch extending from about amino acid residue 341 to about amino acid residue 447 (see Figure 2 ).

[0059] A "functional Fc region" has an "effector function" of a native sequence Fc region. At least one effector function of a polypeptide comprising a variant Fc region of the present invention may be enhanced or reduced relative to a polypeptide comprising a native Fc region or a parent Fc region of a 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), etc. Such effector functions may require that the Fc region be operably linked to a binding domain (e.g., an antibody variable domain) and may be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, target cell depletion in intact or fractionated blood samples, etc.).

[0060] A "native sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region commonly found in nature. An exemplary native sequence canine Fc region is Figure 2 The native sequence of the canine IgG Fc region is shown and included in .

[0061] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region (or a fragment thereof) by at least one "amino acid substitution" as defined herein. In a preferred embodiment, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or in the Fc region of a parent polypeptide, preferably 1, 2, 3, 4 or 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. In an alternative embodiment, a variant Fc region can be produced according to the methods disclosed herein, and this variant Fc region can be fused to a selected heterologous polypeptide, such as an antibody variable domain or a non-antibody polypeptide, such as a binding domain of a receptor or ligand.

[0062] As used herein, in the context of polypeptides, the term "derivative" refers to a polypeptide comprising an amino acid sequence that has been changed by introducing 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 a polypeptide. For example, but not limited to, antibodies can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / capping groups, proteolytic cleavage, linkage with cell ligands or other proteins, etc. Derivative polypeptides can be produced by chemical modification using techniques known to those skilled in the art (including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.). In addition, derivative polypeptides have functions similar to or identical to those of the polypeptides from which they are derived. It should be understood that the polypeptide comprising the variant Fc region of the present invention can be a derivative as defined herein, preferably, derivatization occurs in the Fc region.

[0063] "Substantially of canine origin" as used herein with respect to a polypeptide (e.g., an Fc region or a monoclonal antibody) indicates that the polypeptide has an amino acid sequence that is at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, or even more preferably at least 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence of a native canine amino polypeptide.

[0064] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to an Fc region (e.g., an Fc region of an antibody). A preferred FcR is a native sequence FcR. In addition, a preferred FcR is an FcR that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, FcγRIII subclasses, including allelic variants and alternative splicing 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)). The term "FcR" herein encompasses other FcRs, including those identified in the future.

[0065] The phrases "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) expressing FcRs (e.g., from natural killer ("NK") cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby cause lysis of the target cell. Primary cells used to mediate ADCC NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII.

[0066] As used herein, the phrase "effector cell" refers to a leukocyte (preferably canine) that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least FcγRIII and performs ADCC effector functions. 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., from blood or PBMC).

[0067] A variant polypeptide with an "altered" FcRn binding affinity is a variant polypeptide with enhanced (i.e., increased, greater, or higher) or diminished (i.e., decreased, less, or lower) FcRn binding affinity compared to the variant's parent polypeptide or a polypeptide comprising a native Fc region when measured at pH 6.0. Variant polypeptides that exhibit increased binding to FcRn or increased binding affinity bind to FcRn with greater affinity than the parent polypeptide. Variant polypeptides that exhibit decreased binding to FcRn or decreased binding affinity bind to FcRn with less affinity than their parent polypeptide. Such variants that exhibit decreased binding to FcRn may have little or no significant binding to FcRn, for example, 0 to 20% binding to FcRn compared to the parent polypeptide. A variant polypeptide that binds to FcRn with "enhanced affinity" compared to its parent polypeptide is a variant polypeptide that binds to FcRn with a higher binding affinity than the parent polypeptide when the amounts of the variant polypeptide and the parent polypeptide are substantially the same in a binding assay and all other conditions are the same. For example, a variant polypeptide with enhanced FcRn binding affinity may exhibit an increase in FcRn binding affinity of about 1.10-fold to about 100-fold (more usually about 1.2-fold to about 50-fold) compared to the parent polypeptide, where FcRn binding affinity is determined, for example, in an ELISA assay or other methods available to one of ordinary skill in the art.

[0068] As used herein, "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence by another different "replacement" amino acid residue. One or more replacement residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). The definition of amino acid substitution herein also encompasses substitution with one or more non-naturally occurring amino acid residues. "Non-natural amino acid residues" refer to residues that are capable of covalently binding to adjacent amino acid residues in a polypeptide chain, in addition to those naturally occurring amino acid residues listed above. Examples of non-natural 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).

[0069] The term "assay signal" refers to any method for detecting protein-protein interactions, including but not limited to the output of absorbance measurement, fluorescence intensity or number of decays per minute from a colorimetric assay. The assay format may include ELISA, facs or other methods. The change in "assay signal" may reflect changes in cell viability and / or changes in kinetic dissociation rate, kinetic association rate or both. A "higher assay signal" refers to a measured output number greater than another number (e.g., in an ELISA assay, a variant may have a higher (larger) measured number compared to a parent polypeptide). A "lower assay signal" refers to a measured output number less than another number (e.g., in an ELISA assay, a variant may have a lower (smaller) measured number compared to a parent polypeptide).

[0070] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in concentration units) associated with each Fc receptor-Fc binding interaction. Binding affinity is related to the kinetic dissociation rate (usually expressed in reverse time units, e.g., seconds). -1 The equilibrium dissociation constant is directly related to the ratio of the association rate (usually reported in units of concentration per unit time, e.g., moles / second) divided by the kinetic association rate (usually reported in units of concentration per unit time, e.g., moles / second). In general, it is not possible to unambiguously state whether a change in the equilibrium dissociation constant is due to differences in the association rate, the dissociation rate, or both, unless each of these parameters is determined experimentally (e.g., by BIACORE or SAPIDYNE measurements).

[0071] As used herein, the term "hinge region" refers to the amino acid stretch in canine IgG, for example, extending from position 216 to position 230. Hinge regions of other IgG isotypes can be aligned with the IgG sequence by placing the cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same position.

[0072] "Clq" is a polypeptide that includes the binding site of the Fc region of an immunoglobulin. Clq, together with two serine proteases, Clr and Cls, forms complex Cl, the first component of the CDC pathway.

[0073] As used herein, the term "antibody" is used interchangeably with "immunoglobulin" or "Ig", is used in the broadest sense, and specifically covers 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 activity or functional activity. Single-chain antibodies and chimeric, canine or caninized antibodies, as well as chimeric or CDR-grafted single-chain antibodies, etc., including parts derived from different species, are also covered in the present invention and the term "antibody". The various parts of these antibodies can be chemically linked together in a synthetic manner by conventional techniques, or can be prepared as a continuous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric or caninized chain can be expressed to produce a continuous protein. See, e.g., U.S. Pat. No. 4,816,567; U.S. Pat. No. 4,816,397; WO 86 / 01533; U.S. Pat. No. 5,225,539; and U.S. Pat. Nos. 5,585,089 and 5,698,762. In addition, for primatized antibodies, see Newman, R. et al., BioTechnology, 10: 1455-1460, 1993, and for single-chain antibodies, see Ladner et al., U.S. Pat. No. 4,946,778 and Bird, RE et al., Science, 242: 423-426, 1988. It should be understood that all forms of antibodies comprising an Fc region (or a portion thereof) are encompassed herein within the term "antibody". Additionally, the antibodies can be labeled with a detectable marker, immobilized on a solid phase and / or conjugated to a heterologous compound (eg, an enzyme or toxin) according to methods known in the art.

[0074] As used herein, the term "antibody fragment" refers to a portion of an intact antibody. Examples of antibody fragments may include, but are not limited to, linear antibodies; single-chain antibody molecules; peptides, Fab and Fab fragments of Fc or Fc, and multispecific antibodies formed from antibody fragments. The antibody fragment preferably retains the hinge of the IgG heavy chain and optionally at least a portion of the CH1 region. In other preferred embodiments, the antibody fragment comprises at least a portion of the CH2 region or the entire CH2 region.

[0075] As used herein, the term "functional fragment" when used with reference to a monoclonal antibody is intended to refer to a portion of a monoclonal antibody that still retains functional activity. Functional activity can be, for example, antigen binding activity or specificity, receptor binding activity or specificity, effector function activity, etc. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single-chain Fv (scFv); and Fc fragments. Such terms are described, for example, in Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (Ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Methods in Enzymology, 178: 497-515 (1989) and Day, ED, Advanced Immunochemistry, 2nd ed., Wiley-Liss, Inc., New York York, NY (1990). The term functional fragment is intended to include fragments produced, for example, by protease digestion or reduction of a monoclonal antibody and by recombinant DNA methods known to those skilled in the art.

[0076] As used herein, the term "fragment" refers to a polypeptide comprising an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more consecutive amino acid residues of an amino acid sequence of another polypeptide. In a preferred embodiment, a fragment of a polypeptide retains at least one function of a full-length polypeptide.

[0077] As used herein, the term "chimeric antibody" includes monovalent, divalent or multivalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by the association of a chimeric heavy chain with a chimeric light chain through a disulfide bridge. A divalent chimeric antibody is a tetramer formed by the association of two heavy chain-light chain dimers through at least one disulfide bridge. The chimeric heavy chain for canine antibodies comprises an antigen binding region derived from a heavy chain of a non-canine antibody, which is connected to at least a portion of a canine heavy chain constant region, such as CH1 or CH2. The chimeric light chain for canine antibodies comprises an antigen binding region derived from a light chain of a non-canine antibody, which is connected to at least a portion of a canine light chain constant region (CL). Antibodies, fragments or derivatives of chimeric heavy and light chains with the same or different variable region binding specificities can also be prepared by appropriate association of separate polypeptide chains according to known method steps. Using this method, a host expressing a chimeric heavy chain is cultured separately from a host expressing a chimeric light chain, and the immunoglobulin chains are recovered separately and then associated. Alternatively, the host can be co-cultured and the chains are allowed to spontaneously associate in the culture medium, after which the assembled immunoglobulin or fragment, fragment, or both heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Patents Nos. 6,284,471; 5,807,715; 4,816,567; and 4,816,397).

[0078] As used herein, a "caninized" form of a non-canine (e.g., mouse) antibody (i.e., a caninized antibody) is an antibody containing minimal or no sequences derived from non-canine immunoglobulins. In most cases, a caninized antibody is a canine immunoglobulin (receiver antibody) in which residues from the hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-canine species (donor antibody) having desired specificity, affinity, and capacity such as a mouse, rat, rabbit, human, or non-human primate. In some cases, the framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. In addition, caninized antibodies may be included in residues not found in the recipient antibody or the donor antibody. These modifications are usually performed to further optimize antibody performance. In general, a caninized antibody will include substantially all variable domains of at least one and typically two variable domains, wherein all or substantially all hypervariable loops (CDRs) correspond to those of non-canine immunoglobulins, and all or substantially all FR residues are those of canine immunoglobulin sequences. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin.

[0079] 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 comprises a fusion of an adhesin amino acid sequence that is not an antibody's antigen recognition and binding site (antigen combining site) (i.e., heterologous) with an immunoglobulin constant domain sequence with the desired binding specificity.

[0080] As used herein, the term "ligand binding domain" refers to any natural receptor or any region or derivative thereof that at least retains the qualitative ligand binding ability of the corresponding natural receptor. In certain embodiments, the receptor is from a cell surface polypeptide having an extracellular domain homologous to a member of the immunoglobulin supergene family. Other receptors that are not members of the immunoglobulin supergene family but are still specifically encompassed within this definition are receptors for cytokines, and specifically receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hemopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L- and P-selectins).

[0081] As used herein, the term "receptor binding domain" refers to any natural ligand of a receptor, including, for example, cell adhesion molecules, or any region or derivative of such a natural ligand that retains at least the qualitative receptor binding ability of the corresponding natural ligand.

[0082] As used herein, an "isolated" polypeptide is a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components in its natural environment are materials that will interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteins or non-protein solutes. In certain embodiments, the isolated polypeptide is purified (1) to greater than 95% by weight of the polypeptide, as determined by the Lowry method, and preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated polypeptide includes an in situ polypeptide within a recombinant cell, since at least one component of the natural environment of the polypeptide will not be present. However, the isolated polypeptide will typically be prepared by at least one purification step.

[0083] As used herein, the terms "disorder" and "disease" are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., a pathological condition that predisposes a patient to a particular disorder).

[0084] As used herein, the term "receptor" refers to a polypeptide capable of binding to at least one ligand. Preferably, the receptor is a cell surface receptor or a soluble receptor having an extracellular ligand binding domain and optionally other domains (e.g., a transmembrane domain, an intracellular domain, and / or a membrane anchor). The receptor to be evaluated in the assay described herein can be a complete receptor or a fragment or derivative thereof (e.g., a fusion protein comprising a binding domain of a receptor fused to one or more heterologous polypeptides). In addition, the receptor to be evaluated for its binding properties can be present in a cell, or isolated and optionally coated on an assay plate or some other solid phase, or directly labeled and used as a probe.

[0085] Canine wild-type IgG

[0086] Canine IgG is well known in the art and is fully described in, for example, Bergeron et al., 2014, Veterinary Immunopathology, Vol. 157(1-2), pp. 31-41). In one embodiment, canine IgG is IgG A In another embodiment, canine IgG is IgG B In yet another embodiment, canine IgG is IgG C In another embodiment, canine IgG is IgG D In a specific embodiment, canine IgG is IgG B .

[0087] IgG A IgG B IgG C and IgG D The amino acid and nucleic acid sequences of are also well known in the art.

[0088] In one example, the IgG of the present invention comprises 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 comprises an Fc region, including, for example, a CH2 or CH3 domain, or a combination thereof.

[0089] In a specific example, the wild-type constant domain comprises the amino acid sequence shown in SEQ ID NO.: 1, 2, 3 or 4. In some embodiments, the wild-type IgG constant domain is a homologue, variant, isoform 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.

[0090] IgGs constant domains also include polypeptides having an amino acid sequence substantially similar to the amino acid sequence of the heavy chain and / or light chain. Substantially identical amino acid sequences are defined herein as sequences having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the comparison amino acid sequence as determined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988).

[0091] The present invention also includes nucleic acid molecules encoding IgG or parts thereof as described herein. In one embodiment, the nucleic acid can encode an antibody heavy chain comprising, for example, CH1, CH2, CH3 regions or combinations thereof. In another embodiment, the nucleic acid can encode an antibody heavy chain comprising, for example, any one of the VH regions or portions thereof or any one of the VH CDRs, including any variants thereof. The present invention also includes nucleic acid molecules encoding antibody light chains, the antibody light chains comprising, for example, any one of the CL regions or portions thereof, any one of the VL regions or portions thereof, or any one of the VL CDRs, including any variants thereof. In certain embodiments, nucleic acid encodes both heavy and light chains or portions thereof.

[0092] The amino acid sequence of the wild-type constant domain shown in SEQ ID NO.: 1, 2, 3 or 4 is encoded by its corresponding nucleic acid sequence. For example, the amino acid sequence of the wild-type constant domain shown in SEQ ID NO.: 2 is encoded by the nucleic acid sequence shown in SEQ ID NO.: 5 or 6. In some embodiments, the amino acid sequence of the wild-type constant domain shown in SEQ ID NO.: 1, 2, 3 or 4 is encoded by the nucleic acid sequence shown in SEQ ID NO.: 16, 5, 17 or 18, respectively.

[0093] Modified canine IgG

[0094] The inventors of the present application have found that replacing one or more amino acid residues surprisingly and unexpectedly enhances affinity for FcRn. As used herein, amino acid position numbering refers to positions numbered according to the Eu index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Significance, 5th Edition. U.S. National Institutes of Health Public Health Service, Bethesda, Maryland (1991)).

[0095] Thus, in one embodiment, the present invention provides a modified IgG comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat.

[0096] In some embodiments, the constant domain comprises one or more of the following 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, D312 K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A42 6C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A4 26R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

[0097] In a specific example, the wild-type amino acid sequence of the present invention as shown in SEQ ID NO.: 1, 2, 3 or 4 comprises one or more mutations described herein. In some embodiments, the mutant IgG constant domain is a homologue, title, isomer or functional fragment having one or more mutations described herein. Each possibility represents a separate embodiment of the present invention.

[0098] The amino acid sequence of the mutant constant domain is encoded by its corresponding mutant nucleic acid sequence.

[0099] Methods for preparing the antibody molecules of the invention

[0100] Methods for preparing antibody molecules are well known in the art and are fully described in U.S. Patents 8,394,925; 8,088,376; 8,546,543; 10,336,818; and 9,803,023, and U.S. Patent Application Publication 20060067930, which are incorporated herein by reference in their entirety. Any suitable method, process or technique known to those skilled in the art may be used. Antibody molecules having variant Fc regions of the present invention may be produced according to methods well known in the art. In some embodiments, variant Fc regions may be fused to selected heterologous polypeptides, such as antibody variable domains or the binding domains of receptors or ligands.

[0101] With the advent of molecular biological methods and recombinant technology, those skilled in the art can produce antibodies and antibody-like molecules by recombinant means, and thereby produce gene sequences encoding specific amino acid sequences present in the polypeptide structure of the antibody. Such antibodies can be produced by cloning the gene sequences encoding the polypeptide chains of the antibody or by direct synthesis of the polypeptide chains, wherein the assembly of the synthetic chains forms an active tetramer (H2L2) structure with affinity for specific epitopes and antigenic determinants. This allows the production of antibodies with sequence characteristics of neutralizing antibodies from different species and sources at any time.

[0102] Regardless of the source of the antibody or how it is constructed recombinantly, or how it is in vitro or in vivo using transgenic animals, laboratory or commercial-scale large cell cultures, using transgenic plants, or by direct chemical synthesis, no living organisms are used to synthesize at any stage of the process, all antibodies have a similar overall 3-dimensional structure. The structure is usually given as H2L2, and refers to the fact that antibodies usually contain two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions that can interact with structurally complementary antigenic targets. The region that interacts with the target is called a "variable" or "V" region, and is characterized in that antibodies with different antigenic specificities have differences in amino acid sequence. The variable region of the H chain or L chain contains an amino acid sequence that can specifically bind to the antigenic target.

[0103] As used herein, the term "antigen binding region" refers to the portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antibody for the antigen. The antibody binding region includes the "framework" amino acid residues necessary to maintain the correct configuration of the antigen binding residues. An antigen binding region is provided in the variable region of the H or L chain, which is a smaller sequence called "hypervariable" because it has great variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions explain the basic specificity of antibodies for specific antigenic determinant structures.

[0104] CDR represents the discontinuous extension of amino acid in variable region, but no matter how species, it has been found that the positioning position of these key amino acid sequences in variable heavy chain region and variable light chain region has similar position in the amino acid sequence of variable chain. The variable heavy chain and light chain of all antibodies each have three CDR regions, and each CDR region is not adjacent to other CDR regions. In all mammalian species, antibody peptides contain constant region (that is, highly conserved) and variable region, and in the latter, there is CDR and the so-called "framework region" consisting of the amino acid sequence in the variable region of heavy chain or light chain but outside CDR.

[0105] The present invention further provides a vector comprising at least one of the nucleic acids described above. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which will be able to encode an amino acid. By considering the abnormal base pairing relationship and the frequency of the actual use of a specific codon (to encode a specific amino acid) in a eukaryotic or prokaryotic cell expressing an antibody or part, the probability that a specific oligonucleotide will actually constitute an actual coding sequence can be estimated. Lathe et al., 183 Journal of Molecular Biology (J.Molec.Biol.) 1-12 (1985) discloses such "codon usage rules". Using Lathe's "codon usage rules", a single nucleotide sequence or a set of nucleotide sequences containing a theoretically "most likely" nucleotide sequence that can encode a canine IgG sequence can be identified. It is also hoped that the antibody coding region for the present invention can be provided by using standard molecular biology techniques to change existing antibody genes, and the standard molecular biology techniques produce variants of antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions and substitutions in the amino acid sequences of antibodies or peptides.

[0106] For example, one type of substitution is a conservative amino acid substitution. Such substitutions are those in which a given amino acid in a canine antibody peptide is replaced by another amino acid with similar characteristics. Commonly considered conservative substitutions are substitutions of one by another among the aliphatic amino acids Ala, Val, Leu and lie; hydroxyl residues Ser and Thr interchange, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gin, exchange of basic residues Lys and Arg, substitution among aromatic residues Phe, Tyr, etc. Guidance on which amino acid changes may be phenotypically silent is found in Bowie et al., 247 Science 1306-10 (1990).

[0107] Variant canine antibodies or peptides may have full functionality, or may lack function in one or more activities. Fully functional variants usually contain only conservative changes or changes in non-critical residues or non-critical regions. Functional variants can also contain similar amino acid replacements that do not produce functional changes or produce insignificant functional changes. Alternatively, such replacements can have positive or negative effects on function to some extent. Non-functional variants usually contain one or more non-conservative amino acid replacements, deletions, insertions, inversions or truncations, or replacements, insertions, inversions or deletions in key residues or key regions.

[0108] Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations into each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as anti-epitope binding or in vitro ADCC activity. Sites critical for ligand-receptor binding can also be determined by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 Journal of Mol. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[0109] In addition, polypeptides generally contain amino acids other than twenty kinds of "naturally occurring" amino acids. In addition, many amino acids, including terminal amino acids, can 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 heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositols, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamic acid, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginization and ubiquitination. Such modifications are well known to those skilled in the art and have been described in great detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation are described in most basic texts, such as Proteins-Structure and Molecular Properties (2nd ed., TE Creighton, WH Freeman & Co., NY 1993). Many detailed reviews on this topic are available, for example, Wold, Posttranslational Covalent Modification of Proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983); Seifter et al. 182 Methods in Enzymology 626-46 (1990); and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0110] In another aspect, the invention provides antibody derivatives. "Derivatives" of antibodies contain additional chemical moieties that are not normally part of the protein. Covalent modifications of proteins are included within the scope of the invention. Such modifications can be introduced into the molecule by reacting the targeted 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 with bifunctional agents well known in the art is suitable for crosslinking antibodies or fragments with water-insoluble carrier matrices or other macromolecular carriers.

[0111] Derivatives also include radiolabeled monoclonal antibodies. For example, radioactive iodine (251, 1311), carbon (4C), sulfur (35S), indium, tritium (H 3 ) etc.; a conjugate of a monoclonal antibody having biotin or avidin and an enzyme (such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carboxylic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase); and a conjugate of a monoclonal antibody and a bioluminescent agent (such as luciferase), a chemiluminescent agent (such as acridinium ester) or a fluorescent agent (such as phycobiliprotein).

[0112] Another derivative bifunctional antibody of the present invention is a bispecific antibody produced by combining the parts of two separate antibodies that recognize two different antigen groups. This can be achieved by crosslinking or recombinant technology. In addition, the part can be added to the antibody or a part thereof to increase the half-life in vivo (for example, by extending the time of removal from the bloodstream). Such technology includes, for example, adding a PEG part (also referred to as pegylation), and is well known in the art. See U.S. Patent Application Publication No. 20030031671.

[0113] In certain embodiments, the nucleic acid encoding the subject antibody is directly introduced into the host cell, and the cell is cultivated under conditions sufficient to induce the expression of the encoded antibody. After the subject nucleic acid is introduced into the cell, the cell is cultivated for a period of about 1 to 24 hours under selection, usually at 37°C, to allow the antibody to be expressed. In one embodiment, the antibody is secreted into the supernatant of the culture medium of cell growth. Traditionally, monoclonal antibodies have been produced as natural molecules in mouse hybridoma cell lines. In addition to the technology, the present invention provides recombinant DNA expression of antibodies. This allows the production of antibodies in selected host species, as well as a series of antibody derivatives and fusion proteins.

[0114] The nucleotide sequence encoding at least one antibody, part or polypeptide of the present invention can be recombined with carrier DNA according to conventional techniques, including blunt or staggered ends for connection, restriction enzyme digestion for providing appropriate ends, sticky end filling when appropriate, alkaline phosphatase treatment for avoiding undesirable connection and connection with appropriate ligase. The technology for such manipulation is disclosed in the same documents as those of Maniatis et al., Molecular Cloning Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1982 and 1989) and Ausubel et al. 1993, which can be used to construct the nucleotide sequence encoding antibody molecules or their antigen binding regions.

[0115] If a nucleic acid molecule (such as DNA) contains a nucleotide sequence that includes transcriptional and translational regulatory information, and such sequence is "operably linked" to a nucleotide sequence encoding a polypeptide, then the nucleic acid molecule is said to be "capable of expressing" the polypeptide. An operable connection is a connection in which the regulatory DNA sequence and the DNA sequence for which expression is sought are linked in a manner that allows the gene to be expressed as a recoverable amount of the peptide or antibody moiety. The precise nature of the regulatory region required for gene expression can vary from organism to organism, as is well known in similar fields. See, for example, Sambrook et al., 2001 supra; Osby et al., 1993 supra.

[0116] Therefore, the present invention encompasses expression of antibodies or peptides in prokaryotic or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds and mammalian cells in vivo or in situ, or host cells of mammalian, insect, bird or yeast origin. Mammalian cells or tissues can have human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog or cat origin. Any other suitable mammalian cells known in the art can also be used.

[0117] In one embodiment, the nucleotide sequence of the invention is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. For this purpose, any of a wide variety of vectors may be employed. See, e.g., Osby et al., 1993 supra. Important factors in selecting a particular plasmid or viral vector include: the ease with which recipient cells containing the vector can be identified and selected from those that do not; the number of copies of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0118] Exemplary prokaryotic vectors known in the art include plasmids, such as plasmids capable of replication in E. coli (e.g., pBR322, CoIE1, pSC101, pACYC 184, pi.vX). Such plasmids are disclosed, for example, by Maniatis et al., 1989 supra; Osbe et al., 1993 supra. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan in THE MOLECULAR BIOLOGY OF BACILLI 307-329 (New York Academic Press, 1982). Suitable Streptomyce plasmids include p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)), and Streptomycete phages, such as phLC31 (Chater et al., SIXTH INT'LSYMPOSIUM 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 Osby et al., 1993 supra.

[0119] Alternatively, gene expression elements suitable for expressing cDNA encoding anti-NGF antibodies or peptides include, but are not limited to (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci. USA, 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splicing regions and polyadenylation sites, such as the splicing region and polyadenylation site derived from the SV40 late region (Okayarea et al., 1983) and (c) a polyadenylation site such as that in SV40 (Okayama et al., 1983).

[0120] Immunoglobulin cDNA genes can be expressed using the SV40 early promoter and its enhancer, mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globulin intervening sequence, immunoglobulin and rabbit S-globulin polyadenylation sites, and SV40 polyadenylation elements as expression elements as described by Weidle et al. 51 Gene 21 (1987). For immunoglobulin genes consisting of part cDNA and part genomic DNA (Whittle et al. 1 Protein Engin. 499 (1987)), the transcription promoter can be human cytomegalovirus, the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be native chromosomal immunoglobulin sequences.

[0121] In one embodiment, for expressing cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron of more than 31 bp, and the polyadenylation and transcription terminal regions are derived from natural chromosomal sequences corresponding to synthetic immunoglobulin chains. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements described above to achieve protein expression in mammalian cells.

[0122] Each fusion gene can be assembled in an expression vector or inserted into an expression vector. Recipient cells capable of expressing immunoglobulin chain gene products are then transfected with genes encoding peptides or H chains or L chains alone, or co-transfected with H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the incorporated genes and the expressed immunoglobulin chains or complete antibodies or fragments are recovered from the culture.

[0123] In one embodiment, the fusion gene encoding peptide or H and L chain or its part is assembled in a separate expression vector, and then the expression vector is used for co-transfection recipient cells. Alternatively, the fusion gene encoding H and L chain can be assembled on the same expression vector. For the transfection of expression vector and the production of antibody, the recipient cell line can be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes, and have a mechanism for glycosylation of immunoglobulins. Myeloma cells can grow in culture or in the peritoneal cavity of mice (wherein secreted immunoglobulins can be obtained from ascites). Other suitable recipient cells include lymphoid cells, such as B lymphocytes from canine or non-canine sources, hybridoma cells from canine or non-canine sources, or interspecific heterohybridoma cells.

[0124] The expression vector carrying the antibody construct or polypeptide of the present invention can be introduced into an appropriate host cell by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and the use of polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical methods such as electroporation, direct microinjection and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[0125] Yeast can provide substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeast undergoes post-translational peptide modifications, including glycosylation. A. There are many recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of cloned mammalian gene products and secretes peptides (i.e., propeptides) with the leader sequence. Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0126] The production, secretion and stability levels of peptides, antibodies, fragments and regions thereof of yeast gene expression systems can be routinely evaluated. Any of a series of yeast gene expression systems can be used, and these expression systems have promoters and termination elements from actively expressed genes encoding saccharolytic enzymes produced in large quantities when yeast grows in a medium rich in glucose. Known glycolytic genes can also provide very effective transcription control signals. For example, promoter and terminator signals of phosphoglycerate kinase (PGK) genes can be used. A variety of methods can be used to evaluate the best expression plasmid for immunoglobulin cDNAs expressed in yeast. See Volume II DNA Cloning, 45-66, (Glover, ed.), IRL Press, Oxford, UK, 1985).

[0127] Bacterial strains can also be used as hosts for producing antibody molecules or peptides described in the present invention. Plasmid vectors containing replicons and control sequences derived from species compatible with host cells are used in combination with these bacterial hosts. The vector carries a replication site and a specific gene that can provide phenotypic selection in transformed cells. A large number of methods can be used to evaluate expression plasmids for producing antibodies, fragments and regions or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria (see Glover, 1985, supra; Osby, 1993, supra; Sabrook, 2001, supra; Colligan et al., Current Protocols in Immunology, John Wiley & Sons, NY, New York (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, New York (1997-2001).

[0128] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications for immunoglobulin molecules, including leader peptide removal, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. In addition to the lymphoid cells described above, mammalian cells that can be used as hosts for producing antibody proteins also include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems can be used to express cloned peptide H and L chain genes in mammalian cells (see Glover, 1985 same document). Different methods can be used to obtain complete H2L2 antibodies. H and L chains can be co-expressed in the same cell to achieve intracellular association and connection of H and L chains into complete tetrameric H2L2 antibodies and / or peptides. Co-expression can be performed by using the same or different plasmids in the same host. The genes of H and L chains and / or peptides can be placed in the same plasmid, and then the plasmid is transfected into cells, thereby directly selecting cells expressing two chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the L chain, followed by transfection of the resulting cell line with a plasmid containing a second selectable marker for the H chain. Cell lines producing peptides and / or H2L2 molecules by either route can be transfected with plasmids encoding additional copies of the peptide, H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher yields of assembled H2L2 antibody molecules or increased stability of the transfected cell line.

[0129] In order to produce recombinant antibodies for a long time and in high yield, stable expression can be used. For example, the cell line of stably expressed antibody molecules can be engineered. Host cells can be transformed with immunoglobulin expression cassettes and selectable markers, rather than using expression vectors containing viral replication origins. After introducing foreign DNA, engineered cells can be grown in enriched medium for 1 to 2 days, and then converted to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and allows cells to stably integrate plasmids into their chromosomes and grow to form variant regions (foci), and then the variant regions can be cloned and expanded into cell lines. This engineered cell line can be particularly suitable for screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0130] Once the antibodies of the invention have been produced, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly after protein A with affinity for a specific antigen, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In many embodiments, the antibodies are secreted from the cells into the culture medium and collected from the culture medium.

[0131] Pharmaceutical and veterinary applications

[0132] The present invention also provides a pharmaceutical composition comprising a molecule of the present invention and one or more pharmaceutically acceptable carriers. More specifically, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and an antibody or peptide according to the present invention as an active ingredient.

[0133] "Pharmaceutically acceptable carriers" include any excipient that is nontoxic to cells or animals exposed thereto at the dosages and concentrations employed. Pharmaceutical compositions may include one or additional therapeutic agents.

[0134] "Pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of animals without producing excessive toxicity, irritation, allergic response, or other problematic complications commensurate with a reasonable benefit / risk ratio.

[0135] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents, and absorption delaying agents.

[0136] Pharmaceutically acceptable carriers include water; saline; phosphate-buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphates, citrates and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; EDTA; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN, polyethylene glycol (PEG) and PLURONICS; isotonic agents such as sugars, polyols such as mannitol and sorbitol, and sodium chloride; and combinations thereof.

[0137] The pharmaceutical compositions of the present invention can be formulated in a variety of ways, including, for example, liquid, semisolid or solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills or powders. In some embodiments, the 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, controlled, extended or delayed release composition.

[0138] The composition of the present invention can be administered as a separate therapeutic agent, or in combination with other therapeutic agents. It can be administered alone, but is generally administered together with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein can be performed in any suitable manner, including injection (such as intraperitoneal, subcutaneous or intramuscular injection), oral or by topical administration of the antibody (usually carried in a pharmaceutical formulation) to the airway surface. Topical administration to the airway surface can be performed by intranasal administration (for example, by using a dropper, swab or inhaler). Topical administration of antibodies to the airway surface can also be performed by inhalation administration, such as by generating inhalable particles (including both solid clones and liquid particles) of a pharmaceutical formulation containing antibodies in the form of an aerosol suspension, and then causing the individual to inhale the inhalable particles. Methods and devices for administering inhalable particles of pharmaceutical formulations are well known, and any conventional techniques can be used.

[0139] In some desirable embodiments, the antibody is administered by parenteral injection. For 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 in which the antibody or its mixture is dissolved in an acceptable carrier, such as an aqueous vehicle, such as 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 fixed oils can also be used. These solutions are sterile and generally do not contain particulate matter. These compositions can be sterilized by conventional well-known sterilization techniques. The composition can contain pharmaceutically acceptable auxiliary substances required to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The antibody concentration in these formulations can vary greatly, for example, from less than about 0.5%, usually at or at least about 1% to as much as 15% or 20% by weight, and will be selected based primarily on the volume of fluid, viscosity, etc., according to the specific mode of administration selected. The vehicle or lyophilized powder may contain additives (e.g., sodium chloride, mannitol) to maintain isotonicity and additives (e.g., buffers and preservatives) to maintain chemical stability. The formulation is sterilized by conventional techniques. The actual method for preparing a composition that can be administered parenterally will be known or apparent to those skilled in the art, and is described in more detail in, for example, REMINGTON'S PHARMA. SCI. (15th edition, Mack Pub. Co., Easton, Pa., 1980).

[0140] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technology has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques can be used. It will be appreciated by those skilled in the art that lyophilization and reconstitution can cause varying degrees of antibody activity loss and that the use content may have to be adjusted in order to compensate. Compositions containing antibodies of the present invention or their mixtures can be used to prevent the recurrence of existing diseases and / or therapeutic treatment of existing diseases. Suitable pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in this technical field. In therapeutic applications, compositions are administered to individuals with diseases in an amount sufficient to cure or at least partially inhibit or alleviate the disease and its complications.

[0141] The effective dose of the compositions of the invention for treating a condition or disease as described herein varies depending on many different factors including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the target site; the physiological state of the animal; other drugs being administered; whether the treatment is prophylactic or therapeutic; the age, health, and weight of the recipient; the nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the desired effect.

[0142] Single or multiple administrations of the compositions may be carried out at the dosage level and pattern selected by the treating veterinarian.In any case, the pharmaceutical formulation should provide an amount of one or more antibodies of the invention sufficient to effectively treat the individual.

[0143] Therapeutic doses can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0144] The pharmaceutical compositions of the invention may include a "therapeutically effective amount". A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at dosages and for periods of time necessary. The therapeutically effective amount of a molecule can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

[0145] On the other hand, the compositions of the present invention can be used, for example, to treat various diseases and disorders in dogs. As used herein, the terms "treat" and "treatment" refer to therapeutic treatments, including prophylactic or preventive measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, alleviation of the extent of the disease or condition, stabilization of the disease or condition (i.e., a situation where the disease or condition has not worsened), delay or slowing of the progression of the disease or condition, improvement or alleviation of the disease or condition, and alleviation of the disease or condition (whether partial or complete), whether detectable or undetectable. Situations requiring therapy include situations where a disease or condition is already present, as well as situations where a disease or condition is susceptible to, or situations where a disease or condition is to be prevented.

[0146] All patents and literature references cited in this specification are incorporated herein by reference in their entirety.

[0147] The following examples are provided to supplement the previous disclosure and provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the subject matter described. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and the various modifications or changes made therefrom will be apparent to those skilled in the art and are included in the true scope of the present invention, and can be carried out without departing from the true scope of the present invention.

[0148] Examples

[0149] Example 1: Construction of canine IgG Fc mutants

[0150] Construction of whole canine IgG was performed as described by Bergeron et al. (Bergeron et al., 2014, Veterinary Immunopathology, Vol. 157(1-2), pp. 31-41) Figure 1 ), wherein a plasmid containing sequences encoding the canine constant region of the IgGB (65) subclass was utilized and the VH / VL sequences of each mAb studied herein were inserted upstream and in frame with the nucleotides encoding the constant domains. Mutations were incorporated into each corresponding position of the CH1, CH2, or CH3 domain of each plasmid by direct DNA synthesis of the constant region as a gene fragment ( Figure 2 ) and then subcloned into the corresponding variable region of interest.

[0151] Expression and purification

[0152] Monoclonal antibody (mAb) mutants are expressed in mammalian suspension cell system EXPICHO-S (Chinese hamster ovary) cells obtained from Thermo Fisher. Suspension EXPICHO-S cells are maintained between 0.14 and 8.0 × 10e6 cells / ml in EXPICHO expression medium (Gibco). On the -1 day and transfection day, cells are diluted according to the ExpiCHO protocol user manual. As described in the protocol, the diluted cells are transfected according to the maximum titer conditions using reagents derived from ExpiFectamine CHO transfection kit (Gibco). After cultivating 12 to 14 days, cultures are collected and clarified. Antibodies are purified from clarified supernatants by protein A chromatography on MabSelect Sure LX (GE Healthcare) pre-equilibrated with PBS. After sample loading, the resin is washed with PBS, and then washed with 20mM sodium acetate of pH 5.5. The sample was eluted from the column with 20 mM acetic acid at pH 3.5. After elution, a pool was prepared and neutralized to 4% with the addition of 1 M sodium acetate. Depending on the available volume and intended use, the sample was sometimes exchanged into the final buffer (e.g., PBS, other buffers). The concentration was measured by absorbance at 280 nm.

[0153] SDS-PAGE

[0154] Non-reducing (nr) and reducing sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) were performed using 4-12% Bis-Tris NuPAGE gels in MES-SDS running buffer and SeeBlue Plus 2 standards (both 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 gel was stained with Coomassie blue to detect protein bands.

[0155] Example 2: FcRn binding assay

[0156] Canine FcRn was isolated and prepared, and mutant FcIgG was determined for canine FcRn according to Bergeron et al. discussed above. Canine FcRn-α subunit and β-microglobulin were amplified using standard PCR. FcRn-α subunit and β-microglobulin were co-transfected into HEK 293 cells, and the FcRn complex was purified via a C-terminal His tag by IMAC affinity purification. The FcRn complex was biotin labeled by BirA enzyme biotin protein reaction. KD was measured by Biacore T200 or Biacore 8K (Cytiva life sciences).

[0157] FcRn was captured on the surface of the sensor using a modified SA capture method. 10mM MES; 150mM NaCl; 0.005% Tween20; 0.5mg / mL BSA; pH6 was used as capture method running buffer and titration. 1×HBS-P, 0.5mg / mL BSA; pH7.4 was also used for method running buffer and titration. Fc mutant IgG was flowed over the receptor surface and affinity was determined using T200 evaluation software or Biacore 8K evaluation software. (Table 1). A blank run containing only buffer was subtracted from all runs. The flow cell was regenerated using 50mM Tris pH8. Runs were performed at 15°C.

[0158] Mutations made at the corresponding positions had a significant effect on the affinity of IgG for FcRn at pH 6.

[0159] Binding of wild-type (WT) and mutant IgG to canine FcRn was measured by surface plasmon resonance (Biacore).

[0160] Table 1. Effect of mutants on FcRn binding affinity.

[0161]

[0162]

[0163]

[0164] Mutants are numbered according to the EU index as in Kabat. LS = low signal.

[0165] The results clearly show that mutations made at various positions have a significant impact on the affinity of IgG for FcRn.

[0166] Example 3: FcRn binding validation

[0167] method

[0168] The Fc regions of canine backbone subclasses IgGA and IgGB were first designed using their corresponding CH2 and CH3 regions. The protein modeling feature of Alphafold 2.2 developed by an artificial intelligence company (Deepmind) was implemented to model the 3D structures of the wild-type (WT) and mutant constructs of each canine backbone subclass IgGA and IgGB.

[0169] The Molecular Operating Environment (MOE) (MOE2019.0102), developed by the Chemical Computing Group, provides a flexible and automated graphical user interface for protein modeling. To analyze structural differences, a sequence-to-profile alignment algorithm uses a scoring algorithm to rank sequence templates, and a score above 85% ensures the selection of protein templates with physically realistic structures. The model is then optimized using a systematic pipeline, and the structural stability of the model is verified using Ramachandran Plots, which examine the stereochemical quality of the protein structure.

[0170] result

[0171] The methods described above were 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, Q311 F, 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, A 426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A4 26T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W, and overlapping and mutated residues are shown in ball-and-stick format ( Figure 4 A). RMSD plots were generated to calculate the root mean square deviations (RMSDs) of the two WT structures canine-IgGA_WT and canine-IgGB WT relative to each other. Figure 4 B). The RMSD value of 100 or less was considered as the criterion for considering two structures similar. The results showed that the canine IgGA construct was identical to the canine IgGB subclass, with the average RMSD value of the structure being The RMSD at the five positions subjected to mutation scanning is also noted in Table 3, with values ​​ranging from 0.226 to

[0172] The same procedure was also followed to study the following mutations in both canine subtypes: T286C, T286D, T286E, T286G, T286H, T286I, T286K, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, Q311C, Q311D, Q311E, Q311 F. Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q31 1T, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D3 12L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A426C, A 426D, 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 (Table 3). Similarly, no significant changes in the model were observed at each mutation position between the two subclasses, ranging from 0.226 to As indicated by the RMSD value range of , the proteins were the same as those of WT (Table 3).

[0173] Table 2. Comparison of root mean square deviations (RMSD) of residues T286, Q311, D312, A426 and Y436 in protein models of WT canine IgGA and IgGB.

[0174]

[0175] Table 3. Comparison of root mean square deviations (RMSD) of mutation libraries at residue positions T286, Q311, D312, A426 and Y436 in protein models of canine IgGA and IgGB.

[0176]

[0177]

[0178]

[0179] in conclusion

[0180] Molecular modeling was performed for all mutations in the two subclasses of the canine backbone, IgGA and IgGB, and validated using MOE2019.0102. The Fc fold and residue configurations between subclasses showed less than The RMSD of canine IgG1 and IgG2 protein structures is 1.3 fold higher than that of IgG1, indicating that the canine IgG1 and IgG2 protein structures have extremely high identity at all mutation positions described herein and therefore will function in a similar manner.

[0181] Having described the preferred embodiments of the present invention, it should be understood that the present invention is not limited to the exact embodiments and that various changes and modifications may be implemented therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

1. A modified IgG comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat, and wherein the constant domain comprises one or more of the following 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, A426 C. A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A42 6R, A426S, A426T, A426V, A426W, 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 an IgG having the wild-type canine IgG constant domain.

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

4. The modified IgG according to claim 1, wherein the IgG is IgG A IgG B IgG C or IgG D .

5. The modified IgG according to claim 1, wherein the IgG constant domain is IgG A IgG B IgG C or IgG D constant domain.

6. The modified IgG according to claim 1, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain.

7. The modified IgG according to claim 1, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

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

1.

9. A pharmaceutical composition comprising the modified IgG according to claim 1 and a pharmaceutically acceptable carrier.

10. A kit comprising the modified IgG according to claim 1 in a container and instructions for use.

11. A polypeptide comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 286, 311, 312, 426 or 436 as numbered according to the EU index as in Kabat, and wherein the constant domain comprises one or more of the following 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, A426 C. A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A42 6R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

12. The polypeptide according to claim 11, wherein the affinity of the polypeptide for FcRn is higher than that of an IgG polypeptide having the wild-type canine IgG constant domain.

13. The polypeptide according to claim 11, wherein the polypeptide is a canine or caninized IgG polypeptide.

14. The polypeptide according to claim 11, wherein the IgG is IgG A IgG B IgG C or IgG D .

15. The polypeptide of claim 11, wherein the IgG constant domain is IgG A IgG B IgG C or IgG D constant domain.

16. The polypeptide of claim 11, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain.

17. The polypeptide of claim 11, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

18. The polypeptide of claim 11, wherein the wild-type canine IgG constant domain comprises the amino acid sequence shown in SEQ ID NO.:

1.

19. An antibody comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 286, 311, 312, 426 or 436 as numbered according to the EU index as in Kabat, and wherein the constant domain comprises one or more of the following 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, A426 C. A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A42 6R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

20. The antibody of claim 19, wherein the polypeptide has a higher affinity for FcRn than a polypeptide of IgG having the wild-type canine IgG constant domain.

21. The antibody of claim 19, wherein the polypeptide is a polypeptide of canine or caninized IgG.

22. The antibody according to claim 19, wherein the IgG is IgG A IgG B IgG C or IgG D .

23. The antibody of claim 19, wherein the IgG constant domain is IgG A IgG B IgG C or IgG D constant domain.

24. The antibody of claim 19, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain.

25. The antibody of claim 19, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

26. The antibody of claim 19, wherein the wild-type canine IgG constant domain comprises the amino acid sequence shown in SEQ ID NO.:

1.

27. A pharmaceutical composition comprising the antibody according to claim 19 and a pharmaceutically acceptable carrier.

28. A kit comprising the antibody of claim 19 in a container and instructions for use.

29. A vector comprising a nucleic acid sequence encoding the amino acid sequence of the antibody according to claim 19, wherein the wild-type canine IgG constant domain comprises the amino acid sequence shown in SEQ ID NO.:

1.

30. An isolated cell comprising the vector of claim 29.

31. A method of producing an antibody or molecule, the method comprising: providing a cell according to claim 30; and culturing the cell.

32. A method of producing an antibody, the method comprising: providing the antibody according to any one of claims 19 to 26.

33. A fusion molecule comprising: a canine IgG constant domain comprising at least one amino acid substitution relative to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 286, 311, 312, 426 or 436 as numbered according to the EU index as in Kabat, and wherein the constant domain comprises one or more of the following 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, A426 C. A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A42 6R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

34. The molecule of claim 33, wherein the polypeptide has a higher affinity for FcRn than a polypeptide of IgG having said wild-type canine IgG constant domain.

35. The molecule of claim 33, wherein the polypeptide is a canine or caninized IgG polypeptide.

36. The molecule of claim 33, wherein the IgG is IgG A IgG B IgG C or IgG D .

37. The molecule of claim 33, wherein the IgG constant domain is IgG A IgG B IgG C or IgG D constant domain.

38. The molecule of claim 33, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain.

39. The molecule of claim 33, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

40. The molecule of claim 33, wherein the wild-type canine IgG constant domain comprises the amino acid sequence shown in SEQ ID NO.:

1.

41. A pharmaceutical composition comprising the molecule according to claim 33 and a pharmaceutically acceptable carrier.

42. A kit comprising the molecule of claim 33 in a container and instructions for use.

43. A method for enhancing the binding affinity of canine IgG to a neonatal Fc receptor (FcRn), the method comprising: providing a modified IgG comprising a canine IgG constant domain, the canine IgG constant domain comprising at least one amino acid substitution relative to a 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 as in Kabat, and wherein the canine IgG constant domain comprises one or more of the following mutations: 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, D312 K, D312L, D312M, D312N, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, A42 6C, A426D, A426E, A426G, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A4 26R, A426S, A426T, A426V, A426W, Y436C, Y436D, Y436E, Y436F, Y436G, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

44. The method of claim 43, wherein the canine IgG constant domain has a serum half-life that is longer than an IgG having the wild-type canine IgG constant domain.

45. The method of claim 43, wherein the IgG is IgGA, IgGB, IgGC or IgGD.

46. ​​The method of claim 43, wherein the IgG constant domain is a constant domain of IgGA, IgGB, IgGC, or IgGD.

47. The method of claim 43, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain.

48. The method of claim 43, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

49. The method of claim 43, wherein the wild-type canine IgG constant domain comprises the amino acid sequence shown in SEQ ID NO.: 1, 2, 3 or 4.

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