Bovine antibody mutants

Abstract

The present invention relates generally to bovine antibody mutants and uses thereof. In particular, the present invention relates to mutations in the constant region of bovine antibodies for ameliorating 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 / 370,533, filed on August 5, 2022, which is incorporated herein by reference in its entirety. Technical Field

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

[0004] Bovine IgG monoclonal antibodies (mAbs) can be effective therapeutic agents in veterinary medicine. Several years ago, three subclasses of bovine IgG were identified. However, only limited work has been done to improve the properties of bovine IgG.

[0005] Through the circulation mechanism, the neonatal Fc receptor (FcRn) prolongs the half-life of IgG in the pH-dependent interaction with the IgG fragment crystallizable (Fc) region. 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 a slightly 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] Engineering the Fc region to tailor its interaction with FcRn has become a promising approach for enhancing the activity of therapeutic antibodies.

[0007] Therefore, new bovine IgG Fc region mutations are needed to improve various properties of bovine IgG. Summary of the invention

[0008] The present invention relates to mutant bovine IgGs that exhibit desired properties relative to wild-type bovine IgGs. Specifically, the inventors of the present application have found that replacing the amino acid residue at position 252, 286, 311, 312, 426, 428, 434 or 436 (numbered according to the Eu index as in Kabat) with another amino acid surprisingly and unexpectedly exhibits the desired effect. In an exemplary embodiment, the unexpected desired effect includes, but is not limited to, an increased affinity or decreased affinity for FcRn.

[0009] In one aspect, the present invention provides a modified IgG comprising: a bovine IgG constant domain, which bovine IgG constant domain comprises at least one amino acid substitution relative to a wild-type bovine IgG constant domain, wherein the substitution is at amino acid residue 252, 286, 311, 312, 426, 428, 434 or 436.

[0010] In an exemplary embodiment, the bovine IgG constant domain is an IgG2a constant domain comprising M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G , Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T , Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I , D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V , D312W, D312Y, V426A, V426C, V426D, V426E, V426F, V426G, V426H, V426 I. V426K, V426L, V426M, V426N, V426P, V426Q, V426R, V426S, V426T, V426 W, V426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428 K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428 Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W mutations.

[0011] In another aspect, the present invention provides a polypeptide comprising: a bovine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.

[0012] In yet another aspect, the invention provides an antibody or molecule comprising: a bovine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.

[0013] 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 a nucleic acid sequence encoding an antibody, wherein the antibody comprises a bovine IgG constant domain, which bovine IgG constant domain comprises one or more amino acid substitutions of the invention described herein.

[0014] According to the following detailed description examples and the accompanying 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

[0015] This patent or application file contains at least one drawing printed 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.

[0016] Figure 1 A sequence alignment of bovine IgG subclasses is shown. Three representative allotypes are shown. The CH1, hinge, CH2, and CH3 domains are as follows: CH1: residues 1-98; hinge: 99 to vertical line; CH2: vertical line to 243; CH3: 244-351. Cysteines involved in inter-heavy chain disulfide bonds are in bold and underlined. The 3' extension of the CH1 exon in bovine IgG3a is in italics. The bold amino acids just downstream from the hinge represent the "Winter" or "LALA" site. For bIgG1a and bIgG3a, these sites are LPGG and PLGG, respectively. This site is not present in bIgG2a. The box contains the "PAP" region in bIgG1a and bIgG3a CH2 and the "SAS" region in bIgG2a.

[0017] Figure 2 An alignment of the amino acid sequences of human IgG1, bovine IgG1a, bovine IgG2a and bovine IgG3a is shown. The amino acid residues are numbered according to the Eu index as in Kabat.

[0018] Figure 3 shows protein models of bovine IgG2a and IgG2b Fc regions. Figure 3A : Overlay of protein models, where the enlarged sub-image shows the amino acid residue positions in ball-and-stick form. Figure 3B : Comparison of root mean square deviation (RMSD) of bovine IgG2a and IgG2b wild-type constructs.

[0019] Brief Description of Sequence Listing

[0020] SEQ ID NO.: 1 refers to the amino acid sequence of IgG1a wild type (NCBI ID No. 1S82409).

[0021] SEQ ID NO.: 2 refers to the amino acid sequence of the wild-type IgG1b (NCBI ID No. X16701).

[0022] SEQ ID NO.: 3 refers to the amino acid sequence of the wild-type IgG1c (NCBI ID No. DQ452014.1).

[0023] SEQ ID NO.: 4 refers to the amino acid sequence of IgG1d wild type (NCBI ID No. X62916.1).

[0024] SEQ ID NO.: 5 refers to the amino acid sequence of wild-type IgG2a (NCBI ID No. X16702.1).

[0025] SEQ ID NO.: 6 refers to the amino acid sequence of IgG2b wild type (NCBI ID No. S82407).

[0026] SEQ ID NO.:7 refers to the amino acid sequence of IgG3a wild type.

[0027] SEQ ID NO.: 8 refers to the amino acid sequence of IgG3b wild type.

[0028] SEQ ID NO.: 9 refers to the amino acid sequence of wild-type IgG2a from cow.

[0029] SEQ ID NO.: 10 refers to the amino acid sequence of human IgG1. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] As employed above and throughout the disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise indicated.

[0033] definition

[0034] In this disclosure, the singular forms "a", "an", and "the" include plural references, and reference to a particular value includes at least that particular value unless the context clearly indicates otherwise. Thus, for example, reference to a "molecule" or "compound" is a reference to one or more 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.

[0035] In the specification and claims, the numbering of amino acid residues in immunoglobulin heavy chains is that of the Eu index as in Kabat, Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "Eu index as in Kabat" refers to the residue numbering of IgG antibodies and is reflected in Figure 23 herein.

[0036] 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).

[0037] 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 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 contain 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 the 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.

[0038] 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).

[0039] 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 particular 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 polypeptides in the sample is thereby increased.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 recognized boundaries of the Fc region of an immunoglobulin heavy chain may vary, the bovine IgG heavy chain Fc region is generally defined as, for example, Figure 1 In some embodiments, the variant comprises only a portion of the Fc region and may or may not include the carboxyl terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more constant domains are contemplated. In other embodiments, variants having no such constant domains (or having only a portion of such constant domains) are contemplated.

[0044] The "CH2 domain" of a bovine IgG Fc region refers to, for example, Figure 1243. The CH2 domain is unique in that it is not closely paired with another domain. Two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule.

[0045] The "CH3 domain" of a bovine IgG Fc region is generally the stretch of residues C-terminal to the CH2 domain in the Fc region, e.g. Figure 1 Residue 244 to the C-terminus.

[0046] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); antibody dependent cellular phagocytosis (ADCP); 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, ADCP assays, target cell depletion in whole or fractionated blood samples, etc.).

[0047] 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 bovine Fc region is Figure 1 The region from the vertical line to the C-terminus.

[0048] 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, for example, a binding domain of a receptor or ligand.

[0049] As used herein, in the context of a polypeptide, the term "derivative" refers to a polypeptide comprising an amino acid sequence that has been changed by the introduction of an amino acid residue substitution. 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 by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to cellular ligands or other proteins, etc. Derivatized polypeptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to metabolic synthesis of specific chemical cleavage, acetylation, formylation, tunicamycin, etc. Further, a derived polypeptide has a function similar to or identical to that of a polypeptide derived therefrom. It should be understood that a polypeptide comprising a variant Fc region of the present invention can be a derivative as defined herein, preferably, derivatization occurs within the Fc region.

[0050] "Substantially of bovine 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 bovine amino polypeptide.

[0051] 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 an IgG antibody Fc region, an Fcγ receptor or "FcgR", and includes receptors of the FcγRI (FcgR1), FcγRII (FcgR2), FcγRIII (FcgR3) subclasses, including allelic variants and alternative splicing forms of these receptors and novel bovine Fcγ2R (bFcg2R or bFcg2R). 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 additional FcRs, including those identified in the future.

[0052] The phrases "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) expressing FcgRs (e.g., natural killer ("NK") cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby cause lysis of the target cell. NK cells are the primary cells used to mediate human ADCC and express only FcgR3, while monocytes express FcgR1, FcgR2, and FcgR3. The literature reports that bovine monocytes and macrophages express FcgRs for IgG1 and IgG2 isotypes, while neutrophils express a large number of receptors for IgG2, Fcg2R, but little or no bIgG1 receptor.

[0053] The phrases "antibody-dependent cell-mediated phagocytosis" and "ADCP" refer to a cell-mediated reaction in which phagocytic cells (e.g., macrophages, monocytes, dendritic cells) expressing FcgRs (e.g., FcgR1, FcgR2a, and FcgR3) recognize bound antibody Fc regions on target cells and subsequently trigger a signaling cascade resulting in the phagocytosis of IgG-opsonized particles (e.g., bacteria, dead tissue cells).

[0054] As used herein, the phrase "effector cell" refers to a leukocyte (preferably cattle) that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least FcgR3 and performs ADCC effector functions. Examples of leukocytes that mediate ADCC include PBMC, NK cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be separated from natural sources (e.g., from blood or PBMC). In one example, leukocytes express FcgR1 or other related Fcγ receptors and trigger ADCP function.

[0055] Variant polypeptides with "altered" Fc receptor binding affinity are variant polypeptides with enhanced (i.e., increased, greater or higher) or diminished (i.e., decreased, lesser or lower) FcRn binding affinity compared to the variant's parent polypeptide or a polypeptide comprising a native Fc region. Variant polypeptides that exhibit increased binding to an Fc receptor or increased binding affinity bind to an Fc receptor with greater affinity than the parent polypeptide. Variant polypeptides that exhibit decreased binding to an Fc receptor or decreased binding affinity bind to an Fc receptor with lower affinity than their parent polypeptide. Such variants that exhibit decreased binding to an Fc receptor may have little or no significant binding to an Fc receptor, for example, 0-20% binding to an Fc receptor compared to the parent polypeptide. A variant polypeptide that binds to an Fc receptor with "enhanced affinity" compared to its parent polypeptide is a variant polypeptide that binds to an Fc receptor 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 Fc receptor binding affinity may exhibit an increase in Fc receptor 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 Fc receptor binding affinity is determined, for example, in an ELISA assay or other methods available to one of ordinary skill in the art.

[0056] 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 replacement with one or more non-naturally occurring amino acid residues. "Non-naturally occurring amino acid residues" refers 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-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al. Meth Enzym. 202:301-336 (1991).

[0057] 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).

[0058] 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 dissociation constant (K) is directly related to the ratio of the concentration (reported in units of concentration per unit time, e.g., moles per second) to the kinetic association rate (usually reported in units of concentration per unit time, e.g., moles per second). In general, it is not possible to unambiguously state the equilibrium dissociation constant (K D It is not possible to determine whether changes in KD (or KD) are due to differences in association rate, dissociation rate, or both, unless each of these parameters is determined experimentally (e.g., as measured by BIACORE or SAPIDYNE).

[0059] As used herein, the term "hinge region" refers to the stretch of amino acids that connects the Fab antigen binding region to the Fc region of an antibody. The hinge regions of the IgG subclass can be aligned by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (S—S) bonds in the same position. Figure 1 As shown, for example, the hinge region in the bovine IgG constant region begins at residue 99 and extends to the vertical line.

[0060] "C1q" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q, together with two serine proteases, C1r and C1s, forms the complex C1, which is the first component of the CDC pathway.

[0061] 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, bovine or bovine antibodies, as well as chimeric or CDR-grafted single-chain antibodies, etc., containing 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 chain or a bovine chain can be expressed to produce a continuous protein. See, for example, U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; WO 86 / 01533; U.S. Patent No. 5,225,539; and U.S. Patent Nos. 5,585,089 and 5,698,762. See also Newman, R. et al. BioTechnology, 10: 1455-1460, 1993, regarding privatized antibodies, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, RE et al., Science, 242: 423-426, 1988, regarding single-chain antibodies. It should be understood that all forms of antibodies comprising an Fc region (or a portion thereof) are encompassed herein within the term "antibody". In addition, antibodies can be labeled with a detectable label, can be immobilized on a solid phase and / or combined with a heterologous compound (e.g., an enzyme or a toxin) according to methods known in the art.

[0062] As used herein, the term "antibody fragment" refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. 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.

[0063] 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 in, for example, Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and in Day, ED, Advanced Immunochemistry, 2nd edition, Wiley-Liss, Inc., New York, NY (1990). The term functional fragment is intended to include fragments produced, for example, by protease digestion or reduction of monoclonal antibodies and by recombinant DNA methods known to those skilled in the art.

[0064] 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.

[0065] 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 the antibody of cattle comprises an antigen binding region derived from the heavy chain of a non-bovine antibody, which is connected to at least a portion of the bovine heavy chain constant region, such as CH1 or CH2. The chimeric light chain for the antibody of cattle comprises an antigen binding region derived from the light chain of a non-bovine antibody, which is connected to at least a portion of the bovine 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. Patent Nos. 6,284,471; 5,807,715; 4,816,567; and 4,816,397).

[0066] As used herein, a "bovine" form of a non-bovine (e.g., murine) antibody (i.e., a bovine antibody) is an antibody containing minimal or no sequence derived from a non-bovine immunoglobulin. In most cases, a bovine antibody is a bovine immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-canine species (donor antibody) having the desired specificity, affinity, and capacity, such as mouse, rat, rabbit, human, or non-human primate. In some cases, the framework region (FR) residues of the bovine immunoglobulin are replaced by corresponding non-bovine residues. In addition, the bovine antibody may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are usually performed to further optimize antibody performance. In general, the bovine antibody will contain substantially all of the variable domains of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-bovine immunoglobulin, and all or substantially all of the FR residues are those of a bovine immunoglobulin sequence. The bovinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a bovine immunoglobulin.

[0067] 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.

[0068] 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).

[0069] 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.

[0070] 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 protein 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 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.

[0071] 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).

[0072] 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 separated and can be optionally coated on an assay plate or some other solid phase, or directly labeled and used as a probe.

[0073] As used herein, compared to the parent antibody, the variant polypeptides knocking out or knocking down antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) in the presence of bovine effector cells are polypeptides that mediate ADCC, ADCP and / or CDC activity substantially lower in vitro or in vivo when the amount of the variant polypeptide and the parent antibody used in the assay is substantially the same. For example, such variants cause target cell lysis or phagocytosis of a lower, preferably negligible amount than the parent polypeptide in the same ADCC assay in a given ADCC, ADCP or CDC assay. Such variants can be identified, for example, using ADCC, ADCP or CDC assays, but other assays or methods (e.g., animal models) for determining ADCC, ADCP or CDC activity can also be used. In a preferred embodiment, the variant polypeptides are about 100%, 75%, 50% or 25% lower than the parent polypeptide in mediating ADCC, ADCP and CDC.

[0074] Bovine wild-type IgG

[0075] Bovine IgG is well known in the art and is described, for example, in Symons et al., 1989, Mol. Immunol., Vol. 26(9), pp. 841-850; Kacskovics et al., 1996, Mol. Immunol., Vol. 33(2), pp. 189-195; Saini et al., 2007, Scand. J. Immunol., Vol. 65(1), pp. 32-38; and Rabbani et al., 1997, Immunogenetics, Vol. 46(4), pp. 326-331.

[0076] In one embodiment, the bovine IgG is IgG1. In another embodiment, the bovine IgG is IgG2. In yet another embodiment, the bovine IgG is IgG3. In one example, IgG1 is IgG1a, IgG1b, IgG1c, or IgG1d. In another example, IgG2 is IgG2a or IgG2b. In yet another example, IgG3 is IgG3a or IgG3b.

[0077] The amino acid and nucleic acid sequences of IgG1a, IgG1b, IgG1c, IgG1d, IgG2a, IgG2b, IgG3a, and IgG3b are also well known in the art.

[0078] 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.

[0079] In a specific example, the wild-type constant domain comprises the amino acid sequence listed in SEQ ID NO.: 1-8. In a specific embodiment, the wild-type constant domain of IgG1a, IgG1b, IgG1c, IgG1d, IgG2a, IgG2b, IgG3a and IgG3b comprises the amino acid sequence listed in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7 and 8, respectively. In some embodiments, the wild-type IgG constant domain is a homologue, variant, isomer or functional fragment of any one of SEQ ID NO.: 1-8, but does not have any mutations described herein. Each possibility represents a separate embodiment of the present invention. For example, in one embodiment, in a specific embodiment, the wild-type constant domain of IgG2a comprises the amino acid sequence listed in SEQ ID NO.: 9.

[0080] IgG constant domains also include polypeptides having an amino acid sequence that is 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 following FASTA search method: Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988).

[0081] 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 a combination thereof. In another embodiment, the nucleic acid can encode an antibody heavy chain comprising, for example, any one of the VH region or a portion thereof or any one of the VH CDRs, including any variants thereof. The present invention also includes nucleic acid molecules encoding antibody light chains, which include, for example, any one of the CL region or a portion thereof, any one of the VL region or a portion 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.

[0082] The amino acid sequence of the wild-type constant domain listed in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8 or 9 is encoded by its corresponding nucleic acid sequence.

[0083] Modified bovine IgG

[0084] The inventors of the present application have found that replacing the amino acid residues at positions 252, 286, 311, 312, 426, 428, 434 or 436 with another amino acid surprisingly and unexpectedly exhibits the desired effect. As used herein, the term position refers to the position numbered according to the Eu index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In one embodiment, relative to IgG with wild-type bovine IgG constant domains, the desired effect is a higher affinity for FcRn. In another embodiment, relative to IgG with wild-type bovine IgG constant domains, the desired effect is to eliminate or reduce complement dependent cytotoxicity. In another embodiment, relative to IgG with wild-type bovine IgG constant domains, the desired effect is to eliminate or reduce antibody-dependent cellular phagocytosis. In yet another embodiment, the desired effect is to eliminate or reduce the binding of IgG to Fcγ receptors (bFcgR).

[0085] In one aspect, the invention provides a modified IgG comprising: a bovine IgG constant domain comprising at least one amino acid substitution relative to a wild-type bovine IgG constant domain, wherein the substitution is at amino acid residues 252, 286, 311, 312, 426, 428, 434 or 436 numbered according to the Eu index as in Kabat. The amino acids at these positions can be substituted with any other amino acid. Examples of substituted amino acids include, for example but not limited to, asparagine, histidine, serine, alanine, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid and proline. In some embodiments, the substituted amino acid is a non-natural amino acid.

[0086] The modified bovine IgG of the present invention can be any suitable bovine IgG known to those skilled in the art. Examples of modified bovine IgG include modified variants of IgG1 (e.g., IgG1a, IgG1b, IgG1c or IgG1d), IgG2 (e.g., IgG2a or IgG2b) or IgG3 (IgG3a or IgG3b).

[0087] In an exemplary embodiment, the modified bovine IgG is a modified bovine IgG2, including, for example, a modified IgG2a or a modified IgG2b. In a specific embodiment, the modified bovine IgG2 is a modified IgG2a.

[0088] In another exemplary embodiment, the bovine IgG2 constant domain comprises the substitution mutations M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S , T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311 H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q31 1V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D3 12K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D3 12W, D312Y, V426A, V426C, V426D, V426E, V426F, V426G, V426H, V426I, V 426K, V426L, V426M, V426N, V426P, V426Q, V426R, V426S, V426T, V426W, V426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K , M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428 Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W.

[0089] In another example, the mutant IgG2 constant domain of the present invention comprises one or more mutations described herein. In certain embodiments, the mutant IgG2 constant domain is a homologue, variant, isomer or functional fragment, but has the mutations of the present invention described herein. Each possibility represents a separate embodiment of the present invention.

[0090] The amino acid sequence of the mutant constant domain is encoded by its corresponding mutant nucleic acid sequence.

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

[0092] Methods for preparing antibody molecules are well known in the art and are described in detail 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, the entire contents of which are incorporated herein by reference. Any suitable method, process or technology 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.

[0093] 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.

[0094] 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. This 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.

[0095] 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 for maintaining the correct configuration of the antigen binding residues. Antigen binding regions are provided in the variable region of the H or L chain, which are smaller sequences called "hypervariable" because they have 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.

[0096] 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.

[0097] The present invention further provides a vector, the vector includes 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, it is possible to estimate the probability that a specific oligonucleotide will actually constitute an actual coding sequence. Such "codon usage rules" are disclosed by: Lathe et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe's "codon usage rules", a single nucleotide sequence or a set of nucleotide sequences containing a theoretically "most likely" nucleotide sequence that can encode a bovine 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 the existing antibody gene, 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 sequence of an antibody or peptide.

[0098] For example, one type of substitution is a conservative amino acid substitution. Such substitutions are those in which a given amino acid in a bovine antibody peptide is replaced by another amino acid with similar characteristics. Typically, conservative substitutions are considered to be substitutions in which one of the aliphatic amino acids Ala, Val, Leu, and lie is replaced by another; exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gin, exchange between 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).

[0099] Variant bovine antibodies or peptides may have full functionality, or may lack function in one or more activities. Fully functional variants typically contain only conservative changes or changes in non-critical residues or non-critical regions. Functional variants may also contain replacements of similar amino acids that do not produce functional changes or produce insignificant functional changes. Alternatively, such replacements may have positive or negative effects on function to some extent. Non-functional variants typically 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.

[0100] 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 epitope mapping (e.g., HDX), crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[0101] 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, γ-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., TECreighton, WH Freeman & Co., NY, 1993). Many detailed introductions to this topic are available, such as Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983); Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0102] 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.

[0103] Derivatives also include radiolabeled monoclonal antibodies. For example, radioactive iodine (251, 1311), carbon (4C), sulfur (35S), indium, tritium (H 3) etc.; conjugates of monoclonal antibodies with biotin or avidin with enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carboxylic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and conjugates of monoclonal antibodies with bioluminescent agents such as luciferase, chemiluminescent agents such as acridinium esters or fluorescent agents such as phycobiliproteins.

[0104] Another derivative bifunctional antibody of the present invention is a bispecific antibody produced by combining the parts of two independent 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. Referring to U.S. Patent Application Publication No. 20030031671.

[0105] 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.

[0106] The nucleotide sequence encoding at least one antibody, part or polypeptide of the present invention can be recombined with carrier DNA according to routine techniques, and the routine techniques include blunt end or staggered end for connection, restriction enzyme digestion for providing appropriate end, sticky end filling for carrying out when appropriate, alkaline phosphatase treatment for avoiding undesirable connection and connection with appropriate ligase. The technology of this type of operation is for example disclosed by Maniatis et al., MOLECULAR CLONING, LAB.MANUAL, (Cold Spring Harbor Lab.Press, NY, 1982 and 1989) and Ausubel et al. 1993, which can be used to construct the nucleotide sequence encoding antibody molecule or its antigen binding region.

[0107] 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 portion. 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; Ausubel et al., 1993 supra.

[0108] 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.

[0109] In one embodiment, the nucleotide sequence of the present invention is incorporated into a plasmid or viral vector that can replicate autonomously in a recipient host. Any of a variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993 supra. Important factors in selecting a particular plasmid or viral vector include: the ease with which a recipient cell containing the vector can be recognized and selected from those recipient cells that do not contain the vector; the number of vector copies desired by a particular host; and whether it is necessary to be able to "shuttle" the vector between host cells of different species.

[0110] Exemplary prokaryotic vectors known in the art include plasmids, such as plasmids capable of replication in E. coli (such as, for example, pBR322, CoIE1, pSC101, pACYC 184, pi.vX). Such plasmids are disclosed, for example, by Maniatis et al., 1989 supra; Ausubel et al., 1993 supra. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan in THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)) and Streptomyces phage, such as phLC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986); Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978); and Ausubel et al., 1993 supra.

[0111] Alternatively, gene expression elements that can be used to express cDNA encoding antibodies or polypeptides include, but are not limited to, (a) viral transcriptional 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) splice regions and polyadenylation sites, for example, those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as those in SV40 (Okayama et al., 1983).

[0112] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51Gene 21 (1987) 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. For immunoglobulin genes composed of part cDNA and part genomic DNA (Whittle et al., 1Protein 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.

[0113] 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.

[0114] 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.

[0115] 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 of 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 receptor cells include lymphoid cells, such as B lymphocytes of cattle or non-cattle origin, hybridoma cells of cattle or non-cattle origin, or interspecies heterologous hybridoma cells.

[0116] 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).

[0117] Yeast can provide substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications, including glycosylation. 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).

[0118] Yeast gene expression systems can be routinely evaluated for the production, secretion, and stability levels of peptides, antibodies, fragments, and regions thereof. Any of a range of yeast gene expression systems can be utilized, and these expression systems have promoters and termination elements from actively expressed genes encoding saccharolytic enzymes that are produced in large quantities when yeast is grown in a medium rich in glucose. Known glycolytic genes can also provide very effective transcriptional control signals. For example, promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A variety of methods can be used to evaluate the optimal expression plasmid for expressing cloned immunoglobulin cDNAs in yeast. See Volume II, DNA Cloning, 45-66, (Glover, ed.) IRL Press, Oxford, UK 1985).

[0119] 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 variety of methods can be used to evaluate the expression plasmids that produce antibodies, fragments and regions or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria (see Glover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001 supra; Colligan et al., ed. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001); Colligan et al., ed. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0120] 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 CRL61) cells. Many vector systems can be used to express cloned peptide H and L chain genes in mammalian cells (see Glover, 1985 supra). 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 carried out 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), and the resulting cell line can then be transfected with an H chain plasmid containing a second selectable marker. 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.

[0121] 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 sources.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 gives resistance to selection, and allows cells to stably integrate plasmids into chromosomes and grow to form lesions, and then the lesions 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.

[0122] 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.

[0123] Pharmaceutical and veterinary applications

[0124] 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.

[0125] "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.

[0126] "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.

[0127] 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.

[0128] Pharmaceutically acceptable carriers include water; saline; phosphate-buffered saline; dextrose; glycerol; alcohols such as ethanol, isopropanol; phosphates, citrates and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; 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; isotonicity agents such as sugars, mannitol, polyols such as sorbitol and sodium chloride; and combinations thereof.

[0129] 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 to release the composition immediately, controlled, extended or delayed.

[0130] The composition of the present invention can be administered as a single 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.

[0131] 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 primarily based 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 of preparing the composition for parenteral administration is 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).

[0132] 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.

[0133] The effective dosage of the compositions of the invention for treating the conditions or diseases described herein varies depending on many different factors, including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent and its therapeutic 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 the symptoms, kind of concurrent treatment, frequency of treatment, and the desired effect.

[0134] 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.

[0135] Therapeutic doses can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0136] 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 the dosage and for the period of time necessary. The therapeutically effective amount of a molecule may 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 the therapeutically beneficial effects outweigh any toxic or detrimental effects of the molecule.

[0137] On the other hand, the compositions of the present invention can be used, for example, to treat various diseases and conditions of cattle. As used herein, the terms "treat" and "treatment" refer to therapeutic treatments, including preventive or prophylactic 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 where a disease or condition is to be prevented.

[0138] All patents and literature references cited in this specification are incorporated herein by reference in their entirety.

[0139] 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.

[0140] Examples

[0141] Example 1

[0142] Construction of bovine IgG Fc mutants

[0143] The construction of bovine IgG was performed according to methods well known in the art. A plasmid containing sequences encoding the bovine constant region for the IgG2a subclass was used, and the VH / VL sequences for each mAb studied herein were inserted upstream and in a reading frame with nucleotides encoding the constant domains. Mutations were incorporated into the CH2 or CH3 domain positions of each plasmid by direct DNA synthesis of the constant region as a gene fragment, and then subcloned into the corresponding variable region of interest.

[0144] Expression and purification

[0145] 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 1st day and transfection day, cells are diluted according to the ExpiCHO scheme user manual. As described in the scheme, the reagent derived from ExpiFectamine CHO transfection kit (Gibco) is used to transfect the diluted cells according to the maximum titer condition. After cultivating 12 to 14 days, the culture is collected and the culture is clarified. Antibodies are purified from the clarified supernatant 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. Samples are eluted from the column with 20mM acetic acid of pH 3.5. After elution, pools were prepared and neutralized to 4% by adding 1 M sodium acetate. Depending on available volume and intended use, samples were sometimes exchanged into final buffer (e.g., PBS, other). Concentrations were measured by absorbance at 280 nm.

[0146] SDS-PAGE

[0147] 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.

[0148] Example 2

[0149] FcRn binding assay

[0150] Biacore method for bFcRn:

[0151] The binding affinity of antibodies or fusion proteins based on bovine Fc to bovine FcRn was determined by surface plasmon resonance (SPR). All reported KDs were measured using SA sensors in Biacore T200 (Cytiva, Marlborough, Massachusetts, USA) or Biacore 8K (Cytiva, Marlborough, Massachusetts, USA). Bovine FcRn was captured on the surface of the sensor to obtain the desired surface density. The running buffer used was 20mM MES, 150mM NaCl, 0.005% Tween 20, 0.5mg / mL BSA, pH 6 and / or PBS, 0.0005% Tween 20, pH 7.4. Different concentrations of bovine mAb were titrated in appropriate running buffer and flowed through the receptor surface. Regeneration was performed using 50mM Tris-HCl (pH 8). Kinetic binding affinity was analyzed using Biacore T200 evaluation software (Cytiva, Marlborough, MA, USA) or Biacore 8K Insight evaluation software using a dual reference approach: the reference flow cell was subtracted from the flow cell containing immobilized bovine FcRn, and a blank run containing buffer alone was subtracted from all runs. The resulting curves were fitted with a 1:1 binding model. Runs were performed at 25°C.

[0152] Mutations made at the corresponding positions had a significant effect on the affinity of IgG for FcRn at pH 6.

[0153] Binding of wild-type (WT) and mutant IgG to bovine FcRn was measured by surface plasmon resonance (Biacore).

[0154] Table 1. Effect of mutants on FcRn binding affinity.

[0155]

[0156]

[0157]

[0158]

[0159] Mutants are numbered according to the EU index as in Kabat. LS = low signal.

[0160] NBO = No signal observed.

[0161] The results clearly show that mutations made at various positions have a significant impact on the affinity of IgG for bovine FcRn.

[0162] Example 3

[0163] FcRn binding validation

[0164] method

[0165] The Fc regions of two bovine allotypes IgG2a and IgG2b were first designed using their corresponding CH2 and CH3 regions. The protein modeling feature of Alphafold 2.2 developed by Deepmind was implemented to model the 3D structure of each of the wild-type (WT) and mutant constructs of each bovine allotype IgG2a and IgG2b.

[0166] 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, the sequence map alignment algorithm uses a scoring algorithm to rank sequence templates, and a score above 85% ensures that a protein template with a physically realistic structure is selected. 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.

[0167] result

[0168] The above method was performed on the wild-type (WT) construct and the following mutants: M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286V, 286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q3 11L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D31 2K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y,, M428A, M428C, M428D, M428E, M428F, M42 8G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434 436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W. The positions for the mutant library are Figure 3A RMSD curves were generated to calculate the root mean square deviation (RMSD) of the two WT structures bovine-IgG2a_WT and bovine-IgG2b_WT relative to each other. Figure 3B ). The RMSD value of 100 or less was considered as the criterion for considering two structures similar. The results showed that the bovine IgG2a construct was identical to the bovine IgG2b allotype, and the average RMSD value of the structure was The RMSD at the positions where mutation scanning was performed is also noted in Table 2, with values ​​ranging from 0.226 to

[0169] Table 2. Comparison of root mean square deviations (RMSD) of residues T286, Q311, D312, A426 and Y436 in protein models of WT bovine IgG1 and IgG2.

[0170]

[0171] The same procedure was also followed to study the following mutations in both bovine subclades: M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286V. 6N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311 L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y,, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N 436P, 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 allotypes, ranging from 0.226 to 0.594. As indicated by the RMSD value range of , the proteins were the same as those of WT (Table 3).

[0172] Table 3Ta. Comparison of root mean square deviation (RMSD) of mutation libraries in bovine IgG2a and IgG2b protein models.

[0173]

[0174]

[0175]

[0176]

[0177] in conclusion

[0178] All mutations of the two allotypes IgG2a and IgG2b of the bovine backbone were molecularly modeled and validated using MOE2019.0102. The Fc fold and residue configurations between subclasses showed less than The RMSD of the proteins is very high, indicating that the proteins have very high structural identity and will therefore function in a similar manner.

[0179] 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 bovine IgG constant domain comprising at least one amino acid substitution relative to a wild-type bovine IgG constant domain, wherein the substitution is at amino acid residue 252, 286, 311, 312, 426, 428, 434 or 436 numbered according to the Eu index as in Kabat.

2. The modified IgG of claim 1, wherein the constant domain comprises substitutions M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T 286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I , D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V , D312W, D312Y, V426A, V426C, V426D, V426E, V426F, V426G, V426H, V426 I, V426K, V426L, V426M, V426N, V426P, V426Q, V426R, V426S, V426T, V426 W, V426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M42 8K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M42 Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W.

3. The modified IgG according to claim 1, wherein the modified IgG is bovine or bovine-derived IgG.

4. The modified IgG according to claim 1, wherein the IgG is IgG2. The modified IgG according to claim 4 , wherein the IgG2 is IgG2a or IgG2b.

6. The modified IgG according to any one of the above claims, wherein the modified IgG has a significant effect on its affinity to bovine FcRn relative to the affinity of an IgG with wild-type bovine IgG constant domains.

7. The modified IgG according to any one of the above claims, wherein the modified IgG has an increased half-life compared to an IgG with a wild-type bovine IgG constant domain.

8. The modified IgG according to any one of the above claims, wherein the IgG constant domain comprises a CH1 domain or a hinge region.

9. The modified IgG according to any one of the above claims, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

10. The modified IgG according to any one of the above claims, wherein the wild-type bovine IgG constant domain comprises one of the amino acid sequences listed in SEQ ID NO.: 1-9.

11. A pharmaceutical composition comprising the modified IgG according to any one of the above claims and a pharmaceutically acceptable carrier.

12. A kit comprising the modified IgG according to any one of claims 1 to 10 in a container and instructions for use.

13. A polypeptide comprising the modified IgG according to any one of claims 1 to 10.

14. An antibody comprising the modified IgG according to any one of claims 1 to 10.

15. A vector comprising a nucleic acid sequence encoding the amino acid sequence of the modified IgG according to any one of claims 1 to 10.

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

17. A method of making an antibody or molecule, the method comprising: Providing a cell according to claim 16; and culturing the cells.

18. A method for producing an antibody, the method comprising: An antibody according to claim 14 is provided.

19. A method for increasing the serum half-life of antibodies in livestock, the method comprising: A therapeutically effective amount of the antibody according to claim 14 is administered to the livestock.

20. A fusion molecule comprising: a bovine IgG constant domain fused to an agent, the bovine IgG constant domain comprising the modified IgG according to any one of claims 1 to 10.

21. A method for increasing the serum half-life of antibodies in cattle, the method comprising: A therapeutically effective amount of an antibody comprising a bovine IgG constant domain is administered to the cattle, wherein the bovine IgG constant domain comprises at least one amino acid substitution relative to a wild-type bovine IgG constant domain, wherein the substitution is at amino acid residue 252, 286, 311, 312, 426, 428, 434 or 436 numbered according to the Eu index as in Kabat.

22. The method of claim 21, wherein the bovine IgG constant domain comprises the mutations M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G , Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T , Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312 I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312 V, D312W, D312Y, V426A, V426C, V426D, V426E, V426F, V426G, V426H, V42 6I, V426K, V426L, V426M, V426N, V426P, V426Q, V426R, V426S, V426T, V42 6W, V426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M4 28K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M4 6A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, N436T, Y436V and Y436W.

23. The method of claim 21, wherein the bovine IgG constant domain has a longer serum half-life than an IgG having the wild-type bovine IgG constant domain.

24. The method of claim 21, wherein the IgG is IgG2.

25. The method of claim 24, wherein the IgG is IgG2a or IgG2b.

26. The method of claim 21, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain.

27. The method of claim 21, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

28. The method of claim 21, wherein the wild-type bovine IgG constant domain comprises one of the amino acid sequences listed in SEQ ID NO.: 1-9.

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