Bovine antibody variants

Abstract

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

Description

Related Applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 370,533, filed August 5, 2022, which is incorporated herein by reference in its entirety. [Technical Field]

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

[0003] Bovine IgG monoclonal antibodies (mAbs) can be effective therapeutic agents in veterinary medicine. Several years ago, three bovine IgG subclasses were identified. However, limited research has been conducted on improving the characteristics of bovine IgG.

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

[0005] Engineering the Fc region to modulate its interaction with FcRn has emerged as a promising approach to enhance the activity of therapeutic antibodies.

[0006] Therefore, there is a need for novel bovine IgG Fc region mutations to improve various characteristics of bovine IgG. Summary of the Invention

[0007] The present invention relates to mutant bovine IgGs that exhibit desirable characteristics compared to wild-type bovine IgGs. Specifically, the inventors of the present application have surprisingly and unexpectedly found that substituting amino acid residues at positions 252, 286, 311, 312, 426, 428, 434, or 436 (numbered according to the Eu index in Kabat) with other amino acids exhibits desirable effects. In exemplary embodiments, the unexpected desirable effects include, but are not limited to, increased or decreased affinity for FcRn.

[0008] In one aspect, the invention provides a modified IgG comprising a bovine IgG constant domain which comprises at least one amino acid substitution compared 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.

[0009] In one exemplary embodiment, the bovine IgG constant domain is selected from the group consisting of 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, T286Y ... 6V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q31 1I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311 W, 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, V426I, V426K, V426L, V426M, V426N, V426P, V426Q, V426R, V426S, V426T, V426W, V426Y, M428A, M 428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M 428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N4 and Y436W.

[0010] 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.

[0011] In yet another aspect, the present invention provides an antibody or molecule comprising a bovine IgG constant domain comprising one or more amino acid substitutions of the invention as described herein.

[0012] In a further aspect, the invention provides a method for producing or manufacturing an antibody or molecule, the method comprising providing a vector or host cell comprising a nucleic acid sequence encoding an antibody, wherein the antibody comprises a bovine IgG constant domain comprising one or more amino acid substitutions of the invention as described herein.

[0013] Other features and advantages of the present invention will become apparent from the following detailed description, examples, and drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0014] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0015] [Figure 1]Sequence alignment of bovine IgG subclasses. Three representative allotypes are shown. The CH1, hinge, CH2, and CH3 domains are as follows: CH1: residues 1-98, hinge: 99 to the vertical line, CH2: vertical line to 243, and CH3: 244-351. Cysteines involved in inter-heavy chain disulfide bonds are shown in bold and underlined. The 3' extension of the CH1 exon in bovine IgG3a is shown in italics. The bolded amino acids immediately downstream of the hinge represent the "Winter" or "LALA" site. For bIgG1a and bIgG3a, these sites are LPGG and PLGG, respectively. This site is absent in bIgG2a. Boxes include the "PAP" region in bIgG1a and bIgG3a CH2 and the "SAS" region in bIgG2a. [Figure 2] 1 shows an alignment of the amino acid sequences of human IgG1, bovine IgG1a, bovine IgG2a, and bovine IgG3a. Amino acid residues are numbered according to the Eu index in Kabat. [Figure 2-1] Continued from Figure 2. [Figure 2-2] Continued from Figure 2. [Figure 2-3] Continued from Figure 2. [Figure 2-4] Continued from Figure 2. [Figure 3A] Protein modeling of bovine IgG2a and IgG2b Fc regions. Overlay of protein model with enlarged subpanel showing amino acid residue positions shown in ball-and-stick format. [Figure 3B] Protein modeling of bovine IgG2a and IgG2b Fc regions. Root mean square deviation (RMSD) comparison of wild-type constructs of bovine IgG2a and IgG2b.

[0016] Brief Description of Sequence Listing SEQ ID NO: 1 refers to the amino acid sequence of IgG1a wild type (NCBI ID number 1S82409).

[0017] SEQ ID NO: 2 refers to the amino acid sequence of IgG1b wild type (NCBI ID number X16701).

[0018] SEQ ID NO: 3 refers to the amino acid sequence of IgG1c wild type (NCBI ID number DQ452014.1).

[0019] SEQ ID NO: 4 refers to the amino acid sequence of IgG1d wild type (NCBI ID number X62916.1).

[0020] SEQ ID NO: 5 refers to the amino acid sequence of IgG2a wild type (NCBI ID number X16702.1).

[0021] SEQ ID NO: 6 refers to the amino acid sequence of IgG2b wild type (NCBI ID number S82407).

[0022] SEQ ID NO: 7 refers to the amino acid sequence of IgG3a wild type.

[0023] SEQ ID NO: 8 refers to the amino acid sequence of IgG3b wild type.

[0024] SEQ ID NO: 9 refers to the amino acid sequence of wild-type IgG2a from dairy cows.

[0025] SEQ ID NO: 10 refers to the amino acid sequence of human IgG1. DETAILED DESCRIPTION OF THE INVENTION

[0026]

[0028] The subject matter of the present invention may be more readily understood by reference to the following detailed description, which forms a part of this disclosure: It is to be understood that the present invention is not limited to the specific products, methods, conditions, or parameters described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to limit the claimed invention.

[0027]

[0029] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0028]

[0030] As used above, and throughout this disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings:

[0029] definition

[0031] In this disclosure, the singular forms "a," "an," and "the" include plural referents, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, a reference to a "molecule" or "compound" is a reference to one or more of such molecules or compounds and equivalents thereof known to those of skill in the art. The term "plurality," as used herein, means more than one. When ranges of values are expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0030]

[0032] In this specification and claims, the numbering of amino acid residues in immunoglobulin heavy chains is that of the Eu index in Kabat, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "Eu index in Kabat" refers to the residue numbering of an IgG antibody, as reflected herein in Figure 23.

[0031]

[0033] The term "isolated," when used with reference to a nucleic acid, refers to a nucleic acid that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. An isolated nucleic acid is in a form or setting that is different from that in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from the nucleic acid molecule as it exists in natural cells. Isolated nucleic acid molecules include nucleic acid molecules contained within cells that normally express the encoded polypeptide, e.g., the nucleic acid molecule is in a plasmid or chromosomal location that is different from that of natural cells. Isolated nucleic acids may exist in single-stranded or double-stranded form. When isolated nucleic acid molecules are used to express proteins, the oligonucleotide or polynucleotide will minimally contain a sense or coding strand, but may contain both a sense and an antisense strand (i.e., may be double-stranded).

[0032]

[0034] A nucleic acid molecule is "operably linked" or "operably associated" when it is placed into a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a coding sequence of a nucleic acid if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence of a nucleic acid if it is positioned so as to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof that does not naturally comprise an Fc region) if it is positioned so that the expressed fusion protein contains the heterologous protein or functional fragment thereof adjacent either upstream or downstream of the variant Fc region polypeptide; the heterologous protein may be immediately adjacent to the variant Fc region polypeptide or separated from it by a linker sequence of any length and composition. Similarly, a polypeptide (used interchangeably herein with "protein") molecule is "operably linked" or "operably associated" when it is placed into a functional relationship with another polypeptide.

[0033]

[0035] As used herein, the term "functional fragment," when referring to a polypeptide or protein (e.g., a variant Fc region, or a monoclonal antibody), refers to a fragment of that protein that retains at least one function of the full-length polypeptide. Fragments can range in size from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. Functional fragments of variant Fc region polypeptides of the invention retain at least one "amino acid substitution," as defined herein. Functional fragments of variant Fc region polypeptides retain at least one function known in the art to be associated with an Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, downregulation of a cell surface receptor, or, for example, may increase the in vivo or in vitro half-life of a polypeptide to which it is operably linked).

[0034]

[0036] The term "purified" or "to purify" refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody may be purified by the complete or substantial (at least 90%, 91%, 92%, 93%, 94%, 95%, or more preferably at least 96%, 97%, 98%, or 99%) removal of at least one contaminating non-immunoglobulin protein, and may also be purified by the removal of immunoglobulin proteins that do not bind to the same antigen. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind to a particular antigen results in an increase in the percentage of antigen-specific immunoglobulin in a sample. In another example, a polypeptide (e.g., an immunoglobulin) expressed in a bacterial host cell is purified by the complete or substantial removal of host cell proteins, thereby increasing the percentage of the polypeptide in a sample.

[0035]

[0037] The term "native" referring to a polypeptide (e.g., an Fc region) is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of a polypeptide commonly occurring in nature or a naturally occurring polymorphism thereof. A native polypeptide (e.g., a native Fc region) can be produced by recombinant means or isolated from a natural source.

[0036]

[0038] As used herein, the term "expression vector" refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.

[0037]

[0039] 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.

[0038]

[0040] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. While the generally accepted boundaries of the Fc region of an immunoglobulin heavy chain can vary, the bovine IgG heavy chain Fc region is usually defined, for example, as extending from the vertical line in Figure 1 to the c-terminus. In some embodiments, variants include only a portion of the Fc region, which may or may not include the carboxy terminus. The Fc region of an immunoglobulin generally includes two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants having no such constant domains (or only portions of such constant domains) are contemplated.

[0039]

[0041] The "CH2 domain" of a bovine IgG Fc region, for example, refers to residues beginning at the vertical line in Figure 1 and extending through residue 243. The CH2 domain is unique in that it is not tightly paired with another domain. Two N-linked branched carbohydrate chains are interposed between the two CH2 domains in an intact, native IgG molecule.

[0040]

[0042] The "CH3 domain" of a bovine IgG Fc region is generally a stretch of residues from the C-terminus of the Fc region to the CH2 domain, for example, residues 244 to the C-terminus in FIG.

[0041]

[0043] 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), down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions may require that the Fc region be operably linked to a binding domain (e.g., an antibody variable domain) and may be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, ADCP assays, target cell depletion from whole blood samples or fractionated blood samples, etc.).

[0042]

[0044] A "native sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence identical to that of an Fc region typically found in nature. An exemplary native sequence bovine Fc region is from the vertical line to the c-terminus of Figure 1.

[0043]

[0045] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region (or fragment thereof) by virtue of at least one "amino acid substitution," as defined herein. In preferred embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or 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 alternative embodiments, variant Fc regions may be generated according to the methods disclosed herein, and the variant Fc region may be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a non-antibody polypeptide, e.g., a receptor or ligand binding domain.

[0044]

[0046] As used herein, the term "derivative" in the context of a polypeptide refers to a polypeptide comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions. As used herein, the term "derivative" also refers to a polypeptide that has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, antibodies can be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Derivative polypeptides can be generated by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, derivative polypeptides have similar or identical functions to the polypeptide from which they were derived. It is understood that polypeptides comprising the variant Fc regions of the present invention can be derivatives as defined herein, and preferably, the derivatization occurs within the Fc region.

[0045]

[0047] As used herein with respect to a polypeptide (e.g., an Fc region or a monoclonal antibody), "substantially of bovine origin" indicates that the polypeptide has an amino acid sequence that is at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, or even more preferably at least 95%, 96%, 97%, 98%, or 99% homologous to that of a naturally occurring bovine amino acid polypeptide.

[0046]

[0048] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). A preferred FcR is a native-sequence FcR. Further preferred FcRs are those that bind to the IgG antibody Fc region, Fcγ receptors or "FcgRs (Fc gamma receptors)," including receptors of the FcγRI (FcgR1), FcγRII (FcgR2), FcγRIII (FcgR3) subclasses, including allelic variants and alternatively spliced forms of these receptors, as well as the novel bovine Fcγ2R (bFcg2R or bFcg2R). Another preferred FcR includes the neonatal receptor FcRn, responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0047]

[0049] The phrases "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) that express FcgR (e.g., natural killer ("NK") cells, neutrophils, and macrophages) recognize bound antibody on target cells and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC in humans, express only FcgR3, whereas monocytes express FcgR1, FcgR2, and FcgR3. According to the literature, bovine monocytes and macrophages express FcgRs for IgG1 and IgG2 isotypes, whereas neutrophils express multiple receptors for IgG2, Fcg2R, but little or no expression for bIgG1.

[0048]

[0050] The terms "antibody-dependent cell-mediated phagocytosis" and "ADCP" refer to a cell-mediated reaction in which phagocytes (e.g., macrophages, monocytes, dendritic cells) expressing FcgRs (e.g., FcgR1, FcgR2a, and FcgR3) recognize the bound IgG antibody Fc region on target cells and subsequently trigger a signaling cascade that leads to the phagocytosis of IgG-opsonized particles (e.g., bacteria, dead tissue cells).

[0049]

[0051] As used herein, the phrase "effector cell" refers to a (preferably bovine) leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcgR3 and performs ADCC effector function. Examples of leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources (e.g., blood or PBMCs). In one example, the leukocyte expresses FcgR1 or other related Fcγ receptors and induces ADCC function.

[0050]

[0052] A variant polypeptide with "altered" Fc receptor binding affinity is one that has either improved (i.e., increased, greater, or higher) or decreased (i.e., reduced, decreased, or lower) Fc receptor binding affinity compared to the variant's parent polypeptide or a native Fc-containing polypeptide. A variant polypeptide that exhibits increased binding or increased binding affinity to an Fc receptor binds to an Fc receptor with greater affinity than the parent polypeptide. A variant polypeptide that exhibits decreased binding or decreased binding affinity to an Fc receptor binds to an Fc receptor with lower affinity than the parent polypeptide. Such variants that exhibit decreased binding to an Fc receptor may have little or no appreciable binding to an Fc receptor, e.g., 0-20% binding to an Fc receptor compared to the parent polypeptide. A variant polypeptide that binds to an Fc receptor with "improved affinity" compared to its parent polypeptide is one that binds to an Fc receptor with higher binding affinity than the parent polypeptide, when the amounts of the variant and parent polypeptides in a binding assay are essentially the same and all other conditions are identical. For example, when Fc receptor binding affinity is determined in an ELISA assay or other method available to one of skill in the art, a variant polypeptide with improved Fc receptor binding affinity may exhibit an increase in Fc receptor binding affinity of about 1.10-fold to about 100-fold (more typically, about 1.2-fold to about 50-fold) compared to the parent polypeptide.

[0051]

[0053] As used herein, an "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with another, different, "replacement" amino acid residue. The replacement residue or residues may be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Substitutions with one or more non-naturally occurring amino acid residues are also encompassed within the definition of an amino acid substitution herein. A "naturally occurring amino acid residue" refers to a residue other than the naturally occurring amino acid residues listed above, which is capable of being covalently linked to an adjacent amino acid residue(s) in a polypeptide chain. Examples of 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).

[0052]

[0054] The term "assay signal" refers to the output from any method of detecting protein-protein interactions, including, but not limited to, absorbance measurements from colorimetric assays, fluorescence intensity, or decays per minute. Assay formats can include ELISA, FACS, or other methods. A change in "assay signal" can reflect a change in cell viability and / or a change in kinetic off-rate, kinetic on-rate, or both. A "higher assay signal" refers to a measured output number that is greater than another number (e.g., a variant can have a higher (larger) measured number in an ELISA assay compared to the parent polypeptide). A "lower" assay signal refers to a measured output number that is less than another number (e.g., a variant can have a lower (smaller) measured number in an ELISA assay compared to the parent polypeptide).

[0053]

[0055] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. Binding affinity can also be measured by the kinetic off-rate (generally expressed in units of the reciprocal of time, e.g., seconds). -1 The equilibrium dissociation constant (K) is directly related to the ratio of the equilibrium dissociation constant (K) to the kinetic on-rate (generally reported in units of concentration per unit time, e.g., moles / second). Generally, unless each of these parameters is experimentally determined (e.g., by BIACORE or SAPIDYNE measurements), the equilibrium dissociation constant (K) is D It is not possible to state definitively whether the changes in KD are due to differences in on-rate, off-rate, or both.

[0054]

[0056] 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. Hinge regions of IgG subclasses can be aligned by placing the first and last cysteine residues that form inter-heavy chain disulfide (SS) bonds in the same positions. As shown in Figure 1, for example, the hinge region in a bovine IgG constant region begins at residue 99 and extends to the vertical line.

[0055]

[0057] "C1q" is a polypeptide that contains the binding site for the Fc region of immunoglobulins. C1q, together with two serine proteases, C1r and C1s, forms the complex C1, the first component of the CDC pathway.

[0056]

[0058] As used herein, the term "antibody" is used interchangeably with "immunoglobulin" or "Ig" and is used in the broadest sense to specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological or functional activity. Single-chain antibodies, including portions derived from different species, and chimeric, bovine, or bovinized antibodies, as well as chimeric or CDR-grafted single-chain antibodies, are also encompassed by the present invention and the term "antibody." The various portions of these antibodies can be joined together chemically, synthetically, by conventional techniques, or prepared as a contiguous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric or bovinized chain can be expressed to produce a contiguous protein. See, e.g., 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. Also, regarding primatized antibodies, see Newman, R. et al., BioTechnology, 10:1455-1460, 1993, and regarding single-chain antibodies, see Ladner et al., U.S. Patent No. 4,946,778 and Bird, R. E. et al., Science, 242:423-426, 1988. As used herein, all forms of antibodies containing an Fc region (or a portion thereof) are understood to be encompassed within the term "antibody." Furthermore, the antibodies can be labeled with a detectable label, immobilized on a solid phase, and / or conjugated to a heterologous compound (e.g., an enzyme or a toxin) according to methods known in the art.

[0057]

[0059] As used herein, the term "antibody fragment" refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. Antibody fragments preferably retain at least a portion of the hinge and, optionally, the CH1 region of the IgG heavy chain. In other preferred embodiments, the antibody fragment includes at least a portion of the CH2 region or the entire CH2 region.

[0058]

[0060] As used herein, the term "functional fragment," when used in reference to a monoclonal antibody, is intended to refer to a portion of a monoclonal antibody that still retains functional activity. Functional activity can be, for example, antigen-binding activity or specificity, receptor-binding activity or specificity, effector function activity, etc. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single-chain Fv (scFv); and Fc fragments. Such terms are described, for example, in Harlowe and Lane, *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory, New York (1989), *Molec. Biology and Biotechnology: A Comprehensive Desk Reference* (Myers, RA (ed.), New York: VCH Publishers, Inc.), *Huston et al., *Cell Biophysics*, 22:189-224 (1993), *Pluckthun and Skerra, *Meth. Enzymol.*, 178:497-515 (1989), and *Day, ED, *Advanced Immunochemistry*, Second Ed., *Wiley-Liss, Inc., New York, NY (1990). The term functional fragment is intended to include fragments produced, for example, by protease digestion or reduction of monoclonal antibodies and by recombinant DNA methods known to those skilled in the art.

[0059]

[0061] As used herein, the term "fragment" refers to a polypeptide that contains an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100, or more consecutive amino acid residues of the amino acid sequence of another polypeptide. In preferred embodiments, a fragment of a polypeptide retains at least one function of the full-length polypeptide.

[0060]

[0062] As used herein, the term "chimeric antibody" includes monovalent, divalent, or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through a disulfide bridge with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy-light chain dimers associated through at least one disulfide bridge. The chimeric heavy chain of an antibody for use in bovine animals comprises an antigen-binding region derived from the heavy chain of a non-bovine antibody linked to at least a portion of a bovine heavy chain constant region, such as CH1 or CH2. The chimeric light chain of an antibody for use in bovine animals comprises an antigen-binding region derived from the light chain of a non-bovine antibody linked to at least a portion of a bovine light chain constant region (CL). Antibodies, fragments, or derivatives having chimeric heavy and light chains of the same or different variable region binding specificities can also be prepared by appropriate association of the individual polypeptide chains according to known method steps. Using this approach, hosts expressing the chimeric light chain and hosts expressing the chimeric heavy chain are cultured separately, and the immunoglobulin chains are recovered separately and then associated. Alternatively, the hosts can be co-cultured, the chains allowed to associate spontaneously in the culture medium, and the assembled immunoglobulin or fragment then recovered, or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Patent Nos. 6,284,471, 5,807,715, 4,816,567, and 4,816,397).

[0061]

[0063] As used herein, a "bovine" form of a non-bovine (e.g., murine) antibody (i.e., a bovine antibody) is an antibody that contains minimal or no sequence derived from non-bovine immunoglobulins. In most cases, bovine antibodies are bovine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient have been replaced by residues from a hypervariable region of a non-bovine species (donor antibody) such as mouse, rat, rabbit, human, or non-human primate possessing the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the bovine immunoglobulin are replaced by corresponding non-bovine residues. Furthermore, bovine antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are generally made to further refine antibody performance. Generally, bovine antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-bovine immunoglobulin and all or substantially all of the FR residues are those of a bovine immunoglobulin. The bovine antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a bovine immunoglobulin.

[0062]

[0064] As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding domain of a heterologous "adhesin" protein (e.g., receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of an adhesin amino acid sequence with the desired binding specificity, which is other than the antigen recognition and binding site (antigen binding site) of an antibody (i.e., is "heterologous"), with an immunoglobulin constant domain sequence.

[0063]

[0065] As used herein, the term "ligand-binding domain" refers to any naturally occurring receptor, or any region or derivative thereof, that retains at least qualitative ligand-binding ability of the corresponding naturally occurring receptor. In certain embodiments, the receptor is derived from a cell surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergene family. Other receptors that are not members of the immunoglobulin supergene family but are nevertheless specifically encompassed by this definition are receptors for cytokines, particularly receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L-, and P-selectin).

[0064]

[0066] As used herein, the term "receptor binding domain" refers to any natural ligand of a receptor, e.g., a cell adhesion molecule, or any region or derivative of such a natural ligand that retains at least qualitative receptor binding ability of the corresponding natural ligand.

[0065]

[0067] As used herein, an "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that may interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, an isolated polypeptide is purified (1) to greater than 95% by weight, preferably greater than 99% by weight, of the polypeptide as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining. Since at least one component of the polypeptide's natural environment is absent, an isolated polypeptide includes the polypeptide in situ within recombinant cells. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0066]

[0068] As used herein, the terms "disorder" and "disease" are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder).

[0067]

[0069] As used herein, the term "receptor" refers to a polypeptide capable of binding at least one ligand. Preferred receptors are cell-surface or soluble receptors having an extracellular ligand-binding domain and, optionally, other domains (e.g., a transmembrane domain, an intracellular domain, and / or a membrane anchor). The receptor evaluated in the assays described herein can be the intact receptor, or a fragment or derivative thereof (e.g., a fusion protein comprising the binding domain of the receptor fused to one or more heterologous polypeptides). Furthermore, the receptor evaluated for its binding properties can be present intracellularly or isolated, and can optionally be coated on an assay plate or some other solid phase, or directly labeled and used as a probe.

[0068]

[0070] As used herein, a variant polypeptide that knocks out or knocks down antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) in the presence of bovine effector cells compared to the parent antibody is one that is substantially less active in mediating ADCC, ADCP, and / or CDC in vitro or in vivo when essentially the same amounts of variant polypeptide and parent antibody are used in the assay. For example, such a variant causes a lower, preferably negligible, amount of target cell lysis or phagocytosis in a given ADCC, ADCP, or CDC assay than the parent polypeptide in the same ADCC assay. Such variants may be identified, for example, using an ADCC, ADCP, or CDC assay, although other assays or methods for determining ADCC, ADCP, or CDC activity may also be used (e.g., animal models). In preferred embodiments, the variant polypeptide is about 100, 75, 50, or 25 percent less active in mediating ADCC, ADCP, and CDC than the parent polypeptide.

[0069] Bovine wild-type IgG

[0071] Bovine IgG is well known in the art and is fully described, for example, in Symons et al., 1989, Mol. Immunol., vol. 26(9), pages 841-850; Kacskovics et al., 1996, Mol. Immunol., vol. 33(2), pages 189-195; Saini et al., 2007, Scand. J. Immunol., vol. 65(1), pages 32-38; and Rabbani et al., 1997, Immunogenetics, vol. 46(4), pages 326-331.

[0070]

[0072] 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, the IgG1 is IgG1a, IgG1b, IgG1c, or IgG1d. In another example, the IgG2 is IgG2a or IgG2b. In yet another example, the IgG3 is IgG3a or IgG3b.

[0071]

[0073] The amino acid and nucleic acid sequences of IgG1a, IgG1b, IgG1c, IgG1d, IgG2a, IgG2b, IgG3a, and IgG3b are also well known in the art.

[0072]

[0074] In one example, an IgG of the invention comprises a constant domain, such as a CH1 domain, a CH2 domain, or a CH3 domain, or a combination thereof. In another example, a constant domain of the invention comprises an Fc region, such as a CH2 domain or a CH3 domain, or a combination thereof.

[0073]

[0075] In particular examples, the wild-type constant domain comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1-8. In particular embodiments, the wild-type constant domains of IgG1a, IgG1b, IgG1c, IgG1d, IgG2a, IgG2b, IgG3a, and IgG3b comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, respectively. In some embodiments, the wild-type IgG constant domain is a homolog, variant, isomer, or functional fragment of any one of SEQ ID NOs: 1-8, but does not have any of the mutations described herein. Each possibility represents a separate embodiment of the invention. For example, in one embodiment, the wild-type constant domain of IgG2a comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0074]

[0076] IgG contant domains also include polypeptides having amino acid sequences substantially similar to those of the heavy and / or light chains, where substantially the same amino acid sequence is defined herein as a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence being compared, as determined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988).

[0075]

[0077] The present invention also includes nucleic acid molecules encoding the IgGs described herein or portions thereof. In one embodiment, the nucleic acid may encode an antibody heavy chain, for example, comprising a CH1, CH2, or CH3 region, or a combination thereof. In another embodiment, the nucleic acid may encode an antibody heavy chain, for example, comprising any one of a VH region or a portion thereof, or any one of a VH CDR, including any variant thereof. The present invention also includes nucleic acid molecules encoding an antibody light chain, for example, comprising any one of a CL region or a portion thereof, a VL region or a portion thereof, or any one of a VL CDR, including any variant thereof. In certain embodiments, the nucleic acid encodes both a heavy chain and a light chain, or portions thereof.

[0076]

[0078] The amino acid sequence of the wild-type constant domain set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9 is encoded by the corresponding nucleic acid sequence.

[0077] Modified Bovine IgG

[0079] The inventors of the present application have surprisingly and unexpectedly found that substituting the amino acid residues at positions 252, 286, 311, 312, 426, 428, 434, or 436 with another amino acid exhibited the desired effect. As used herein, the term "position" refers to a position numbered according to the Eu index in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In one embodiment, the desired effect is higher affinity for FcRn compared to IgGs having a wild-type bovine IgG constant domain. In another embodiment, the desired effect is elimination or reduction of complement-dependent cytotoxicity compared to IgGs having a wild-type bovine IgG constant domain. In another embodiment, the desired effect is elimination or reduction of antibody-dependent cellular phagocytosis compared to IgGs having a wild-type bovine IgG constant domain. In yet another embodiment, the desired effect is to eliminate or reduce binding of IgG to Fcγ receptors (bFcgR).

[0078]

[0080] In one embodiment, the present invention provides a modified IgG comprising a bovine IgG constant domain comprising at least one amino acid substitution compared 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, as numbered according to the Eu index in Kabat. The amino acids at these positions may be substituted with any other amino acid. Examples of substituted amino acids include, but are 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 an unnatural amino acid.

[0079]

[0081] 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).

[0080]

[0082] In exemplary embodiments, 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.

[0081]

[0083] 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, 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, V426I, V426K, 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, M428Y, and one or more of N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

[0082]

[0084] In another example, a mutant IgG2 constant domain of the invention comprises one or more mutations described herein. In some embodiments, the mutant IgG2 constant domain is a homolog, variant, isomer, or functional fragment, but does not have any of the mutations of the invention described herein. Each possibility represents a separate embodiment of the invention.

[0083]

[0085] The amino acid sequence of the variant constant domain is encoded by its corresponding variant nucleic acid sequence.

[0084] Methods for producing antibody molecules of the invention

[0086] Methods for generating antibody molecules are well known in the art and are fully described in U.S. Patent Nos. 8,394,925, 8,088,376, 8,546,543, 10,336,818, and 9,803,023, and U.S. Patent Application Publication No. 20060067930, which are incorporated herein by reference in their entireties. Any suitable method, process, or technique known to those of skill in the art can be used. Antibody molecules with variant Fc regions of the present invention can be produced according to methods well known in the art. In some embodiments, the variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or binding domain of a receptor or ligand.

[0085]

[0087] With the advent of molecular biology methods and recombinant technology, those skilled in the art can produce antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences that encode the specific amino acid sequences found in the polypeptide structure of an antibody. Such antibodies can be produced either by cloning the gene sequences encoding the antibody polypeptide chains or by directly synthesizing the polypeptide chains and assembling the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This has allowed the ready production of antibodies with sequences characterized by neutralizing antibodies from different species and sources.

[0086]

[0088] Regardless of the source of the antibody or how it is recombinantly constructed or synthesized—in vitro or in vivo, using transgenic animals, laboratory- or commercial-sized large cell cultures, transgenic plants, or by direct chemical synthesis without using living organisms at any stage of the process—all antibodies have a similar overall three-dimensional structure. This structure is often provided as H2L2, referring to the fact that antibodies generally contain two light (L) chain amino acids and two heavy (H) chain amino acids. Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting regions are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence between antibodies of different antigen specificities. The variable region of either the H or L chain contains amino acid sequences capable of specifically binding to antigen targets.

[0087]

[0089] As used herein, the term "antigen-binding region" refers to a portion of an antibody molecule containing amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody-binding region contains "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the heavy or light chains that provide the antigen-binding region, there are smaller sequences called "hypervariable" regions, which are responsible for the extreme variability between antibodies of different specificities. Such hypervariable regions are also called "complementarity-determining regions" or "CDR regions." These CDR regions are responsible for the basic specificity of an antibody for a particular antigenic determinant structure.

[0088]

[0090] Although CDRs represent non-contiguous stretches of amino acids within the variable region, the locations of these important amino acid sequences within the variable heavy and light chain regions have been found to have similar positions within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) regions and variable regions, within the latter of which there are CDRs and so-called "framework regions" composed of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

[0089]

[0091] The present invention also provides vectors containing at least one of the above-described nucleic acids. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences may be identified, each of which may be capable of encoding the amino acid. The probability that a particular oligonucleotide will actually constitute an actual coding sequence can be estimated by considering unusual base-pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in the eukaryotic or prokaryotic cell expressing the antibody or moiety. Such "codon usage rules" are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Lathe's "codon usage rules" can be used to identify a single nucleotide sequence or set of nucleotide sequences containing a theoretical "most likely" nucleotide sequence capable of encoding a bovine IgG sequence. It is also contemplated that antibody coding regions for use in the present invention may be provided by modifying existing antibody genes using standard molecular biology techniques to generate variants of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequences of the antibody or peptide.

[0090]

[0092] For example, one type of substitution is a conservative amino acid substitution. Such a substitution replaces a given amino acid in a bovine antibody peptide with another amino acid with similar characteristics. Typical conservative substitutions include the substitution of aliphatic amino acids Ala, Val, Leu, and Lie for each other; the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the exchange of amide residues Asn and Gin, the exchange of basic residues Lys and Arg, and the exchange of aromatic residues Phe and Tyr. Guidance on which amino acid changes are likely to be phenotypically silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0091]

[0093] Variant bovine antibodies or peptides may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative mutations or mutations in non-critical residues or non-critical regions. Functional variants may also contain substitutions of similar amino acids that do not change or only slightly change function. Alternatively, such substitutions may have a positive or negative effect to some extent. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions in critical residues or critical regions.

[0092]

[0094] Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites important for ligand-receptor binding can also be determined by structural analysis, such as 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).

[0093]

[0095] Furthermore, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Furthermore, 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 a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those skilled in the art and are well described in the scientific literature. Some particularly common modifications, such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in most basic textbooks, such as, for example, Proteins - Structure and Molecular Properties (2nd ed., TECreighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews on this subject are available, such as those by Wold, Posttranslational Covalent Modification of Proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. Meth. Enzymol. 626-46 (1990), and Rattan et al. Ann. NY Acad. Sci. 48-62 (1992).

[0094]

[0096] In another aspect, the present invention provides antibody derivatives. A "derivative" of an antibody contains additional chemical moieties that are not normally part of the protein. Covalent modifications of proteins are included within the scope of the present invention. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to water-insoluble support matrices or other macromolecular carriers.

[0095]

[0097] Derivatives also include radiolabeled monoclonal antibodies, for example, labeled with radioactive iodine (251, 1311), carbon (C), sulfur (S), indium, tritium (H 3 ); conjugates of monoclonal antibodies containing biotin or avidin with enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters), or fluorescent agents (such as phycobyl proteins) are used.

[0096]

[0098] Another derivative bifunctional antibody of the present invention is a bispecific antibody, generated by combining portions of two separate antibodies that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques. Additionally, moieties can be added to the antibody or portions thereof to increase its half-life in vivo (e.g., by extending the time until clearance from the bloodstream). Such techniques include, for example, the addition of PEG moieties (also called pegylation), and are well known in the art. See U.S. Patent Application Publication No. 20030031671.

[0097]

[0099] In some embodiments, nucleic acids encoding the subject antibodies are directly introduced into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded antibody. After the subject nucleic acids are introduced into the cells, the cells are typically incubated for a period of about 1 to 24 hours, usually at 37°C, sometimes under selection, to allow expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the culture medium in which the cells are growing. Traditionally, monoclonal antibodies are produced as natural molecules in murine hybridoma lines. In addition to that technology, the present invention provides for recombinant DNA expression of antibodies, allowing for the production of antibodies in a selected host species and the generation of a spectrum of antibody derivatives and fusion proteins.

[0098] [000100] Nucleic acid sequences encoding at least one antibody, portion, or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques, including restriction enzyme digestion to provide blunt or cohesive ends for ligation, suitable termini, appropriate filling of sticky ends, alkaline phosphatase treatment to avoid undesired ligations, and ligation with an appropriate ligase. Techniques for such manipulations are disclosed, for example, by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 (supra), and can be used to construct nucleic acid sequences encoding antibody molecules or antigen-binding regions thereof.

[0099] [000101] A nucleic acid molecule, such as DNA, comprises a nucleotide sequence containing transcriptional and translational regulatory information, and is said to be "capable of expressing" a polypeptide when such a sequence is "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the DNA sequence to be expressed are connected in such a way as to permit gene expression as a peptide or antibody moiety in recoverable amounts. The precise nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 2001 (supra); Ausubel et al., 1993 (supra).

[0100] [000102] Thus, the present invention encompasses expression of antibodies or peptides in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacterial, yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or in host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin. Any other suitable mammalian cells known in the art can also be used.

[0101] [000103] In one embodiment, the nucleotide sequences of the present invention will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be used for this purpose. See, e.g., Ausubel et al., 1993 (supra). Important factors in selecting a particular plasmid or viral vector include the ease with which vector-containing recipient cells can be recognized and selected from vector-free recipient cells; the number of vector copies desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0102] [000104] Examples of prokaryotic vectors known in the art include plasmids such as those capable of replicating in E. coli (e.g., pBR322, CoIE1, pSC101, pACYC184, .pi.vX, etc.) and are disclosed 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 Streptomyces bacteriophages such as p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)) and phLC31 (Chater et al., SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986)). Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986), Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978), and Ausubel et al., 1993 (supra).

[0103] [000105] Alternatively, gene expression elements useful for expressing antibody or peptide encoding cDNA include, but are not limited to, (a) viral transcription promoters and 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 such as those derived from the SV40 late region (Okayama et al., 1983), and (c) polyadenylation sites such as those from SV40 (Okayama et al., 1983).

[0104] [000106] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987) using the SV40 early promoter and enhancer, mouse immunoglobulin heavy chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements as expression elements. For immunoglobulin genes composed of cDNA portions and genomic DNA portions (Whittle et al., 1 Protein Engin. 499 (1987)), the transcription promoter can be human cytomegalovirus, the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be native chromosomal immunoglobulin sequences.

[0105] [000107] In one embodiment, for expression of cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements listed above to achieve protein expression in mammalian cells.

[0106] [000108] Each fusion gene can be assembled into or inserted into an expression vector. Recipient cells capable of expressing the immunoglobulin chain gene products are then transfected with the peptide or H or L chain encoding genes singly or co-transfected with the H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the integrated genes, and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.

[0107] [000109] In one embodiment, fusion genes encoding peptides or heavy and light chains, or portions thereof, are then assembled into separate expression vectors used to cotransfect recipient cells. Alternatively, fusion genes encoding heavy and light chains can be assembled on the same expression vector. The recipient cell line for transfection of the expression vector and production of antibodies can be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin genes and possess the machinery for immunoglobulin glycosylation. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, from which secreted immunoglobulins can be obtained from ascites. Other suitable recipient cells include lymphoid cells, such as B lymphocytes of bovine or non-bovine origin, hybridoma cells of bovine or non-bovine origin, or interspecies heterohybridoma cells.

[0108] [000110] Expression vectors carrying the antibody constructs or polypeptides of the present invention can be introduced into appropriate host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[0109] [000111] Yeast may offer substantial advantages over bacteria in the production of immunoglobulin heavy and light chains. Yeast carries out post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies now exist that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes peptides bearing the leader sequence (i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0110] [000112] Yeast gene expression systems can be routinely evaluated for levels of production, secretion, and stability of peptides, antibodies, fragments, and regions thereof. Any of a range of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium can be utilized. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Several approaches can be selected to evaluate the optimal expression plasmid for expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985).

[0111] [000113] Bacterial strains can also be utilized as hosts for the production of the antibody molecules or peptides described by this invention. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these bacterial hosts. The vector carries a replication site as well as specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be selected to evaluate expression plasmids for the production in bacteria of antibodies, fragments, and regions, or antibody chains, encoded by cloned immunoglobulin cDNAs (see Glover, 1985 (supra); Ausubel, 1993 (supra); Sambrook, 2001 (supra); Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001)).

[0112] [000114] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, folding and assembly of hand L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. Mammalian cells that may be useful as hosts for antibody protein production include cells of fibroblast origin, such as Vero (ATCC CRL81) or CHO-K1 (ATCC CRL61) cells, in addition to the lymphoid origin cells mentioned above. Many vector systems are available for the expression of cloned peptide hand L chain genes in mammalian cells (see Glover, 1985, supra). Different approaches can be followed to obtain complete H2L2 antibodies. Hand L chains can be co-expressed in the same cell to achieve intracellular assembly and linkage of hand L chains into complete tetrameric H2L2 antibodies and / or peptides. Co-expression can be achieved by using either the same or different plasmids in the same host. Genes for both the hand L chain and / or peptide can be placed on the same plasmid, which can then be transfected into cells, thereby directly selecting for cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the L chain, and the resulting cell line subsequently transfected with an H chain plasmid containing a second selectable marker. To generate cell lines with improved properties, such as higher production of assembled H2L2 antibody molecules or improved stability of the transfected cell line, cell lines producing peptide and / or H2L2 molecules via either route can be transfected with plasmids encoding additional copies of the peptide, H, L, or H plus L chain, along with additional selectable markers.

[0113] [000115] For long-term, high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. Following introduction of the foreign DNA, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow, forming populations (foci) that can then be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful in screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0114] [000116] Once an antibody of the invention is produced, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly for a specific antigen following Protein A, and sizing column chromatography), centrifugation, absorbance differential solubility, or any other standard technique for the purification of proteins. In many embodiments, the antibody is secreted from the cells into the culture medium and harvested from the culture medium.

[0115] Pharmaceutical and veterinary uses [000117] The present invention also provides pharmaceutical compositions comprising a molecule of the invention and one or more pharmaceutically acceptable carriers. More specifically, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent and an antibody or peptide according to the invention as an active ingredient.

[0116] [000118] A "pharmaceutically acceptable carrier" includes any excipient that is nontoxic to cells or animals exposed at the dosages and concentrations employed. The pharmaceutical composition may include one or additional therapeutic agents.

[0117] [000119] "Pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of an animal without undue toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit / risk ratio.

[0118] [000120] 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.

[0119] [000121] Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphates, citrates, and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; and other carbohydrates including monosaccharides, disaccharides, and glucose, mannose, or dextrins; EDTA; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®; isotonic agents such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride; and combinations thereof.

[0120] [000122] The pharmaceutical compositions of the present invention can be formulated in a variety of ways, including, for example, liquid, semi-solid, or solid dosage forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the compositions are in the form of an injectable or infusible solution. The compositions may be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The compositions may be formulated as immediate-release, controlled-release, sustained-release, or delayed-release compositions.

[0121] [000123] The compositions of the present invention can be administered either as individual therapeutic agents or in combination with other therapeutic agents. While they can be administered alone, they are generally administered with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein can be carried out to respiratory tract surfaces by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of the antibody (typically in a pharmaceutical formulation). Topical administration to respiratory tract surfaces can be carried out by intranasal administration (e.g., using a dropper, swab, or inhaler). Topical administration of antibodies to respiratory tract surfaces can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibody as an aerosol suspension and then having a subject inhale the respirable particles. Methods and devices for administering respirable particles of a pharmaceutical formulation are well known, and any conventional techniques can be used.

[0122] [000124] In some desirable embodiments, antibodies are administered by parenteral injection. For parenteral administration, antibodies or molecules can be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle can be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier. Such vehicles include water, saline, Ringer's solution, dextrose solution, trehalose, or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, and the like. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting agents and buffers, toxicity adjusters, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of antibody in these formulations can vary widely, for example, from less than about 0.5% by weight, usually from about 1% or more to up to 15% or 20% by weight, and will be selected primarily based on fluid volume, viscosity, etc., according to the particular mode of administration selected. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[0123] [000125] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution technique can be used. Those skilled in the art will appreciate that lyophilization and reconstitution may result in varying degrees of antibody activity loss, which may require adjustment of use levels to compensate. Compositions containing the present antibodies or a cocktail thereof can be administered for the prevention of recurrence and / or therapeutic treatment of existing disease. Suitable pharmaceutical carriers are described in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference textbook in the art. In therapeutic applications, the compositions are administered to a subject already suffering from a disease in an amount sufficient to cure, or at least partially arrest or alleviate, the disease and its complications.

[0124] [000126] Effective doses of the compositions of the invention for treating the conditions or diseases described herein will vary depending on many different factors, including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the target site; the physiological condition of the animal; other pharmaceutical agents being administered; whether the treatment is prophylactic or therapeutic; the age, health, and weight of the recipient; the nature and extent of the type of symptom; the type of concurrent treatment, the frequency of treatment, and the desired effect.

[0125] [000127] Single or multiple administrations of the compositions can be carried out with the dose level and pattern being selected by the treating veterinarian. In any event, the pharmaceutical formulation should provide a quantity of the antibody of the invention sufficient to effectively treat the subject.

[0126] [000128] Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0127] [000129] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount." A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary depending on factors such as the disease state, age, sex, and weight in the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

[0128] [000130] In another aspect, the compositions of the present invention can be used to treat various diseases and disorders, for example, in bovine animals. As used herein, the terms "treat" and "treatment" refer to therapeutic procedures, including prophylactic or preventative measures, where the subject is one in whom undesired physiological changes associated with a disease or condition are to be prevented or slowed (alleviated). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease or condition, stabilization of the disease or condition (i.e., the disease or condition does not worsen), delay or slowing of the progression of the disease or condition, improvement or alleviation of the disease or condition, and remission (partial or complete) of the disease or condition. Those in need of treatment include those already with the disease or condition, as well as those prone to have the disease or condition, or those in whom the disease or condition is to be prevented.

[0129] [000131] All patents and literature references cited herein are incorporated by reference in their entirety.

[0130] [000132] The following examples are provided to supplement the foregoing disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the subject matter described. The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or changes in light thereof, which are apparent to those skilled in the art, are within the true scope of the present invention and can be made without departing from the true scope of the present invention. [Example]

[0131] Example 1 Construction of bovine IgG Fc mutants [000133] Construction of bovine IgGs was performed according to methods well known in the art. Plasmids containing sequences encoding bovine constant regions for the IgG2a subclass were used, and the VH / VL sequences of each mAb investigated herein were inserted upstream and in frame with nucleotides encoding the constant domains. Mutations were incorporated into either the CH2 or CH3 domain positions of each plasmid by direct DNA synthesis of the constant regions as gene fragments, which were then subcloned into the respective variable regions of interest.

[0132] Expression and purification [000134] Monoclonal antibody (mAb) variants were expressed in mammalian suspension cell line EXPICHO-S (Chinese Hamster Ovary) cells obtained from Thermo Fisher Scientific. Suspension EXPICHO-S cells were maintained at 0.14–8.0 × 10e6 cells / ml in EXPICHO Expression Medium (Gibco). Cells were diluted according to the ExpiCHO protocol user manual on day -1 and the day of transfection. Following maximum titer conditions, diluted cells were transfected as described in the protocol using reagents supplied with the ExpiFectamine CHO Transfection Kit (Gibco). After 12–14 days of incubation, the culture was harvested and clarified. Antibodies were purified from the clarified supernatant via Protein A chromatography using a MabSelect Sure LX (GE Healthcare) pre-equilibrated with PBS. After sample loading, the resin was washed with PBS and then with 20 mM sodium acetate, pH 5.5. Samples were eluted from the column with 20 mM acetic acid, pH 3.5. After elution, a pool was made and neutralized by adding 1 M sodium acetate to 4%. Depending on the available volume and intended use, samples were occasionally exchanged into a final buffer (e.g., PBS, etc.). Concentration was measured by absorbance at 280 nm.

[0133] SDS-PAGE [000135] Non-reduced (nr) and reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4-12% Bis-Tris NuPAGE gels in MES-SDS running buffer and SeeBlue Plus 2 standards (all from Invitrogen). For non-reduced samples, 1 mM of the alkylating agent N-ethylmaleimide (NEM) was added, and for reduced samples, the reducing agent dithiothreitol (DTT) was added. Gels were stained with Coomassie blue to detect protein bands.

[0134] Example 2 FcRn binding assay Biacore method for bFcRn: [000136] The binding affinity of bovine Fc-based antibodies or fusion proteins to bovine FcRn was determined by surface plasmon resonance (SPR). All reported KDs were measured using a Biacore T200 (Cytiva, Marlborough, MA, USA) or Biacore 8K (Cytiva, Marlborough, MA, USA) with an SA sensor. Bovine FcRn was captured on the sensor surface to the desired surface density. The running buffer used was 20 mM MES, 150 mM NaCl, 0.005% Tween® 20, 0.5 mg / mL BSA, pH 6, and / or PBS, 0.0005% Tween® 20, pH 7.4. Various concentrations of bovine mAb were titrated in the appropriate running buffer and flowed over the receptor surface. Regeneration was performed with 50 mM Tris-HCl, pH 8. Dynamic binding affinity was analyzed using the Biacore T200 Evaluation software (Cytiva, Marlborough, MA, USA) or Biacore 8K Insight Evaluation software using the double-referencing method. A reference flow cell was subtracted from the flow cell containing immobilized bovine FcRn, and a blank experiment containing buffer alone was subtracted from all experiments. The resulting curves were fitted to a 1:1 binding model. Experiments were performed at 25°C.

[0135] [000137] Mutations made at each position have a significant effect on the affinity of IgG to FcRn at pH 6.

[0136] [000138] Binding of wild-type (WT) and mutant IgGs to bovine FcRn was measured by surface plasmon resonance (Biacore). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0137] [000139] The results clearly show that mutations made at various positions have a significant effect on the affinity of IgG to bovine FcRn.

[0138] Example 3 FcRn binding validation method [000140] First, the Fc regions of two bovine allotypes, IgG2a and IgG2b, were designed using their respective CH2 and CH3 regions. The protein modeling function of Alphafold2.2, developed by Deepmind, was implemented to model the 3D structures of wild-type (WT) and mutant constructs of each of the bovine allotypes, IgG2a and IgG2b.

[0139] [000141] The Molecular Operating Environment (MOE) (MOE2019.0102), developed by the Chemical Computing Group, provides a flexible, automated graphical user interface for protein modeling. To analyze structural differences, a sequence-to-profile alignment algorithm ranks sequence templates using a scoring algorithm, with a score above 85% ensuring the selection of a protein template with a physically realistic structure. The same pipeline was then used to optimize the model, and the structural stability of the model was verified using a Ramachandran plot, which checks the stereochemical quality of the protein structure.

[0140] result [000142] 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, T286R, T286S, T286V, T286W, T286Y ... 86M, 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, D3 12K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, M428A, M428C, M428D, M428E, M428F, M428 G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N Mutations were performed on the following amino acids: 434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W. The positions of the mutation library are represented in ball-and-stick format in Figure 3A. An RMSD plot was generated to calculate the root mean square deviation of the two WT structures (bovine-IgG2a_WT and bovine-IgG2b_WT) relative to each other (Figure 3B). An RMSD value of 2.0 Å or less is considered the criterion for considering two structures as similar.The results showed that the bovine IgG2a construct was identical to the bovine IgG2b allotype, with an average RMSD value of 0.57 Å for the structures. The RMSD values at the positions where the mutation scan was performed are also shown in Table 2, and ranged from 0.226 to 0.997 Å. [Table 2]

[0141] [000143] We also investigated the following mutations in both bovine subclasses following the same procedure: 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, T286J, T286K ... 86K, 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, , M428A, M428C, M428D, M428E, M4 28F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N 434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W (Table 3). Again, similar to the WT, no dramatic variation in the models was observed at each of the mutation positions between the two allotypes, as indicated by the RMSD value range of 0.226–0.997 Å (Table 3 ). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0142] conclusion [000144] Molecular modeling of all mutations across the two allotypes, bovine backbone IgG2a and IgG2b, was performed and validated using MOE2019.0102. The Fc fold and residue conformations between the subclasses were shown to have an RMSD of less than 2 Å, indicating that the protein structures have a high degree of identity and may therefore function in a similar manner.

[0143] [000145] Although preferred embodiments of the present invention have been described, it should be understood that the invention is not limited to the precise embodiments and that various changes and modifications may be made by those 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 compared to a wild-type bovine IgG constant domain, said substitution being at amino acid residue 252, 286, 311, 312, 426, 428, 434, or 436, as numbered according to the Eu index in Kabat.

2. The constant domain may be any of the following 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, T286S, T286V, T286W, T286 Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q31 1L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D31 2A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D3 12N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, V426A, V4 26C, V426D, V426E, V426F, V426G, V426H, V426I, V426K, V426L, V426M, V 426N, V426P, V426Q, V426R, V426S, V426T, V426W, V426Y, M428A, M428C, M 428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, 2. The modified IgG of claim 1, comprising one or more of N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, 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 of claim 1 , wherein the modified IgG is a bovine or bovinized IgG.

4. The modified IgG of claim 1 , wherein the IgG is IgG2.

5. The modified IgG of claim 4, wherein the IgG2 is IgG2a or IgG2b.

6. 10. The modified IgG of any one of the preceding claims, wherein the modified IgG has a significant effect on its affinity for bovine FcRn compared to the affinity of an IgG having the wild-type bovine IgG constant domain.

7. 10. The modified IgG of any one of the preceding claims, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type bovine IgG constant domain.

8. 10. The modified IgG of claim 1, wherein the IgG constant domain comprises a CH1 domain or a hinge region.

9. 10. The modified IgG of claim 1, wherein the IgG constant domain comprises an Fc constant region having a CH2 domain and a CH3 domain.

10. 10. The modified IgG according to any one of the preceding claims, wherein the wild-type bovine IgG constant domain comprises one of the amino acid sequences set forth in SEQ ID NOs: 1-9.

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

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

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

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

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

16. An isolated cell comprising the vector of claim 15.

17. 17. A method for producing an antibody or molecule, said method comprising providing a cell according to claim 16 and culturing said cell.

18. 15. A method for producing an antibody, said method comprising providing the antibody of claim 14.

19. 15. A method for increasing antibody serum half-life in livestock, said method comprising administering to said livestock a therapeutically effective amount of the antibody of claim 14.

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

21. 1. A method for increasing antibody serum half-life in a bovine animal, said method comprising administering to said bovine animal a therapeutically effective amount of an antibody comprising a bovine IgG constant domain which comprises at least one amino acid substitution compared to a wild-type bovine IgG constant domain, said substitution being at amino acid residue 252, 286, 311, 312, 426, 428, 434, or 436, as numbered according to the Eu index in Kabat.

22. The bovine IgG constant domain may be any of the following 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, T286Y ... 86W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q 311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q 311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D 312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, V 426A, V426C, V426D, V426E, V426F, V426G, V426H, V426I, V426K, 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, M428Y, N434A, N434C, 22. The method of claim 21 , comprising one or more of N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W.

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

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

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

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

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

28. 22. The method of claim 21, wherein the wild-type bovine IgG constant domain comprises one of the amino acid sequences set forth in SEQ ID NOs: 1-9.

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