Pig antibody mutants

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 378,740, filed on October 7, 2022, which is incorporated herein by reference in its entirety. Technical Field

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

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

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

[0006] Engineering the Fc region to tailor its interaction with FcRn has become a promising approach for enhancing the activity of therapeutic antibodies.

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

[0008] The present invention relates to mutant pig IgGs that exhibit desired properties relative to wild-type pig IgGs. Specifically, the inventors of the present application have found that replacing the amino acid residue at position 233, 234, 235, 236, 238, 252, 254, 256, 265, 286, 293, 297, 307, 311, 312, 322, 329, 330, 331, 378, 426, 428, 434 or 436 (numbered according to the Eu index as in Kabat) with another amino acid surprisingly and unexpectedly exhibits the desired effect. In an exemplary embodiment, the unexpected desired effect includes, but is not limited to, an enhanced affinity for FcRn and a change in effector function.

[0009] In one aspect, the present invention provides a modified IgG comprising: a porcine IgG constant domain comprising at least one amino acid substitution relative to a wild-type porcine IgG constant domain, wherein the substitution is at amino acid residue 233, 234, 235, 236, 238, 252, 254, 256, 265, 286, 293, 297, 307, 311, 312, 322, 329, 330, 331, 378, 426, 428, 434 or 436.

[0010] In an exemplary embodiment, the porcine IgG constant domain comprises mutations E233P, G234A, V234A, A235L, P235L, P235A, G236A, G236L, P238A, M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, S254T, T256E, T256F, T256N, D265A, T286A, T286C, T286D, T286E, T2 86F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, T289A, R290A, K292A, E293 deleted, N297G, P307Q, E311A, E311C, E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E3 11N, E311P, E311Q, E311R, E311S, E311T, E311V, E311W, E311Y, D312A, D312C , D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D 312Q, D312R, D312S, D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P32 9L, A330S, P331S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426H , A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A4 26V, A426W, A426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428 I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M 428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N43 4L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A,One or more of Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

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

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

[0013] In another aspect, the present invention provides a method for producing or manufacturing an antibody or molecule, the method comprising: providing a vector or host cell having a nucleic acid sequence encoding an antibody, wherein the antibody comprises a porcine IgG constant domain, which porcine IgG constant domain comprises one or more amino acid substitutions of the invention described herein.

[0014] According to the following detailed description examples and drawings, other features and advantages of the present invention will become apparent. However, it should be understood that although the detailed description and specific examples indicate the preferred embodiments of the present invention, they are only given by way of illustration, because according to the detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] This patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] Figure 1 An alignment of the amino acid sequences of human IgG1 and porcine IgG1a, IgG2, IgG3, IgG4a, IgG5a and IgG6a is shown. The CH1, hinge, CH2 and CH3 domains are as follows: CH1: residues 118-215; hinge: 216-230; CH2: 231-340; CH3: 341-447. The amino acid residues are numbered according to the Eu index as in Kabat.

[0017] Figure 2 Cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc subclass CTLA4 fusion protein is shown.

[0018] Figure 3A and 3B show the cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) activity of porcine wild-type Fc subclass CTLA4 fusion protein.

[0019] Figure 4 Cell-based antibody-dependent cell-mediated phagocytosis (ADCP) of porcine wild-type Fc subclass CTLA4 fusion protein is shown.

[0020] Figure 5 A, 5B and 5C show the cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc IgG6 subclass CTLA4 fusion protein and Fc mutants of this subclass.

[0021] Figure 6 A, 6B and 6C show the cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) activity of porcine wild-type Fc IgG6 subclass CTLA4 fusion protein and mutations of this Fc subclass.

[0022] Figure 7 A and 7B show the cell-based antibody-dependent cell-mediated phagocytosis (ADCP) activity of porcine wild-type Fc IgG6 subclass CTLA4 fusion protein and mutations of this Fc subclass.

[0023] Figure 8 A and 8B show the cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc IgG4a and 4b subclass CTLA4 fusion proteins and Fc mutants of these subclasses.

[0024] Fig. 9 Cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) activities of porcine wild-type Fc IgG4a and IgG4b subclass CTLA4 fusion proteins and SAP mutations of these Fc subclasses are shown.

[0025] Fig.10 Cell-based antibody-dependent cellular phagocytosis (ADCP) activity of porcine wild-type Fc IgG4a subclass CTLA4 fusion protein and SAP and WinPG mutations of this Fc subclass is shown.

[0026] Fig.11 Cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc IgG2 subclass CTLA4 fusion protein and Fc mutants of this subclass is shown.

[0027] Fig.12 The cell-based antibody-dependent cellular cytotoxicity (ADCC) activity of porcine wild-type IgG2 subclass CTLA4 fusion protein and Fc mutants of this subclass is shown.

[0028] Fig.13 A and 13B show the cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc IgG1a and 1b subclass CTLA4 fusion proteins and Fc mutants of these subclasses.

[0029] Fig.14 Cell-based antibody-dependent cellular cytotoxicity (ADCC) activities of porcine wild-type IgG1a and 1b subclass CTLA4 fusion proteins and Fc mutants of these subclasses are shown.

[0030] Fig.15 A and 15B. Cell-based antibody-dependent cell-mediated phagocytosis (ADCP) activity of porcine wild-type and corresponding mutants of the IgG6 subclass.

[0031] Fig.16 A and 16B. Cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc IgG4a and 4b subclasses, CTLA4 fusion proteins, and Fc mutants of these subclasses.

[0032] Fig.17 A and 17B. Cell-based antibody-dependent cellular phagocytosis (ADCP) activity of porcine wild-type Fc IgG4a subclass mutations of this Fc subclass.

[0033] Fig.18 A and 18B. Cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild-type Fc IgG2 subclass CTLA4 fusion protein and Fc mutants of this subclass.

[0034] Fig.19 A and 19B. Cell-based antibody-dependent phagocytosis of porcine wild-type IgG2 subclass and Fc mutants of this subclass.

[0035] Fig. 20 A and 20B. Cell-based complement-dependent cytotoxic activity of porcine wild-type Fc IgG1a subclass CTLA4 fusion proteins and Fc mutants of these subclasses.

[0036] Fig.21 Cell-based antibody-dependent phagocytosis of porcine wild-type IgG1a proteins and Fc mutants of these subclasses.

[0037] Fig. 22 Cell-based antibody-dependent phagocytosis of porcine wild-type IgG1a proteins and Fc mutants of these subclasses.

[0038] Fig.23A) Overlay of protein models of porcine IgG1a and IgG1b Fc regions showing amino acid residue positions important for effector functions in ball-and-stick format B) Protein model of porcine IgG2 (2a and 2b Fc are identical) Fc region showing amino acid residue positions important for effector functions in ball-and-stick format.

[0039] Fig.24 A) Overlay of protein model of porcine IgG4a and IgG4b Fc region showing amino acid residue positions important for effector function in ball-and-stick format B) Protein model of porcine IgG6a and IgG6b Fc region showing amino acid residue positions important for effector function in ball-and-stick format.

[0040] Fig.25 Comparison of root mean square deviation (RMSD) of wild-type constructs of porcine IgG1a, IgG1b, IgG2, IgG4a, IgG4b, IgG6a, and IgG6b.

[0041] Brief Description of Sequence Listing

[0042] SEQ ID NO.: 1 refers to the amino acid sequence of the wild-type constant region of porcine IgG1a.

[0043] SEQ ID NO.: 2 refers to the amino acid sequence of the wild-type constant region of porcine IgG1b.

[0044] SEQ ID NO.: 3 refers to the amino acid sequence of the wild-type constant region of porcine IgG2a.

[0045] SEQ ID NO.: 4 refers to the amino acid sequence of the wild-type constant region of porcine IgG2b.

[0046] SEQ ID NO.: 5 refers to the amino acid sequence of the wild-type constant region of porcine IgG3.

[0047] SEQ ID NO.: 6 refers to the amino acid sequence of the wild-type constant region of porcine IgG4a.

[0048] SEQ ID NO.: 7 refers to the amino acid sequence of the wild-type constant region of porcine IgG4b.

[0049] SEQ ID NO.: 8 refers to the amino acid sequence of the wild-type constant region of porcine IgG5a.

[0050] SEQ ID NO.: 9 refers to the amino acid sequence of the wild-type constant region of porcine IgG5b.

[0051] SEQ ID NO.: 10 refers to the amino acid sequence of the wild-type constant region of porcine IgG6a.

[0052] SEQ ID NO.: 11 refers to the amino acid sequence of the wild-type constant region of porcine IgG6b.

[0053] SEQ ID NO.: 12 refers to the amino acid sequence of the wild-type constant region of human IgG1. DETAILED DESCRIPTION

[0054] The subject matter of the present invention may be more readily understood by reference to the following detailed description which forms a part of the present disclosure. It should be understood that the present invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is only for the purpose of describing specific embodiments by way of example and is not intended to limit the claimed invention.

[0055] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular.

[0056] As employed above and throughout the disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise indicated.

[0057] definition

[0058] In this disclosure, the singular forms "a", "an", and "the" include plural references, and reference to a particular value includes at least that particular value unless the context clearly indicates otherwise. Thus, for example, reference to a "molecule" or "compound" is a reference to one or more of such molecules or compounds and equivalents thereof known to those skilled in the art, and so on. As used herein, the term "plurality" means more than one. When a range of values ​​is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0059] In the specification and claims, the numbering of amino acid residues in immunoglobulin heavy chains is the Eu index as in Kabat, Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "Eu index as in Kabat" refers to the residue numbering of IgG antibodies, and is used herein Figure 1 reflected in.

[0060] When used with respect to nucleic acids, the term "isolated" refers to a nucleic acid that is identified and separated from at least one contaminant nucleic acid that is usually associated with it in its natural source. The isolated nucleic acid is in a form or environment different from the form or environment in which it exists in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acid molecule present in natural cells. The isolated nucleic acid molecule includes nucleic acid molecules contained in cells that are usually expressed in polypeptides encoded therein, wherein, for example, the nucleic acid molecule is located in a plasmid or chromosomal position different from the position of natural cells. The isolated nucleic acid can exist in single-stranded or double-stranded form. When the isolated nucleic acid molecule is used to express a protein, the oligonucleotide or polynucleotide will contain at least a sense strand or a coding strand, but can contain both a sense strand and an antisense strand (that is, can be double-stranded).

[0061] A nucleic acid molecule is "operably linked" or "operably attached" when it is in a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a coding sequence of a nucleic acid if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence of a nucleic acid if it is positioned to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof that does not contain an Fc region when present in nature) if the nucleic acid molecule is positioned so that the expressed fusion protein comprises a heterologous protein or functional fragment thereof abutting the variant Fc region polypeptide upstream or downstream; the heterologous protein may be immediately adjacent to the variant Fc region polypeptide or may be separated therefrom by a linking sequence of any length and composition. Similarly, a polypeptide (used synonymously with "protein" herein) molecule is "operably linked" or "operably attached" when it is in a functional relationship with another polypeptide.

[0062] As used herein, when referring to a polypeptide or protein (e.g., a variant Fc region or a monoclonal antibody), the term "functional fragment" refers to a fragment of the protein that retains at least one function of the full-length polypeptide. The size of the fragment can range from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. The functional fragment of the variant Fc region polypeptide of the present invention retains at least one "amino acid substitution" as defined herein. The functional fragment of the variant Fc region polypeptide retains at least one function associated with the Fc region known in the art (e.g., ADCC, CDC, Fc receptor binding, Clq binding, cell surface receptor downregulation, or can, for example, increase the in vivo or in vitro half-life of the polypeptide to which it is operably attached).

[0063] The term "purified" or "purification" refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody can be purified by complete or substantial removal (at least 90%, 91%, 92%, 93%, 94%, 95%, or more preferably at least 96%, 97%, 98%, or 99%) of at least one contaminating non-immunoglobulin; it can also be purified by the removal of immunoglobulins that do not bind to the same antigen. Removal of non-immunoglobulins and / or removal of immunoglobulins that do not bind to a particular antigen increases the percentage of antigen-specific immunoglobulins in the sample. In another example, a polypeptide (e.g., immunoglobulin) expressed in a bacterial host cell is purified by complete or substantial removal of host cell proteins; thus, the percentage of polypeptide in the sample is increased.

[0064] The term "native" when it refers to a polypeptide (e.g., an Fc region) is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of the polypeptide (in the case where the polypeptide normally occurs in nature) or a naturally occurring polymorph thereof. A native polypeptide (e.g., a native Fc region) can be produced by recombinant means or can be isolated from a naturally occurring source.

[0065] As used herein, the term "expression vector" refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences required for the expression of the operably linked coding sequence in a particular host organism.

[0066] 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.

[0067] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the recognized boundaries of the Fc region of an immunoglobulin heavy chain may vary, the porcine IgG heavy chain Fc region is generally defined as, for example, Figure 1 In some embodiments, the variant comprises only a portion of the Fc region and may or may not include the carboxyl terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more constant domains are contemplated. In other embodiments, variants having no such constant domains (or having only a portion of such constant domains) are contemplated.

[0068] The "CH2 domain" of the porcine IgG Fc region refers to, for example, Figure 1The CH2 domain is unique in that it is not tightly paired with another domain.

[0069] The "CH3 domain" of a porcine IgG Fc region is generally the stretch of residues C-terminal to the CH2 domain in the Fc region, e.g. Figure 1 Residue 341 to the C-terminus.

[0070] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); antibody dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require that the Fc region be operably linked to a binding domain (e.g., an antibody variable domain) and may be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, ADCP assays, target cell depletion in whole or fractionated blood samples, etc.).

[0071] A "native sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region commonly found in nature. Figure 1 In the exemplary native sequence porcine Fc region is from residue 231 to the c-terminus.

[0072] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region (or a fragment thereof) by at least one "amino acid substitution" as defined herein. In a preferred embodiment, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or in the Fc region of a parent polypeptide, preferably 1, 2, 3, 4 or 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. In an alternative embodiment, a variant Fc region can be produced according to the methods disclosed herein, and this variant Fc region can be fused to a selected heterologous polypeptide, such as an antibody variable domain or a non-antibody polypeptide, for example, a binding domain of a receptor or ligand.

[0073] As used herein, in the context of a polypeptide, the term "derivative" refers to a polypeptide comprising an amino acid sequence that has been changed by the introduction of an amino acid residue substitution. As used herein, the term "derivative" also refers to a polypeptide that has been modified by the covalent attachment of any type of molecule to a polypeptide. For example, but not limited to, an antibody may be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization using known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, and the like. Derivatized polypeptides may be prepared by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, tunicamycin-mediated metabolic synthesis, and the like. In addition, a derived polypeptide has a function similar to or identical to that of a polypeptide prior to its derivatization. It should be understood that a polypeptide comprising a variant Fc region of the present invention may be a derivative as defined herein, preferably, derivatization occurs within the Fc region.

[0074] "Substantially of porcine origin" as used herein with respect to a polypeptide (e.g., an Fc region or a monoclonal antibody) indicates that the polypeptide has an amino acid sequence that is at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, or even more preferably at least 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence of a native porcine amino polypeptide.

[0075] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to an Fc region (e.g., an Fc region of an antibody). A preferred FcR is a native sequence FcR. In addition, a preferred FcR is an FcR that binds to an IgG antibody Fc region, an Fcγ receptor or "FcgR", and includes receptors of the FcγRI (FcgR1), FcγRII (FcgR2), FcγRIII (FcgR3) subclasses, which include allelic variants and alternative splicing forms of these receptors and novel porcine Fcγ2R. Another preferred FcR includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). The term "FcR" herein encompasses other FcRs, including those identified in the future.

[0076] The phrases "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) expressing FcgRs (e.g., natural killer ("NK") cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby cause lysis of the target cell. NK cells are the primary cells used to mediate ADCC in humans and express only FcgR3, while monocytes express FcgR1, FcgR2, and FcgR3.

[0077] The phrases "antibody-dependent cell-mediated phagocytosis" and "ADCP" refer to a cell-mediated reaction in which phagocytic cells (e.g., macrophages, monocytes, dendritic cells) expressing FcgRs (e.g., FcgR1, FcgR2a, and FcgR3) recognize bound IgG antibody Fc regions on target cells and subsequently trigger a signaling cascade resulting in the phagocytosis of IgG-opsonized particles (e.g., bacteria, dead tissue cells).

[0078] As used herein, the phrase "effector cell" refers to a leukocyte (preferably pig) that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least FcgR3 and performs ADCC effector functions. Examples of leukocytes that mediate ADCC include PBMC, NK cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be separated from natural sources (e.g., from blood or PBMC). In one example, leukocytes express FcgR1 or other related Fcγ receptors and trigger ADCP function.

[0079] Variant polypeptides with "altered" Fc receptor binding affinity are variant polypeptides with enhanced (i.e., increased, greater or higher) or diminished (i.e., decreased, lesser or lower) Fc receptor binding affinity compared to the variant's parent polypeptide or a polypeptide comprising a native Fc region. Variant polypeptides that exhibit increased binding to an Fc receptor or increased binding affinity bind to an Fc receptor with greater affinity than a parent polypeptide. Variant polypeptides that exhibit reduced binding to an Fc receptor or reduced binding affinity bind to an Fc receptor with lower affinity than their parent polypeptide. Such variants that exhibit reduced binding to an Fc receptor may have little or no significant binding to an Fc receptor, for example, 0-20% binding to an Fc receptor Fc receptor compared to a parent polypeptide. A variant polypeptide that binds to an Fc receptor with "enhanced affinity" compared to its parent polypeptide is a variant polypeptide that binds to an Fc receptor with a higher binding affinity than a parent polypeptide when the amounts of the variant polypeptide and the parent polypeptide are substantially the same in a binding assay and all other conditions are the same. For example, a variant polypeptide with enhanced Fc receptor binding affinity may exhibit an increase in Fc receptor binding affinity of about 1.10-fold to about 100-fold (more usually about 1.2-fold to about 50-fold) compared to the parent polypeptide, where Fc receptor binding affinity is determined, for example, in an ELISA assay or other methods available to one of ordinary skill in the art.

[0080] As used herein, "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence by another different "replacement" amino acid residue. The replacement residue can be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and is selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (H is); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). The definition of amino acid substitution herein also encompasses replacement with one or more non-naturally occurring amino acid residues. "Non-naturally occurring amino acid residues" refers to residues that are capable of covalently binding to adjacent amino acid residues in a polypeptide chain, in addition to those naturally occurring amino acid residues listed above. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991).

[0081] The term "assay signal" refers to any method for detecting protein-protein interactions, including but not limited to the output of absorbance measurement, fluorescence intensity or number of decays per minute from a colorimetric assay. The assay format may include ELISA, FACS or other methods. The change in "assay signal" may reflect changes in cell viability and / or changes in kinetic dissociation rate, kinetic association rate or both. A "higher assay signal" refers to a measured output number greater than another number (e.g., in an ELISA assay, a variant may have a higher (larger) measured number compared to a parent polypeptide). A "lower" assay signal refers to a measured output number less than another number (e.g., in an ELISA assay, a variant may have a lower (smaller) measured number compared to a parent polypeptide).

[0082] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in concentration units) associated with each Fc receptor-Fc binding interaction. Binding affinity is related to the kinetic dissociation rate (usually expressed in reverse time units, e.g., seconds). -1 The dissociation constant (K) is directly related to the ratio of the concentration (reported in units of concentration per unit time, e.g., moles per second) to the kinetic association rate (usually reported in units of concentration per unit time, e.g., moles per second). In general, it is not possible to unambiguously state the equilibrium dissociation constant (K DIt is not possible to determine whether changes in KD (or KD) are due to differences in association rate, dissociation rate, or both, unless each of these parameters is determined experimentally (e.g., as measured by BIACORE or SAPIDYNE).

[0083] As used herein, the term "hinge region" refers to the stretch of amino acids that connects the Fab antigen binding region to the Fc region of an antibody. The hinge regions of the IgG subclass can be aligned by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (S—S) bonds in the same position. Figure 1 As shown, for example, the hinge region in the porcine IgG constant region starts at residue 216 and extends to residue 230.

[0084] "C1q" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q, together with two serine proteases, C1r and C1s, forms the complex C1, which is the first component of the CDC pathway.

[0085] As used herein, the term "antibody" is used interchangeably with "immunoglobulin" or "Ig", is used in the broadest sense, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity or functional activity. Single-chain antibodies and chimeric, pig or swine antibodies, as well as chimeric or CDR-grafted single-chain antibodies, etc., containing parts derived from different species, are also covered in the present invention and the term "antibody". The various parts of these antibodies can be chemically linked together in a synthetic manner by conventional techniques, or can be prepared as a continuous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric chain or a swine chain can be expressed to produce a continuous protein. See, for example, U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; WO 86 / 01533; U.S. Patent No. 5,225,539; and U.S. Patent Nos. 5,585,089 and 5,698,762. See also Newman, R. et al. BioTechnology, 10: 1455-1460, 1993, regarding privatized antibodies, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, RE et al., Science, 242: 423-426, 1988, regarding single-chain antibodies. It should be understood that all forms of antibodies comprising an Fc region (or a portion thereof) are encompassed herein within the term "antibody". In addition, antibodies can be labeled with a detectable label, can be immobilized on a solid phase and / or combined with a heterologous compound (e.g., an enzyme or a toxin) according to methods known in the art.

[0086] As used herein, the term "antibody fragment" refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. The antibody fragment preferably retains the hinge of the IgG heavy chain and optionally at least a portion of the CH1 region. In other preferred embodiments, the antibody fragment comprises at least a portion of the CH2 region or the entire CH2 region.

[0087] As used herein, the term "functional fragment" when used with reference to a monoclonal antibody is intended to refer to a portion of a monoclonal antibody that still retains functional activity. Functional activity can be, for example, antigen binding activity or specificity, receptor binding activity or specificity, effector function activity, etc. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single-chain Fv (scFv); and Fc fragments. Such terms are described, for example, in Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and in Day, ED, Advanced Immunochemistry, 2nd edition, Wiley-Liss, Inc., New York, NY (1990). The term functional fragment is intended to include fragments produced, for example, by protease digestion or reduction of monoclonal antibodies and by recombinant DNA methods known to those skilled in the art.

[0088] As used herein, the term "fragment" refers to a polypeptide comprising an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more consecutive amino acid residues of an amino acid sequence of another polypeptide. In a preferred embodiment, a fragment of a polypeptide retains at least one function of a full-length polypeptide.

[0089] As used herein, the term "chimeric antibody" includes monovalent, divalent or multivalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by the association of a chimeric heavy chain with a chimeric light chain through a disulfide bridge. A divalent chimeric antibody is a tetramer formed by the association of two heavy chain-light chain dimers through at least one disulfide bridge. The chimeric heavy chain for the antibody of pigs comprises an antigen binding region derived from the heavy chain of a non-porcine antibody, which is connected to at least a portion of a porcine heavy chain constant region, such as CH1 or CH2. The chimeric light chain for the antibody of pigs comprises an antigen binding region derived from the light chain of a non-porcine antibody, which is connected to at least a portion of a porcine light chain constant region (CL). Antibodies, fragments or derivatives of chimeric heavy and light chains with the same or different variable region binding specificities can also be prepared by appropriate association of separate polypeptide chains according to known method steps. Using this method, a host expressing a chimeric heavy chain is cultured separately from a host expressing a chimeric light chain, and the immunoglobulin chains are recovered separately and then associated. Alternatively, the host can be co-cultured and the chains allowed to spontaneously associate in the culture medium, after which the assembled immunoglobulin or fragment is 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).

[0090] As used herein, a "porcine" form of a non-porcine (e.g., murine) antibody (i.e., a porcine antibody) is an antibody containing minimal or no sequence derived from a non-porcine immunoglobulin. In most cases, a porcine antibody is a porcine immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-porcine species (donor antibody) having the desired specificity, affinity, and capacity, such as a mouse, rat, rabbit, human, or non-human primate. In some cases, the framework region (FR) residues of the porcine immunoglobulin are replaced by corresponding non-porcine residues. In addition, the porcine antibody may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are usually performed to further optimize antibody performance. In general, a porcine antibody will contain substantially all variable domains of at least one and typically two variable domains, wherein all or substantially all hypervariable loops (CDRs) correspond to those of a non-porcine immunoglobulin, and all or substantially all FR residues are those of a porcine immunoglobulin sequence. The porcinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a porcine immunoglobulin.

[0091] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding domain of a heterologous "adhesin" protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an adhesin amino acid sequence that is not an antibody's antigen recognition and binding site (antigen combining site) (i.e., "heterologous") with an immunoglobulin constant domain sequence with the desired binding specificity.

[0092] As used herein, the term "ligand binding domain" refers to any natural receptor or any region or derivative thereof that at least retains the qualitative ligand binding ability of the corresponding natural receptor. In certain embodiments, the receptor is from a cell surface polypeptide having an extracellular domain homologous to a member of the immunoglobulin supergene family. Other receptors that are not members of the immunoglobulin supergene family but are still specifically encompassed within this definition are receptors for cytokines, and specifically receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hemopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L- and P-selectins).

[0093] As used herein, the term "receptor binding domain" refers to any natural ligand of a receptor, including, for example, cell adhesion molecules, or any region or derivative of such a natural ligand that retains at least the qualitative receptor binding ability of the corresponding natural ligand.

[0094] As used herein, an "isolated" polypeptide is a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components in its natural environment are materials that will interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other protein or non-protein solutes. In certain embodiments, the isolated polypeptide is purified (1) to greater than 95% by weight of the polypeptide, as determined by the Lowry method, and preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated polypeptide includes the polypeptide in situ within a recombinant cell, since at least one component of the natural environment of the polypeptide will not be present. However, the isolated polypeptide will typically be prepared by at least one purification step.

[0095] As used herein, the terms "disorder" and "disease" are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., a pathological condition that predisposes a patient to a particular disorder).

[0096] As used herein, the term "receptor" refers to a polypeptide capable of binding to at least one ligand. Preferably, the receptor is a cell surface receptor or a soluble receptor having an extracellular ligand binding domain and optionally other domains (e.g., a transmembrane domain, an intracellular domain, and / or a membrane anchor). The receptor to be evaluated in the assay described herein can be a complete receptor or a fragment or derivative thereof (e.g., a fusion protein comprising a binding domain of a receptor fused to one or more heterologous polypeptides). In addition, the receptor to be evaluated for its binding properties can be present in a cell, or isolated and optionally coated on an assay plate or some other solid phase, or directly labeled and used as a probe.

[0097] 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 porcine effector cells compared to the parent antibody is a polypeptide that mediates ADCC, ADCP, and / or CDC activity in vitro or in vivo substantially lower when the amount of the variant polypeptide and the parent antibody used in the assay is substantially the same. 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 can be identified, for example, using ADCC, ADCP, or CDC assays, but other assays or methods (e.g., animal models) for determining ADCC, ADCP, or CDC activity can also be employed. In a preferred embodiment, the activity of the variant polypeptide in mediating ADCC, ADCP, and CDC is about 100%, 75%, 50%, or 25% lower than that of the parent polypeptide.

[0098] Porcine wild-type IgG

[0099] Porcine IgG is well known in the art and is fully described in, for example, Butler et al., 2009, Immunogenetics, Vol. 61(3): pp. 209-30; and Paudyal et al., 2022, Front Immunol, Vol. 13, pp. 903755. In one embodiment, porcine IgG is IgG1. In another embodiment, porcine IgG is IgG2. In another embodiment, porcine IgG is IgG3. In another embodiment, porcine IgG is IgG4. In another embodiment, porcine IgG is IgG5. In another embodiment, porcine IgG is IgG6.

[0100] The allotypes of the porcine IgG subclasses shown in Table 1 below are also well known in the art. The IgG1 described herein can be, for example, IgG1a or 1b; the IgG2 described herein can be, for example, IgG2a or 2b; the IgG4 described herein can be, for example, IgG4a or 4b; the IgG5 described herein can be, for example, IgG5a or 5b; and the IgG6 described herein can be, for example, IgG6a or 6b. In a specific example, the porcine IgG is IgG6a.

[0101] The amino acid and nucleic acid sequences of IgG are also well known in the art.

[0102] In one example, the IgG of the present invention comprises a constant domain, such as a CH1, CH2 or CH3 domain, or a combination thereof. In another example, the constant domain of the present invention comprises an Fc region, including, for example, a CH2 or CH3 domain, or a combination thereof.

[0103] In a specific example, the wild-type constant domain comprises the amino acid sequence listed in SEQ ID NO.: 1-11. In specific embodiments, the wild-type constant domain of IgG1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a and 6b comprises the amino acid sequence listed in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, respectively. In some embodiments, the wild-type IgG constant domain is a homolog, variant, isomer or functional fragment of any one of SEQ ID NO.: 1-11, but does not have any mutations described herein. Each possibility represents a separate embodiment of the present invention.

[0104] IgG constant domains also include polypeptides having an amino acid sequence that is substantially similar to the amino acid sequence of the heavy chain and / or light chain. Substantially identical amino acid sequences are defined herein as sequences having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the comparison amino acid sequence, as determined by the following FASTA search method: Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988).

[0105] The present invention also includes nucleic acid molecules encoding IgG or parts thereof as described herein. In one embodiment, the nucleic acid can encode an antibody heavy chain comprising, for example, CH1, CH2, CH3 regions or a combination thereof. In another embodiment, the nucleic acid can encode an antibody heavy chain comprising, for example, any one of the VH region or a portion thereof or any one of the VH CDRs, including any variants thereof. The present invention also includes nucleic acid molecules encoding antibody light chains, which include, for example, any one of the CL region or a portion thereof, any one of the VL region or a portion thereof, or any one of the VL CDRs, including any variants thereof. In certain embodiments, nucleic acid encodes both heavy and light chains or portions thereof.

[0106] The amino acid sequence of the wild-type constant domain listed in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 is encoded by its corresponding nucleic acid sequence.

[0107] Modified porcine IgG

[0108] The inventors of the present application have found that replacing an amino acid residue at position 233, 234, 235, 236, 238, 252, 254, 256, 265, 286, 293, 297, 307, 311, 312, 322, 329, 330, 331, 378, 426, 428, 434 or 436 with another amino acid surprisingly and unexpectedly exhibits a desired effect. As used herein, the term position refers to a position numbered according to the Eu index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In one embodiment, the desired effect is a higher affinity for FcRn relative to an IgG with a wild-type porcine IgG constant domain. In another embodiment, the desired effect is elimination or reduction of complement dependent cytotoxicity (CDC) relative to an IgG with a wild-type porcine IgG constant domain. In another embodiment, the desired effect is elimination or reduction of antibody-dependent cell-mediated cytotoxicity (ADCC) relative to an IgG having a wild-type porcine IgG constant domain. In another embodiment, the desired effect is elimination or reduction of antibody-dependent cellular phagocytosis (ADCP) relative to an IgG having a wild-type porcine IgG constant domain. In yet another embodiment, the desired effect is elimination or reduction of IgG binding to Fcγ receptors (pFcgR).

[0109] In one embodiment, the present invention provides a modified IgG comprising: a porcine IgG constant domain comprising at least one amino acid substitution relative to a wild-type porcine IgG constant domain, wherein the substitution is at amino acid residues 233, 234, 235, 236, 238, 252, 254, 256, 265, 286, 293, 297, 307, 311, 312, 322, 329, 330, 331, 378, 426, 428, 434 or 436 numbered according to the Eu index as in Kabat. The amino acids at these positions can be substituted with any other amino acid. Examples of substituted amino acids include, for example, but not limited to, asparagine, histidine, serine, alanine, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid and proline. In some embodiments, the substituted amino acid is a non-natural amino acid.

[0110] The modified porcine IgG of the present invention can be any suitable porcine IgG known to those skilled in the art. Examples of modified porcine IgG include modified variants of IgG1 (e.g., IgG1a or 1b), IgG2 (e.g., IgG2a or 2b), IgG3, IgG4 (e.g., IgG4a or 4b), IgG5 (e.g., IgG5a or 5b) or IgG 6 (e.g., IgG6a or 6b).

[0111] In another exemplary embodiment, the porcine IgG constant domain comprises substitution mutations E233P, G234A, V234A, A235L, P235L, P235A, G236A, G236L, P238A, M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, S254T, T256E, T256F, T256N, D265A, T286A, T286C, T286D, T286E, T 286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, T289A, R290A, K292A, E293 deleted, N297G, P307Q , E311A, E311C, E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E 311N, E311P, E311Q, E311R, E311S, E311T, E311V, E311W, E311Y, D312A, D312 C. D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P32 9L, A330S, P331S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426H , A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A4 26V, A426W, A426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428 I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M 428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N43 4L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A,One or more of Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

[0112] In one embodiment, the modified porcine IgG is an IgG1a constant domain comprising one or more of the substitutions selected from the group consisting of: (i) P329S and A330S; (ii) D265A, P329G and A330S; (iii) V234A, A235L, G236A, P329L and A330S; (iv) V234A, A235L, G236A and P329G; (v) E233P, A330S and P331S; (vi) K322A and P331A; and (vii) P329S.

[0113] In another embodiment, the modified porcine IgG is an IgG1b constant domain comprising one or more of the substitutions selected from the group consisting of: (i) V234A, A235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; and (v) K322A and P331A.

[0114] In another embodiment, the modified porcine IgG is an IgG2a constant domain comprising one or more of the substitutions selected from the group consisting of: (i) V234A, A235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

[0115] In another embodiment, the modified porcine IgG is an IgG2b constant domain comprising one or more of the substitutions selected from the group consisting of: (i) V234A, A235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

[0116] In another embodiment, the modified porcine IgG is an IgG4a constant domain comprising one or more of the substitutions selected from the group consisting of: (i) G234A, P235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

[0117] In another embodiment, the modified porcine IgG is an IgG4b constant domain comprising one or more of the substitutions selected from the group consisting of: (i) G234A, P235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

[0118] In another embodiment, the modified porcine IgG is an IgG6a constant domain comprising one or more of the substitutions selected from the group consisting of: (i) D265A, N297G, P329G and A330S; (ii) G234A, P235L, G236A and P329G; (iii) E233P, A330S and P331S; (iv) P235A; G236L ; and P238A; (v) D265A and N297G; (vi) P329G; (vii) P331A; (viii) K322A; (ix) E233P; (x) P329S and A330S; (xi) G234A, P235L, G236A, P329L and A330S; (xii) K322A and P331A; and (xiii) P329S.

[0119] In another embodiment, the modified porcine IgG is an IgG6b constant domain comprising one or more of the substitutions selected from the group consisting of: (i) G234A, P235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

[0120] In another example, the mutant IgG constant domain of the present invention comprises one or more mutations described herein. In certain embodiments, the mutant IgG constant domain is a homologue, variant, isomer or functional fragment, but has a mutation of the present invention described herein. Each possibility represents a separate embodiment of the present invention.

[0121] The amino acid sequence of the mutant constant domain is encoded by its corresponding mutant nucleic acid sequence.

[0122] Methods for preparing the antibody molecules of the invention

[0123] Methods for preparing antibody molecules are well known in the art and are described in detail in U.S. Patents 8,394,925; 8,088,376; 8,546,543; 10,336,818; and 9,803,023 and U.S. Patent Application Publication 20060067930, the entire contents of which are incorporated herein by reference. Any suitable method, process or technology known to those skilled in the art may be used. Antibody molecules having variant Fc regions of the present invention may be produced according to methods well known in the art. In some embodiments, variant Fc regions may be fused to selected heterologous polypeptides, such as antibody variable domains or the binding domains of receptors or ligands.

[0124] With the advent of molecular biological methods and recombinant technology, those skilled in the art can produce antibodies and antibody-like molecules by recombinant means, and thereby produce gene sequences encoding specific amino acid sequences present in the polypeptide structure of the antibody. Such antibodies can be produced by cloning the gene sequences encoding the polypeptide chains of the antibody or by direct synthesis of the polypeptide chains, wherein the assembly of the synthetic chains forms an active tetramer (H2L2) structure with affinity for specific epitopes and antigenic determinants. This allows the production of antibodies with sequence characteristics of neutralizing antibodies from different species and sources at any time.

[0125] Regardless of the source of the antibody or how it is constructed recombinantly, or how it is in vitro or in vivo using transgenic animals, laboratory or commercial-scale large cell cultures, using transgenic plants, or by direct chemical synthesis, no living organisms are used to synthesize at any stage of the process, all antibodies have a similar overall 3-dimensional structure. The structure is usually given as H2L2, and refers to the fact that antibodies usually contain two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions that can interact with structurally complementary antigenic targets. The region that interacts with the target is called a "variable" or "V" region, and is characterized in that antibodies with different antigenic specificities have differences in amino acid sequence. The variable region of the H chain or L chain contains an amino acid sequence that can specifically bind to the antigenic target.

[0126] As used herein, the term "antigen binding region" refers to the portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antibody for the antigen. The antibody binding region includes the "framework" amino acid residues necessary to maintain the correct configuration of the antigen binding residues. An antigen binding region is provided in the variable region of the H or L chain, which is a smaller sequence called "hypervariable" because it has great variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions explain the basic specificity of antibodies for specific antigenic determinant structures.

[0127] CDR represents the discontinuous extension of amino acid in variable region, but no matter how species, it has been found that the positioning position of these key amino acid sequences in variable heavy chain region and variable light chain region has similar position in the amino acid sequence of variable chain. The variable heavy chain and light chain of all antibodies each have three CDR regions, and each CDR region is not adjacent to other CDR regions. In all mammalian species, antibody peptides contain constant region (that is, highly conserved) and variable region, and in the latter, there is CDR and the so-called "framework region" consisting of the amino acid sequence in the variable region of heavy chain or light chain but outside CDR.

[0128] The present invention further provides a vector comprising at least one of the nucleic acids described above. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which will be able to encode an amino acid. By considering the abnormal base pairing relationship and the frequency of the actual use of a specific codon (to encode a specific amino acid) in a eukaryotic or prokaryotic cell expressing an antibody or part, it is possible to estimate the probability that a specific oligonucleotide will actually constitute an actual coding sequence. Such "codon usage rules" are disclosed by: Lathe et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe's "codon usage rules", a single nucleotide sequence or a set of nucleotide sequences containing a theoretically "most likely" nucleotide sequence capable of encoding a pig IgG sequence can be identified. It is also hoped that the antibody coding region for the present invention can be provided by using standard molecular biology techniques to change the existing antibody gene, which produces variants of antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions and substitutions in the amino acid sequence of an antibody or peptide.

[0129] For example, one type of substitution is a conservative amino acid substitution. Such substitutions are those in which a given amino acid in a pig antibody peptide is replaced by another amino acid with similar properties. Substitutions are generally considered conservative substitutions, with one replacing another in aliphatic amino acids Ala, Val, Leu and lie; exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gin, exchange between basic residues Lys and Arg, substitution among aromatic residues Phe, Tyr, etc. Guidance on which amino acid changes may be phenotypically silent is found in Bowie et al., 247 Science 1306-10 (1990).

[0130] Variant pig antibodies or peptides may be fully functional, or may lack function in one or more activities. Fully functional variants typically contain only conservative changes or changes in non-critical residues or non-critical regions. Functional variants may also contain replacements of similar amino acids that do not produce functional changes or produce insignificant functional changes. Alternatively, such replacements may have a positive or negative impact on function to some extent. Non-functional variants typically contain one or more non-conservative amino acid replacements, deletions, insertions, inversions or truncations, or replacements, insertions, inversions or deletions in key residues or key regions.

[0131] Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations into each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as anti-epitope binding or in vitro ADCC activity. Sites critical for ligand-receptor binding can also be determined by structural analysis, such as epitope mapping (e.g., HDX), crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[0132] In addition, polypeptides generally contain amino acids other than twenty kinds of "naturally occurring" amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositols, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginization and ubiquitination. Such modifications are well known to those skilled in the art and have been described in great detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation are described in most basic texts, such as Proteins-Structure and Molecular Properties (2nd ed., TECreighton, WH Freeman & Co., NY, 1993). Many detailed introductions to this topic are available, such as Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983); Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0133] In another aspect, the invention provides antibody derivatives. "Derivatives" of antibodies contain additional chemical moieties that are not normally part of the protein. Covalent modifications of proteins are included within the scope of the invention. Such modifications can be introduced into the molecule by reacting the targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art is suitable for crosslinking antibodies or fragments with water-insoluble carrier matrices or other macromolecular carriers.

[0134] Derivatives also include radiolabeled monoclonal antibodies that are labeled, for example, using radioactive iodine (251, 1311), carbon (4C), sulfur (35S), indium or tritium (H 3) etc.; conjugates of monoclonal antibodies with biotin or antigenic proteins, with enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carboxylic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridinium esters) or fluorescent agents (such as phycobiliproteins).

[0135] Another derivative bifunctional antibody of the present invention is a bispecific antibody produced by combining the parts of two independent antibodies that recognize two different antigen groups. This can be achieved by crosslinking or recombinant technology. In addition, part can be added to the antibody or a part thereof to increase the half-life in vivo (for example, by extending the time of removal from the bloodstream. Such technology includes, for example, adding a PEG part (also referred to as pegylation), and is well known in the art. Referring to U.S. Patent Application Publication No. 20030031671.

[0136] In certain embodiments, the nucleic acid encoding the subject antibody is directly introduced into the host cell, and the cell is cultivated under conditions sufficient to induce the expression of the encoded antibody. After the subject nucleic acid is introduced into the cell, the cell is cultivated for a period of about 1 to 24 hours under selection, usually at 37°C, to allow the antibody to be expressed. In one embodiment, the antibody is secreted into the supernatant of the culture medium of cell growth. Traditionally, monoclonal antibodies have been produced as natural molecules in mouse hybridoma cell lines. In addition to the technology, the present invention provides recombinant DNA expression of antibodies. This allows the production of antibodies in selected host species, as well as a series of antibody derivatives and fusion proteins.

[0137] The nucleotide sequence of at least one antibody, part or polypeptide of the present invention can be recombined with carrier DNA according to routine techniques, and the routine techniques include blunt end or staggered end for connection, restriction enzyme digestion for providing appropriate end, sticky end filling for carrying out when appropriate, alkaline phosphatase treatment for avoiding connection and connection with appropriate ligase for avoiding undesirable connection. The technology of such operation is for example by Maniatis et al., MOLECULAR CLONING, LAB.MANUAL, (Cold Spring Harbor Lab.Press, NY, 1982 and 1989), and Ausubel et al. 1993 the same disclosure, can be used for constructing the nucleotide sequence encoding antibody molecule or its antigen binding domain.

[0138] If a nucleic acid molecule (such as DNA) contains a nucleotide sequence that includes transcriptional and translational regulatory information, and such sequence is "operably linked" to a nucleotide sequence encoding a polypeptide, then the nucleic acid molecule is said to be "capable of expressing" the polypeptide. An operable connection is a connection in which the regulatory DNA sequence and the DNA sequence for which expression is sought are linked in a manner that allows the gene to be expressed as a recoverable amount of the peptide or antibody portion. The precise nature of the regulatory region required for gene expression can vary from organism to organism, as is well known in similar fields. See, for example, Sambrook et al., 2001 supra; Ausubel et al., 1993 supra.

[0139] Therefore, the present invention encompasses expression of antibodies or peptides in prokaryotic or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds and mammalian cells in vivo or in situ, or host cells of mammalian, insect, bird or yeast origin. Mammalian cells or tissues can have human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog or cat origin. Any other suitable mammalian cells known in the art can also be used.

[0140] In one embodiment, the nucleotide sequence of the invention is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a variety of vectors may be used for this purpose. See, for example, Ausubel et al., 1993 supra. Important factors in selecting a particular plasmid or viral vector include: the ease with which a recipient cell containing the vector can be identified and selected from those that do not contain the vector; the number of copies of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0141] Exemplary prokaryotic vectors known in the art include plasmids, such as plasmids capable of replication in E. coli (such as, for example, pBR322, CoIE1, pSC101, pACYC 184, .pi.vX). Such plasmids are disclosed, for example, by Maniatis et al., 1989 supra; Ausubel et al., 1993 supra. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan in THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987) and Streptomyces phage, such as phLC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ONACTINOMYCETALES 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.

[0142] Alternatively, gene expression elements that can be used to express cDNA encoding antibodies or polypeptides include, but are not limited to (a) viral transcriptional promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985); (b) splice regions and polyadenylation sites, for example, those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as those in SV40 (Okayama et al., 1983).

[0143] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51Gene 21 (1987) using the SV40 early promoter and its enhancer, mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globulin intervening sequence, immunoglobulin and rabbit S-globulin polyadenylation sites, and SV40 polyadenylation elements as expression elements. For immunoglobulin genes composed of part cDNA and part genomic DNA (Whittle et al., 1Protein Engin. 499 (1987)), the transcription promoter can be human cytomegalovirus, the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be native chromosomal immunoglobulin sequences.

[0144] In one embodiment, for expressing cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron of more than 31 bp, and the polyadenylation and transcription terminal regions are derived from natural chromosomal sequences corresponding to synthetic immunoglobulin chains. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements described above to achieve protein expression in mammalian cells.

[0145] Each fusion gene can be assembled in an expression vector or inserted into an expression vector. Recipient cells capable of expressing immunoglobulin chain gene products are then transfected with genes encoding peptides or H chains or L chains alone, or co-transfected with H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the incorporated genes and the expressed immunoglobulin chains or complete antibodies or fragments are recovered from the culture.

[0146] In one embodiment, the fusion gene encoding peptide or H and L chain or its part is assembled in a separate expression vector, and then the expression vector is used for co-transfection recipient cells. Alternatively, the fusion gene encoding H and L chain can be assembled on the same expression vector. For the transfection of expression vector and the production of antibody, the recipient cell line can be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes, and have a mechanism for glycosylation of immunoglobulins. Myeloma cells can grow in culture or in the peritoneal cavity of mice (wherein secreted immunoglobulins can be obtained from ascites). Other suitable receptor cells include lymphoid cells, such as B lymphocytes of pig or non-pig origin, hybridoma cells of pig or non-pig origin, or inter-species heterologous hybridoma cells.

[0147] The expression vector carrying the antibody construct or polypeptide of the present invention can be introduced into an appropriate host cell by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and the use of polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical methods such as electroporation, direct microinjection and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[0148] Yeast can provide substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications, including glycosylation. There are many recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of cloned mammalian gene products and secretes peptides (i.e., propeptides) with the leader sequence. Hitzman et al., 11th edition Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0149] Yeast gene expression systems can be routinely evaluated for the production, secretion, and stability levels of peptides, antibodies, fragments, and regions thereof. Any of a range of yeast gene expression systems can be utilized, and these expression systems have promoters and termination elements from actively expressed genes encoding saccharolytic enzymes that are produced in large quantities when yeast is grown in a medium rich in glucose. Known glycolytic genes can also provide very effective transcriptional control signals. For example, promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A variety of methods can be used to evaluate the optimal expression plasmid for expressing cloned immunoglobulin cDNAs in yeast. See Volume II, DNA Cloning, 45-66, (Glover, ed.), IRL Press, Oxford, UK, 1985).

[0150] Bacterial strains can also be used as hosts for producing antibody molecules or peptides described in the present invention. Plasmid vectors containing replicons and control sequences derived from species compatible with host cells are used in combination with these bacterial hosts. The vector carries a replication site and a specific gene that can provide phenotypic selection in transformed cells. A variety of methods can be used to evaluate the expression plasmids that produce antibodies, fragments and regions or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria (see Glover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001 supra; Colligan et al., ed. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001); Colligan et al., ed. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0151] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications for immunoglobulin molecules, including leader peptide removal, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. In addition to the lymphoid cells described above, mammalian cells that can be used as hosts for producing antibody proteins also include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL61) cells. Many vector systems can be used to express cloned peptide H and L chain genes in mammalian cells (see Glover, 1985 supra). Different methods can be used to obtain complete H2L2 antibodies. H and L chains can be co-expressed in the same cell to achieve intracellular association and connection of H and L chains into complete tetrameric H2L2 antibodies and / or peptides. Co-expression can be carried out by using the same or different plasmids in the same host. The genes of H and L chains and / or peptides can be placed in the same plasmid, and then the plasmid is transfected into cells, thereby directly selecting cells expressing two chains. Alternatively, cells can be first transfected with a plasmid encoding one chain (e.g., the L chain), and the resulting cell line can then be transfected with an H chain plasmid containing a second selectable marker. Cell lines producing peptides and / or H2L2 molecules by either route can be transfected with plasmids encoding additional copies of the peptide, H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher yields of assembled H2L2 antibody molecules or increased stability of the transfected cell line.

[0152] In order to produce recombinant antibodies for a long time and in high yield, stable expression can be used.For example, the cell line of stably expressed antibody molecules can be engineered.Host cells can be transformed with immunoglobulin expression cassettes and selectable markers, rather than using expression vectors containing viral replication sources.After introducing foreign DNA, engineered cells can be grown in enriched medium for 1 to 2 days, and then converted to selective medium.The selectable marker in the recombinant plasmid gives resistance to selection, and allows cells to stably integrate plasmids into chromosomes and grow to form lesions, and then the lesions can be cloned and expanded into cell lines.This engineered cell line can be particularly suitable for screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0153] Once the antibodies of the invention have been produced, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly after protein A with affinity for a specific antigen, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In many embodiments, the antibodies are secreted from the cells into the culture medium and collected from the culture medium.

[0154] Pharmaceutical and veterinary applications

[0155] The present invention also provides a pharmaceutical composition comprising a molecule of the present invention and one or more pharmaceutically acceptable carriers. More specifically, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and an antibody or peptide according to the present invention as an active ingredient.

[0156] "Pharmaceutically acceptable carriers" include any excipient that is nontoxic to cells or animals exposed thereto at the dosages and concentrations employed. Pharmaceutical compositions may include one or additional therapeutic agents.

[0157] "Pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of animals without producing excessive toxicity, irritation, allergic response, or other problematic complications commensurate with a reasonable benefit / risk ratio.

[0158] 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.

[0159] 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 polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or glucose; EDTA; salts that constitute counterions such as sodium; and / or nonionic surfactants such as TWEEN, polyethylene glycol (PEG) and PLURONICS; isotonic agents such as sugars, polyols (such as mannitol and sorbitol) and sodium chloride; and combinations thereof.

[0160] The pharmaceutical compositions of the present invention can be formulated in a variety of ways, including, for example, liquid, semisolid or solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills or powders. In some embodiments, the composition is in the form of an injectable or infusible solution. The composition can be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical or transdermal administration. The composition can be formulated to release the composition immediately, controlled, extended or delayed.

[0161] The composition of the present invention can be administered as a single therapeutic agent, or in combination with other therapeutic agents. It can be administered alone, but is generally administered together with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice. The administration of the antibodies disclosed herein can be carried out in any suitable manner, including parenteral injection (such as intraperitoneal, subcutaneous or intramuscular injection), oral administration, or by topical administration of the antibody (usually carried in a pharmaceutical formulation) to the airway surface. Topical administration to the airway surface can be performed by intranasal administration (for example, by using a dropper, swab or inhaler). Topical administration of antibodies to the airway surface can also be performed by inhalation administration, such as by generating inhalable particles (including both solid clones and liquid particles) of a pharmaceutical formulation containing antibodies in the form of an aerosol suspension, and then causing the individual to inhale the inhalable particles. The methods and devices for administering inhalable particles of pharmaceutical formulations are well known, and any conventional techniques can be used.

[0162] In some desirable embodiments, the antibody is administered by parenteral injection. For parenteral administration, the antibody or molecule can be formulated as a solution, suspension, emulsion, or lyophilized powder associated with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle can be a solution in which the antibody or its mixture is dissolved in an acceptable carrier, such as an aqueous vehicle, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, etc. Liposomes and non-aqueous vehicles such as fixed oils can also be used. These solutions are sterile and generally do not contain particulate matter. These compositions can be sterilized by conventional well-known sterilization techniques. The composition can contain pharmaceutically acceptable auxiliary substances required to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The antibody concentration in these formulations can vary greatly, for example, from less than about 0.5%, usually at or at least about 1% to as much as 15% or 20% by weight, and will be selected based primarily on the volume of fluid, viscosity, etc., according to the specific mode of administration selected. The vehicle or lyophilized powder may contain additives (e.g., sodium chloride, mannitol) to maintain isotonicity and additives (e.g., buffers and preservatives) to maintain chemical stability. The formulation is sterilized by conventional techniques. The actual method of preparing the composition for parenteral administration is known or apparent to those skilled in the art, and is described in more detail in, for example, REMINGTON'S PHARMA. SCI. (15th edition, Mack Pub. Co., Easton, Pa., 1980).

[0163] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technology has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques can be used. It will be appreciated by those skilled in the art that lyophilization and reconstitution can cause varying degrees of antibody activity loss and that the use content may have to be adjusted in order to compensate. Compositions containing antibodies of the present invention or their mixtures can be used to prevent the recurrence of existing diseases and / or therapeutic treatment of existing diseases. Suitable pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in this technical field. In therapeutic applications, compositions are administered to individuals with diseases in an amount sufficient to cure or at least partially inhibit or alleviate the disease and its complications.

[0164] The effective dosage of the compositions of the invention as described herein for treating a condition or disease varies depending on many different factors, such factors including, for example, but not limited to, the pharmacodynamic characteristics of the particular drug and its mode and route of administration; the target site; the physiological state of the animal; other drugs administered; whether the treatment is prophylactic or therapeutic; the age, health, and weight of the recipient; the nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the desired effect.

[0165] Single or multiple administrations of the compositions may be carried out with the dosage level and pattern selected by the treating veterinarian. In any case, the pharmaceutical formulation should provide an amount of one or more antibodies of the invention sufficient to effectively treat the individual.

[0166] Therapeutic doses can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0167] The pharmaceutical compositions of the invention may include a "therapeutically effective amount". A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the dosage and for the period of time necessary. The therapeutically effective amount of a molecule may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the molecule.

[0168] In another aspect, the compositions of the invention can be used, for example, to treat various diseases and conditions of pigs. As used herein, the terms "treat" and "treatment" refer to therapeutic treatments, including prophylactic or preventive measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in the extent of the disease or condition, stabilization of the disease or condition (i.e., a situation where the disease or condition has not worsened), delay or slowing of the progression of the disease or condition, improvement or alleviation of the disease or condition, and alleviation of the disease or condition (whether partial or complete), whether detectable or undetectable. Situations in which therapy is needed include situations in which the disease or condition is already present, as well as situations in which the disease or condition is susceptible to or is to be prevented.

[0169] All patents and literature references cited in this specification are incorporated herein by reference in their entirety.

[0170] The following examples are provided to supplement the previous disclosure and provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the subject matter described. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and the various modifications or changes made therefrom will be apparent to those skilled in the art and are included in the true scope of the present invention, and can be carried out without departing from the true scope of the present invention.

[0171] Examples

[0172] Example 1

[0173] Porcine IgG and FcRn

[0174] Isolated porcine olfactory tissue, primary cells from porcine olfactory epithelium (OEPC), and the human cell line RPMI 2650 were used for evaluation. Comparable IgG permeability was observed for human and porcine IgG in OEPC, which showed the highest expression of FcRn. Only trace amounts of porcine IgG were recovered at the basolateral compartment in isolated olfactory tissue, while human IgG levels were much higher.

[0175] The porcine FcRn cDNA has a length of 1,577 bp (GenBank Accession No.: AAP49846.1) and contains a 1,077 bp open reading frame (ORF) encoding a polypeptide of 356 amino acids. The 3' end of the sequence contains a poly(A) segment preceded by a putative polyadenylation signal AATAAA (nucleotides 1523-1529). Blast analysis revealed that the mRNA sequence of the porcine FcRn gene has 79.4%, 66.3% and 83.9% nucleotide identities with the corresponding genes of humans, mice and cattle, respectively. The complete porcine FcRn genomic DNA sequence spans 8,900 bp (GenBank Accession No.: HQ026019), and it consists of 5 introns separating 6 exons. The intron / exon organization of the porcine (5 introns and 6 exons) is the same as that of the human and mouse FcRn genes.

[0176] The porcine IgGs used to assess in vitro FcRn binding are provided in Table 1.

[0177] Table 1. Porcine IgG subtypes and allotypes

[0178]

[0179] Porcine FcRn was recombinantly produced. Porcine beta-2-microglobulin (B2M) small subunit, which binds to FcRn to form a functional complex, was also recombinantly produced and used in surface plasmon resonance (SPR) binding affinity experiments.

[0180] The DNA of porcine FcRn / B2M (Genbank accession number: AAP49846.1 / NP_999143.1) and all porcine mAb genes were codon optimized for mammalian expression and the constructs were transiently expressed in HEK 293 cells using a standard liposome transfection protocol (Invitrogen Life Technologies, Carlsbad, CA, USA) or in CHO cells using the ExpiCHO transient system (ThermoFisher Scientific) kit protocol. ExpiCHO expression follows the protocol outlined by ThermoFisher for mAb or FcRn / B2M transfection. For mAb, a plasmid containing the gene sequence encoding the IgG kappa light chain was co-transfected with a plasmid encoding the IgG heavy chain. For HEK293 expression, equal amounts of heavy chain plasmids and kappa chain plasmids were co-transfected by weight. For FcRn / B2M, two plasmids encoding each were transfected. Cells were grown for 7 days (HEK293) or 12 days (CHO), after which supernatants were collected for protein purification. mAbs bound to protein A or protein G sensors were screened via Octet QKe quantification (PallForteBio Corp, Menlo Park, CA, USA). Expression was quantified on Octet using a standard curve with protein A or protein G sensors, and mAbs were purified using protein G or protein A / G affinity chromatography. For all protein constructs, sodium acetate pH 5.5 was used as binding and washing buffer, and eluted at pH 3.4. Purified protein was neutralized and dialyzed into 20mM sodium acetate (pH 5.5), 140mM NaCl for further analysis. FcRn plasmids contain c-terminal His tags, so FcRn / B2M complexes are purified by IMAC affinity purification. The concentration of mAbs and FcRn / B2M proteins was measured at 280nm via NanoDrop. Protein quality was assessed via analytical SEC and standard coomassie protein gels.

[0181] The purified FcRn / B2M was biotinylated as follows. The purified FcRn / B2M protein was dialyzed into 10mM Tris-HCl (pH 8.0) and concentrated using AmiconUltra, 10KMWCO (EMD Millipore, Billerica, MA). The biotin receptor peptide (BAP) AGLNDIFEAQKIEWHE expressed at the receptor c-terminal end allowed the use of biotin ligase BirA to transfer biotin to this section of amino acids. Biotinylation was performed as described in the manufacturer's protocol (Avidity, LLC, Aurora, CO). The FcRn / B2M receptor was then dialyzed into PBS to remove residual biotin.

[0182] Example 2

[0183] Construction of porcine IgG Fc mutants

[0184] A plasmid containing the sequence encoding the porcine constant region for IgG6a was used, and the VH / VL sequences for each mAb studied herein were inserted upstream and in frame with the nucleotides encoding the constant domains. Mutations were incorporated into the CH2 or CH3 domain positions of each plasmid by direct DNA synthesis of the constant region as gene fragments, and then subcloned into the corresponding variable region of interest.

[0185] Expression and purification

[0186] Monoclonal antibody (mAb) mutants are expressed in mammalian suspension cell system EXPICHO-S (Chinese hamster ovary) cells obtained from Thermo Fisher (Thermo Fisher).Suspension EXPICHO-S cells are maintained between 0.14 and 8.0x106 cells / ml in EXPICHO expression culture medium (Gibco).At -1 day and transfection day, cells are diluted according to the ExpiCHO scheme user manual.As described in the scheme, the reagent derived from ExpiFectamine CHO transfection kit (Gibco) is used to transfect diluted cells according to the maximum titer condition.After cultivating 12 to 14 days, culture is collected and culture is clarified.Antibodies are purified from the supernatant of clarification by protein A chromatography on MabSelect Sure LX (GE Healthcare) pre-balanced with PBS.After sample loading, resin is washed with PBS, and then washed with 20mM sodium acetate of pH 5.5.Sample is eluted from post with 20mM acetic acid of pH 3.5. After elution, pools were prepared and neutralized to 4% with the addition of 1 M sodium acetate. Depending on available volume and intended use, samples were sometimes exchanged into final buffer (e.g., PBS, other). Concentrations were measured by absorbance at 280 nm.

[0187] SDS-PAGE

[0188] Non-reducing (nr) and reducing sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) were performed using 4-12% Bis-Tris NuPAGE gels in MES-SDS running buffer and SeeBlue Plus 2 standards (both from Invitrogen). For non-reducing samples, 1 mM alkylating agent N-ethylmaleimide (NEM) was added, and for reducing samples, reducing agent dithiothreitol (DTT) was added. The gel was stained with Coomassie blue to detect protein bands.

[0189] Example 3

[0190] FcRn binding assay

[0191] Biacore method for pFcRn:

[0192] The binding affinity of porcine Fc-based antibodies or fusion proteins to porcine FcRn was determined by surface plasmon resonance (SPR). All reported KDs were measured using SA sensors in Biacore T200 (Cytiva, Marlborough, MA, USA) or Biacore 8K (Cytiva, Marlborough, MA, USA). Porcine FcRn was captured on the surface of the sensor to obtain the desired surface density. The running buffer used was 20mM MES, 150mM NaCl, 0.005% Tween 20, 0.5mg / mL BSA, pH 6 and / or PBS, 0.0005% Tween 20, pH 7.4. Different concentrations of porcine mAbs were titrated in appropriate running buffer and flowed over the receptor surface. Regeneration was performed using 50mM Tris-HCl (pH 8). Kinetic binding affinity was analyzed using Biacore T200 evaluation software (Cytiva, Marlborough, MA, USA) or Biacore 8K Insight evaluation software using a dual reference approach: the reference flow cell was subtracted from the flow cell containing immobilized porcine FcRn, and a blank run containing only buffer was subtracted from all runs. The resulting curves were fitted with a 1:1 binding model. Runs were performed at 25°C.

[0193] Mutations made at the corresponding positions had a significant effect on the affinity of IgG for FcRn at pH 6.

[0194] The binding of wild-type (WT) and mutant IgG to porcine FcRn was measured by surface plasmon resonance (Biacore). The results are shown in Table 2 below.

[0195] Table 2. Effect of mutants on pFcRn binding affinity.

[0196]

[0197]

[0198]

[0199]

[0200] Mutants are numbered according to the EU index as in Kabat. LS = low signal.

[0201] The results clearly show that mutations made at various positions have a significant impact on the affinity of IgG to porcine FcRn.

[0202] Example 4

[0203] Effector functions and their modulation

[0204] Antibodies can exhibit their therapeutic function by blocking antigens through "neutralization" or by mediating effector functions. Antibody effector functions are an important component of the humoral immune response and are induced via the constant (Fc) region of antibodies, which can interact with complement proteins and specialized Fc receptors. The best-known Fc-mediated antibody effector functions are antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0205] All three effector functions described above require two major components to neutralize the target in a specific and potent manner - the right Fc region and its corresponding Fc receptor. The isotype and subclass / allotype of the Fc region determine the binding capacity of IgG to mediate effector functions.

[0206] Little is known about IgG subclasses in pigs, as subclasses were not defined until recently. The lack of progress is mainly due to the lack of IgG subclass reagents for immunoassays and the lack of subclass knockout animals.

[0207] To date, six porcine IgG subclasses are known - IgG1, G2, G3, G4, G5 and G6. These subclasses are further divided into several allotypes based on the relative occurrence, differences in intronic sequences and sequence similarities of the CH2-CH3 domain complexes of these subclasses - IgG1a, IgG1b, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a and IgG6b.

[0208] The sequences of porcine IgG subtypes and allotypes are well known in the art. Table 1 lists porcine IgG subtypes, allotypes and their associated sequence identifiers from the public NCBI database.

[0209] Generation of Fcγ receptors

[0210] Recombinant porcine FcgR1, FcgR2b, FcgR3a and Fcg3b DNA were codon optimized for mammalian expression and synthesized based on sequences from the NCBI database, as shown in Table 3.

[0211] Table 3: NCBI accession numbers of Fcγ receptors

[0212]

[0213]

[0214] The DNA was cloned into the pcDNA3.1(+) vector and engineered with a c-terminal 6x His+BAP tag (AGLNDIFEAQKIEWHE). All FcgRs were transfected into HEK 293 or Expi-CHO cells and the FcgRs were purified by IMAC affinity purification via the c-terminal His tag.

[0215] The purified FcR is biotinylated as follows. The purified Fc receptor protein is dialyzed into 10mM Tris-HCl (pH8.0) and concentrated using AmiconUltra, 10KMWCO (EMD Millipore, Billerica, MA). The biotin receptor peptide (BAP) AGLNDIFEAQKIEWHE expressed at the receptor c-terminal allows the use of biotin ligase BirA to transfer biotin to this section of amino acids. According to the biotinylation reaction described in the manufacturer's scheme (Avidity, LLC, Aurora, CO). The receptor is then dialyzed into PBS to remove residual biotin.

[0216] The Biacore SPR binding assay was designed to test the affinity of porcine IgG subclasses and mutants to pFcgR1, pFcgR2b, pFcgR3a, pFcg3b.

[0217] Generation of Fc fusion proteins and mAbs

[0218] Recombinant CTLA4-Fc fusions were constructed by inserting the canine CTLA4 gene (NCBI NM_001003106.1) into pcDNA3.1(+) mammalian expression vectors containing pIgG1a, pIgG2a, pIgG3, pIgG4a, pIgG5a, or pIgG6a. The Fc begins just upstream of the heavy chain hinge region. No additional linker is required.

[0219] Recombinant mAbs with pIgG1a, pIgG2a, pIgG3, pIgG4a, pIgG5a or pIgG6a Fc regions were constructed by inserting VH sequences upstream and in frame with nucleotides encoding constant domains in the pcDNA3.1(+) mammalian expression vector. Similarly, light chains were constructed by inserting VL sequences upstream and with the porcine kappa allele 1 constant region (NCBIAAA03520.1).

[0220] In CTLA4 Fc fusion and complete mAb formats, mutations were introduced into the three wild-type subclasses to knock out binding to FcgR, knock out CDC and / or ADCP. Mutations were incorporated into various positions on each wild-type plasmid using Agilent's QuikChange II mutagenesis and the associated Agilent primer design tool for single-shot site-directed mutagenesis (www.agilent.com / store / primerDesignProgram.jsp).

[0221] DNA for all CTLA4 fusions and mAb genes is codon optimized for mammalian expression, and constructs are transiently expressed in HEK 293 cells using standard liposome transfection protocols (Invitrogen Life Technologies, Carlsbad, CA, USA), or in CHO cells using ExpiCHO transient system (ThermoFisherScientific) kit protocols. ExpiCHO expression follows the protocol for mAb or CTLA4Fc fusion transfection outlined by ThermoFisher. For mAb, a plasmid containing a gene sequence encoding an IgG κ light chain is cotransfected with a plasmid encoding an IgG heavy chain. For HEK293 expression, cotransfection is performed with equal amounts of heavy chain plasmids and κ chain plasmids by weight. For Fc fusions, a single plasmid is transfected. Cells are allowed to grow for 7 days (HEK293) or 12 days (CHO), and supernatants are then collected for protein purification. CTLA4 Fc fusion and mAb were screened quantitatively via OctetQKe (Pall ForteBio Corp, Menlo Park, CA, USA) for binding to protein A or protein G sensors. Expression was quantified on Octet using a standard curve with protein A or protein G sensors, and mAb / fusions were purified using protein G or protein A / G affinity chromatography. For all protein constructs, sodium acetate pH 5.5 was used as binding and washing buffer, and eluted at pH 3.4. Purified protein was neutralized and dialyzed into 20mM sodium acetate (pH 5.5), 140mM NaCl for further analysis. The concentration of mAb and fusion protein was measured at 280nm via NanoDrop. Protein quality was assessed via analysis of SEC and standard coomassie protein gel.

[0222] SPR method for biotinylated pFcgR1, pFcgR2, and pFcgR3

[0223] The Biacore SPR binding assay was designed to test the affinity of bovine IgG subclasses to pFcgR1, pFcgR2, and pFcgR3. All reported KDs were measured by Biacore (cytiva, Marlborough, MA, USA) using a series of SSA sensors. Biotinylated bovine FcgR1, R2, and R3 were captured on the sensor surface using a modified SA capture method to achieve the desired surface density. 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% v / v surfactant P20, pH 7.4 buffer was used as the running and titration buffer. Various concentrations of porcine CTLA4-Fc fusions or mAbs were titrated and flowed on the receptor surface, and the affinity was determined using Biacore T200 evaluation software (cytiva, Marlborough, MA, USA) and a 1:1 binding model. A double referencing approach has been applied, where the reference flow cell was subtracted from the flow cell containing the immobilized receptor and a blank injection containing buffer only was subtracted from all injections. The flow cell was regenerated using 10 mM glycine pH 1.5. Injections were performed at 15 °C.

[0224] CDC assay

[0225] A CDC cell-based assay was developed and employed to characterize the effectiveness of nine CTLA4 porcine IgG subclass Fc fusion proteins in mediating CDC and to investigate Fc region mutations within the subclasses. This will help define key residues in the Fc region that determine CDC activity for porcine IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 that binds to CTLA4 on the Fc fusion protein. These target cells have been used in past canine ADCC assays and were used in CDC assays due to their reliability.

[0226] Incubation of the fusion protein-bound target cells with complement-preserved serum may result in binding of Fc on the fusion protein to complement component C1q, which initiates the complement cascade, culminating in the formation of the membrane attack complex. The pore-formed complex mediates cell lysis of the target cell as measured by loss of cell viability. If there is no binding of Fc to C1q, there is no resulting cell lysis / death.

[0227] Briefly, CHO cells expressing CD80 (target cells) were plated at 40,000 cells / well in CD CHO medium in a round-bottom 96-well plate. Titrated fusion protein in CD CHO medium was added to the target cells and allowed to bind for 60 minutes at 37°C. Porcine complement-retained serum (20% in CD CHO medium) was added to the plate for 45 minutes at 37°C. Cell viability was then measured using CellTiter-Glo, and data were expressed as "% cell viability of control" calculated using serum control without fusion protein + complement retention.

[0228] like Figure 2 As shown in Table 4, all porcine wild-type Fc subclasses, except IgG3 and IgG5a, showed strong and efficient CDC activity. The EC50 values ​​ranged from 0.010 to 0.044 μg / mL.

[0229] Table 4. CDC effect elicited by porcine IgG Fc wild-type constructs.

[0230]

[0231] Our results showed that porcine IgG 1a, 1b, 2a, 4a, 4b, 6a and 6b Fc CTLA4 fusion proteins all showed CDC activity. IgG 5a showed a decrease in CDC activity, while IgG3 had almost no CDC activity.

[0232] ADCC assay

[0233] An ADCC cell-based assay was developed and employed to characterize the effectiveness of nine CTLA4 porcine IgG subclass Fc fusion proteins in mediating ADCC and to study Fc region mutations within the subclasses. This will help define key residues in the Fc region that determine ADCC activity of porcine IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 that binds to CTLA4 on the Fc fusion protein. These target cells have been used in past canine ADCC assays and are used in CDC assays due to their reliability.

[0234] Incubation of fusion protein-bound target cells with cultured activated porcine PBMCs may result in Fc binding to FcγRIII-mediated granzyme binding on the fusion protein and perforin release to form NK cells in the PBMC population. The action of these proteins will result in cytotoxicity of fusion protein-bound target cells measured by flow cytometry quantification of dead target cells. In the absence of Fc binding to FcγRIII, there is no resulting target cell death.

[0235] Briefly, CHO cells expressing CD80 (target cells) were plated at 20,000 cells / well in CD CHO medium in a round-bottom 96-well plate. Titrated fusion proteins in CD CHO medium were added to target cells and allowed to bind for 60 minutes at 37°C. Single donor porcine PBMCs (effector cells) were added to the plate for 18 to 20 hours at 37°C using an effector target cell ratio (E:F ratio) of 40-50:1, which was cultured overnight in RPMI 1640 medium + IL-2 and IL-15. Cells were then stained, fixed and analyzed by flow cytometry to quantify the live / dead stains of target cells. Data are expressed as individual fusion proteins (minus effector PBMCs) normalized to "% dead target cells".

[0236] Figure 3 A and 3B show the results of cell-based antibody-dependent cell-mediated cytotoxicity activity of porcine wild-type Fc subclass CTLA4 fusion protein. Figure 3 All porcine wild-type Fc subclasses, except IgG3, showed some ADCC activity as shown in A and 3B and Table 5. The extent and potency of ADCC varied with EC50 values ​​ranging from 0.031 to 0.477 μg / mL.

[0237] Our results show that porcine IgG 1a, 1b, 2a, 4a, 4b, 6a and 6b Fc CTLA4 fusion proteins all showed ADCC activity with varying potency. IgG3 showed no ADCC activity.

[0238] Table 5. ADCC effect caused by porcine IgG Fc wild-type CTLA4 fusion protein

[0239]

[0240] ADCP assay

[0241] The antibody-dependent cellular phagocytosis (ADCP) assay utilizes CHO target cells engineered to express canine CD80 bound to CTLA4 on an Fc fusion protein. The Fc region of the fusion protein can then bridge this complex to Fcγ receptors on alveolar macrophage effector cells, which have the ability to phagocytose target cells. ADCP is measured by the signal intensity and frequency of a pH-sensitive fluorescent dye within a population of effector macrophages in co-culture, where fluorescent cells indicate that the effector cells have successfully internalized the target cells into acidic lysosomes.

[0242] Briefly, canine CD80-expressing CHO cells (CD80 target cells) or wild-type CHO cells that do not express CD80 (parental target cells) were stained with pHrodo red dye for 30 minutes at 37°C. The stained cells were then incubated with CTLA4-Fc fusion protein for 20 minutes to mediate CTLA4:CD80 binding. 60,000 target cells were then added to 30,000 pre-plated porcine alveolar macrophages that had previously been stained with a cell marker (CellTrace Violet, CTV) to aid in subsequent identification. Co-culture was maintained at 37C for 5 to 6 hours and then harvested and analyzed by flow cytometry to identify effector cells (CTV+) that successfully performed ADCP (pHrodo+).

[0243] Figure 4 The results of cell-based antibody-dependent cell-mediated phagocytosis of pig wild-type Fc subclass CTLA4 fusion protein are shown. In the CTLA4: Fc construct comprising wild-type pig Fc domains, IgG1a, IgG1b, IgG2a, IgG4a, IgG4b, IgG5a, IgG6a and IgG6b all show ADCP activity. Only pig IgG3a is not shown ADCP activity. Among those with activity, IgG4b and IgG6b show the lowest (most effective) EC50 at 2.6ng / mL and 5.9ng / mL respectively. When each construct is incubated with parental target cells that do not express canine CD80, and therefore not combined with CTLA4: Fc construct, no increase in phagocytosis is observed (the range of phagocytosis observed in WT ChoK1 target cells is shown as horizontal gray bars on the chart, and is shown as spots displayed on average).

[0244] pIgG6 mutation knocks out effector function

[0245] CDC assay

[0246] like Figure 5 As shown, the wild-type IgG6 subclass showed strong and efficient CDC activity. The Fc mutations studied all showed different effects on CDC activity, ranging from no effect to complete knockout of CDC effector function.

[0247] Both A and B allotypes of porcine IgG6 Fc CTLA4 fusion proteins show CDC activity. Mutations in Fc show a range of effects on CDC activity, ranging from no effect to moderate effect to complete knockout of effector function. Mutations SSP, WIN-LSP, WIN-PG, PG, EP-PSS, KAPA, KA, PA and PG appear to be most effective in knocking out the CDC effector function of the pIgG6a allotype. In addition, alanine substitutions T289A, R290A and K292A knock out the CDC effector function of IgG6a. Mutations DANG-SAP, WIN-PG and KAPA appear to be most effective in knocking out the CDC effector function of the pIgG6b allotype. Mutation SAP shows partial knockout of the CDC effector function of both 6a and 6b allotypes.

[0248] ADCC assay

[0249] like Figure 6 As shown, wild-type IgG6a or IgG6b subclasses showed strong and effective ADCC activity. IgG6a_WIN, IgG6a_WIN-LSP, IgG6a_WIN-PG, IgG6a_PG, IgG6a_DANG, IgG6a_EP, IgG6a_EP-PSS, IgG6a_SAP and IgG6a_SSP significantly knocked out ADCC. SAP Fc mutations showed significant knockout of the ADCC effector function of IgG6b.

[0250] ADCP assay

[0251] In the case of CTLA4: Fc constructs containing wild-type porcine Fc domains, IgG1a, IgG1b, IgG2a, IgG4a, IgG4b, IgG5a, IgG6a, and IgG6b all showed ADCP activity. Only porcine IgG3a did not show ADCP activity. Of those with activity, IgG4b and IgG6b showed the lowest (most effective) EC50 at 2.6 ng / mL and 5.9 ng / mL, respectively. When each construct was incubated with parental target cells that did not express canine CD80, and therefore did not bind to the CTLA4: Fc construct, no increase in phagocytosis was observed (the range of phagocytosis observed in WT ChoK1 target cells is shown as horizontal gray bars on the graph, and is shown as spots displayed as averages)

[0252] Of the IgG6a mutants tested, PG, WIN2, WIN-PG, DANG, DANG-GSP, EP, EP-PSS, KA, SAP, and PA mutants did not show significant amounts of ADCP. WIN-LSP, SSP, KAPA, and T289A mutations knocked out ADCP.

[0253] Table 6A below lists the various constructs, their mutations and their corresponding codon usage. Table 6B below summarizes the effector function results of pIgG6 WT and mutations.

[0254] Table 6A. Constructs, mutations and their codons

[0255]

[0256]

[0257]

[0258] Table 6B. Summary of effector functions of pIgG6 WT and mutants

[0259]

[0260]

[0261] PA = partial activity; KO = knockout; NT = not tested, NC = no change, EE = enhanced effect.

[0262] pIgG4 mutation knocks out effector function

[0263] CDC assay

[0264] Figure 8 and 16 Shown are the results of cell-based CDC activity of porcine wild-type Fc IgG4a and 4b subclass CTLA4 fusion proteins and Fc mutants of these subclasses.

[0265] Porcine IgG4a and 4b Fc CTLA4 fusion proteins showed potent CDC activity. Mutations in the Fc showed modest knockout of CDC activity, but no mutations appeared to substantially knockout CDC activity for either subclass.

[0266] ADCC assay

[0267] Fig. 9 Shown are the results of cell-based ADCC activity of porcine wild-type Fc IgG4a and IgG4b subclass CTLA4 fusion proteins and SAP mutants of these Fc subclasses.

[0268] Wild-type pIgG4a and pIgG4b constructs showed ADCC activity with SAP mutation knockout in each of these two allotypes.

[0269] ADCP assay

[0270] Fig.10 , 17A and 17B show the cell-based ADCP activity of porcine wild-type Fc IgG4a subclass CTLA4 fusion protein and SAP and WinPG mutations of this Fc subclass.

[0271] The wild-type pIgG4a construct showed robust activity that was completely knocked out using mutations of SAP, KAPA, GSP, EP-PAS, DANG, SSP, WIN2, and WIN-PG. Mutations EP, PA, PG, R290A, T289A, and DANG-SAP showed partial knockout of ADCP.

[0272] Table 7 below summarizes the effector function results of pIgG4 WT and mutants.

[0273] Table 7. Summary of effector functions of pIgG4 WT and mutants

[0274]

[0275] PA = partial activity; KO = knockout; NT = not tested, NC = no change, EE = enhanced effect.

[0276] pIgG2 mutations knock out effector function

[0277] CDC assay

[0278] Fig.11 The results of cell-based CDC activity of porcine wild-type Fc IgG2 subclass CTLA4 fusion protein and Fc mutants of this subclass are shown. Fig.11 As shown, the wild-type IgG2 Fc subclass showed strong and efficient CDC activity. The Fc mutations studied all showed varying degrees of reduction in CDC activity.

[0279] Porcine IgG2 Fc CTLA4 fusion protein showed CDC activity. Mutations in Fc showed knockout of CDC activity using GSP mutations, which appeared to be the most effective mutation to knock out CDC activity.

[0280] ADCC assay

[0281] Fig.12 The results of the cell-based antibody-dependent cellular cytotoxicity activity of porcine wild-type IgG2 subclass CTLA4 fusion protein and Fc mutants of this subclass are shown.

[0282] The wild-type pIgG2 construct showed ADCC activity which was knocked out using the SAP mutation.

[0283] ADCP assay

[0284] The wild-type pIgG2 construct showed ADCP activity that was knocked out using the SAP, KAPA, GSP, EP-PAS, DANG, PG, SSP, and WIN-PG mutations. Mutations EP, KA, PA, R290A, T289A, and WIN2 showed partial knockout of ADCP. See Fig.19 A and 19B.

[0285] The results for effector function of pIgG2 WT and mutations are summarized below in Table 8. The results support both pIgG2a and 2b allotypes, as 2a and 2b are identical in both constant region domains CH2 and CH3.

[0286] Table 8. Summary of effector functions of pIgG2 WT and mutants

[0287]

[0288] PA = partial activity; KO = knockout; NT = not tested, NC = no change, EE = enhanced effect.

[0289] pIgG1 mutations knock out effector function

[0290] CDC assay

[0291] Fig.13 Results are shown for the cell-based complement-dependent cytotoxic activity of porcine wild-type Fc IgG1a and 1b subclass CTLA4 fusion proteins and Fc mutants of these subclasses. Fig.13 As shown, wild-type IgG1a and IgG1b Fc subclasses showed strong and efficient CDC activity. The investigated Fc mutations all showed varying degrees of reduction in CDC activity.

[0292] Both porcine IgG1a and 1b Fc CTLA4 fusion proteins showed CDC activity. Mutations in Fc showed knockout of CDC activity using the KAPA mutation, which appeared to be the most effective mutation to knock out CDC activity.

[0293] ADCC assay

[0294] Fig.14 Cell-based antibody-dependent cellular cytotoxicity activities of porcine wild-type IgG1a and 1b subclass CTLA4 fusion proteins and Fc mutants of these subclasses are shown.

[0295] Both porcine IgG1a and 1b Fc CTLA4 fusion proteins showed ADCC activity. Fig.12 As shown, SAP mutation significantly knocked out ADCC function.

[0296] ADCP assay

[0297] Porcine IgG1a showed ADCP activity. Fig.15 As shown, SAP, DANG, PG, SSP, WIN2 and WIN-PG mutations significantly knocked out ADCP function. Mutations EP, KA, PA, R290A and T289A showed partial knockout of ADCP.

[0298] Table 9 below summarizes the effector function results for pIgG1 WT and mutations.

[0299] Table 9. Summary of effector functions of pIgG1 WT and mutants

[0300]

[0301]

[0302] PA = partial activity; KO = knockout; NT = not tested, NC = no change, EE = enhanced effect.

[0303] Example 5

[0304] FcRn binding validation

[0305] method

[0306] First, the Fc regions of four porcine subclasses and their allotypes IgG1 (IgG1a, IgG1b), IgG2, IgG4 (IgG4a and IgG4b), IgG6 (IgG6a and IgG6b) were designed using their respective CH2 and CH3 regions. The protein modeling feature of Alphafold 2.2 developed by Deepmind was implemented to model the 3D structure of porcine FcRn and each of the wild-type (WT) and mutant constructs of each porcine allotype.

[0307] The Molecular Operating Environment (MOE) (MOE2019.0102), developed by the Chemical Computing Group, provides a flexible and automated graphical user interface for protein modeling. To analyze structural differences, a sequence-to-profile alignment algorithm uses a scoring algorithm to rank sequence templates, and a score above 85% ensures the selection of protein templates with physically realistic structures. The model is then optimized using a systematic pipeline, and the structural stability of the model is verified using Ramachandran Plots, which examine the stereochemical quality of the protein structure.

[0308] result

[0309] The methods described above were performed on porcine wild-type (WT) constructs and the following mutants of IgG1a, IgG1b, IgG2a, IgG2b, IgG4a, IgG4b, IgG6a, and IgG6b: E233P, G234A, P235L, P235A, G236A, G236L, P238A, D265A, T289A, R290A, K292A, N297G, K322A, P329G, P329L, P329S, A330S, P331S, P331A. Fig.23 and Fig.24 The positions of the mutation libraries are represented in ball-and-stick format.

[0310] RMSD plots were generated to calculate the root mean square deviations (RMSDs) of the WT structure, porcine IgG1a, porcine IgG1b, porcine IgG2, porcine IgG4a, porcine IgG4b, porcine IgG6a, and porcine IgG6b relative to each other. Fig.25 ). The RMSD value of 100 Å or less was considered as the criterion for considering two structures similar. The results indicated that the protein fold of the porcine IgG4a construct was identical to that of the porcine IgG4b allotype, with an average RMSD value of 100 Å for the structure. (RMSD of each position in Table 10). The porcine IgG6a construct is identical to the porcine IgG6b allotype, with an average RMSD of (RMSD of each position in Table 11). Allotypes IgG1a, IgG1b, and IgG2 also show RMSD of 0.2 and The RMSD at the positions where the mutation scans were performed are noted in Tables 10 to 13.

[0311] Table 10. Comparison of root mean square deviation (RMSD) of residues in protein models of WT pig IgG4a and IgG4b.

[0312]

[0313] Table 11. Comparison of root mean square deviation (RMSD) of residues in protein models of WT pig IgG6a and IgG6b.

[0314]

[0315] Table 12. Comparison of root mean square deviation (RMSD) of residues in protein models of WT pig IgG1a and IgG1b.

[0316]

[0317]

[0318] Table 13. Comparison of root mean square deviation (RMSD) of residues in protein models of WT pig IgG1a and IgG2.

[0319]

[0320] in conclusion

[0321] All mutations of the two allotypes of porcine backbone, IgG6a and IgG6b, were molecularly modeled and validated using MOE2019.0102 and AlphaFold2.2. Similarly, the molecular models of all mutations of the two allotypes of each subclass of porcine backbone (IgG4a vs IgG4b, IgG1a vs IgG1b, and IgG2) were compared and validated using MOE2019.0102 and AlphaFold2.2. The Fc fold and residue configurations between subclasses showed less than The RMSD of the proteins is very high, indicating that the proteins have very high structural identity and will therefore function in a similar manner.

[0322] Having described the preferred embodiments of the present invention, it should be understood that the present invention is not limited to the exact embodiments and that various changes and modifications may be implemented therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0323] Sequence Listing

[0324]

[0325]

[0326]

[0327]

Claims

1. A method for modulating or regulating effector function in a porcine subject, the method comprising: administering to the subject a fusion molecule comprising a modified IgG, wherein the modified IgG comprises: a porcine IgG constant domain comprising at least one amino acid substitution relative to a wild-type porcine IgG constant domain, wherein the substitution is at amino acid residue 233, 234, 235, 236, 238, 252, 254, 256, 265, 286, 293, 297, 307, 311, 312, 322, 329, 330, 331, 378, 426, 428, 434 or 436 as numbered according to the Eu index as in Kabat.

2. The method of claim 1, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or a combination thereof.

3. The method of claim 1, wherein the constant domain comprises the substitutions E233P, G234A, V234A, A235L, P235L, P235A, G236A, G236L, P238A, M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, S254T, T256E, T256F, T256N, D265A, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, T289A, R290A, K292A, E293 deleted, N297G, P307 Q. E311A, E311C, E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E311N, E311P, E311Q, E311R, E311S, E311T, E311V, E311W, E311Y, D312A, D31 2C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P , D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P3 29L, A330S, P331S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426 H, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A 426V, A426W, A426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M42 8I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N43 4L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A,One or more of Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

4. The method according to any one of the above claims, wherein the modified IgG is porcine or porcine IgG.

5. The method according to any one of the above claims, wherein the IgG is IgG1a, IgG1b, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a or IgG6b.

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

7. The method of any of the above claims, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

8. The method according to any one of the above claims, wherein the wild-type porcine IgG constant domain comprises one of the amino acid sequences listed in SEQ ID NO.: 1-11.

9. A modified IgG comprising: a porcine IgG constant domain comprising at least one amino acid substitution relative to a wild-type porcine IgG constant domain, wherein the substitution is at amino acid residue 233, 234, 235, 236, 238, 252, 254, 256, 265, 286, 293, 297, 307, 311, 312, 322, 329, 330, 331, 378, 426, 428, 434 or 436 as numbered according to the Eu index as in Kabat.

10. The modified IgG of claim 9, wherein the constant domain comprises substitutions E233P, G234A, V234A, A235L, P235L, P235A, G236A, G236L, P238A, M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, S254T, T256E, T256F, T256N, D265A, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, E293 deleted, N297G, P307Q, E311A, E311 C. E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E311N, E311P, E311Q, E311R, E311S, E311T, E311V, E311W, E311Y, D312A, D312C, D312E, D31 2F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R , D312S, D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P329L, A330S, P3 31S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426H, A426I, A426 K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, A 426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M42 8L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N43 4P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D,One or more of Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

11. The modified IgG according to any one of claims 9 to 10 above, wherein the modified IgG is porcine or porcine derived IgG.

12. The modified IgG according to any one of claims 9 to 11 above, wherein the IgG is IgG1a, IgG1b, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a or IgG6b.

13. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG1a, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) P329S and A330S; (ii) D265A, P329G and A330S; (iii) V234A, A235L, G236A, P329L and A330S; (iv) V234A, A235L, G236A and P329G; (v) E233P, A330S and P331S; (vi) K322A and P331A; and (vii) P329S.

14. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG1b, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) V234A, A235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; and (v) K322A and P331A.

15. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG2, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) V234A, A235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

16. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG4a, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) G234A, P235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

17. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG4b, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) G234A, P235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

18. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG6a, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) D265A, N297G, P329G and A330S; (ii) G234A, P235L, G236A and P329G; (iii) E233P, A330S and P331S; (i (v) P235A; G236L; and P238A; (v) D265A and N297G; (vi) P329G; (vii) P331A; (viii) K322A; (ix) E233P; (x) P329S and A330S; (xi) G234A, P235L, G236A, P329L and A330S; (xii) K322A and P331A; and (xiii) P329S.

19. A modified IgG according to any one of claims 9 to 12 above, wherein the IgG is IgG6b, and wherein the constant domain comprises one or more of the substitutions selected from the group consisting of: (i) G234A, P235L, G236A and P329G; (ii) D265A, N297G and P329S; (iii) P329G and A330S; (iv) E233P and P331S; (v) K322A and P331A; and (vi) P329S.

20. The modified IgG according to any one of claims 9 to 19 above, wherein the modified IgG has a higher affinity for FcRn than an IgG having the wild-type porcine IgG constant domain.

21. The modified IgG according to any one of claims 9 to 20 above, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type porcine IgG constant domain.

22. The modified IgG according to any one of claims 9 to 21 above, wherein the IgG constant domain comprises a CH1 domain or a hinge region.

23. The modified IgG according to any one of claims 9 to 22 above, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains.

24. The modified IgG according to any one of claims 9 to 23 above, wherein the wild-type porcine IgG constant domain comprises one of the amino acid sequences listed in SEQ ID NO.: 1-11.

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

26. A kit comprising the modified IgG according to any one of claims 9 to 24 in a container and instructions for use.

27. A polypeptide comprising the modified IgG according to any one of claims 9 to 24.

28. An antibody comprising the modified IgG according to any one of claims 9 to 24.

29. A vector comprising a nucleic acid sequence encoding the amino acid sequence of the modified IgG according to any one of claims 9 to 24.

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

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

32. A method for producing an antibody, the method comprising: An antibody according to claim 28 is provided.

33. A fusion molecule comprising: a porcine IgG constant domain fused to an agent, wherein the porcine IgG constant domain comprises the modified IgG according to any one of claims 9 to 24.

34. A method for enhancing the binding affinity of porcine IgG to FcRn, the method comprising: A modified IgG according to any one of claims 9 to 24 is provided.

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