WO2024077207A2 - Porcine antibody mutants - Google Patents
Porcine antibody mutants Download PDFInfo
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- WO2024077207A2 WO2024077207A2 PCT/US2023/076184 US2023076184W WO2024077207A2 WO 2024077207 A2 WO2024077207 A2 WO 2024077207A2 US 2023076184 W US2023076184 W US 2023076184W WO 2024077207 A2 WO2024077207 A2 WO 2024077207A2
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- igg
- porcine
- antibody
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Abstract
The invention relates generally to porcine antibody mutants and uses thereof. Specifically, the invention relates to mutations in the constant region of porcine antibody for improving various characteristics.
Description
ZP000432A PORCINE ANTIBODY MUTANTS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and the benefit of United States Provisional Patent Application 63/378,740, filed on October 7, 2022, which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION [0002] The invention relates generally to porcine antibody mutants and uses thereof. Specifically, the invention relates to one or more mutations in the Fc constant region of porcine antibody for improving various characteristics. BACKGROUND OF THE INVENTION [0003] Porcine IgG monoclonal antibodies (mAbs) can be effective therapeutics in veterinary medicine. Several years ago, six porcine IgG subclasses were identified. However, only a limited work has been done to improve the characteristics of porcine IgGs. [0004] Through a recycling mechanism, the neonatal Fc receptor (FcRn) prolongs the half-life of an IgG in a pH-dependent interaction with its fragment crystallizable (Fc) region. Specifically, the Fc region spanning the interface of CH2 and CH3 domains interacts with the FcRn on the surface of cells to regulate IgG homeostasis. This interaction is favored by an acidic interaction after IgG pinocytosis and thus IgG is protected from degradation. The endocytosed IgG is then recycled back to the cell surface and released into the blood stream at a slightly alkaline pH thereby maintaining sufficient serum IgG for proper function. Accordingly, the pharmacokinetic profile of IgGs depend on the structural and functional properties of their Fc regions. [0005] Engineering Fc regions to tune their interactions with FcRn has emerged as a promising approach for enhancing the activity of therapeutic antibodies. [0006] Accordingly, there exists a need for novel porcine IgG Fc region mutations to improve various characteristics of porcine IgGs. SUMMARY OF THE INVENTION [0007] The invention relates to mutant porcine IgGs that exhibit desired characteristics, relative to wild-type porcine IgGs. Specifically, the inventors of the instant application have
ZP000432A found that substituting 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 (numbered according to the Eu index as in Kabat) with another amino acid surprisingly and unexpectedly exhibited a desired effect. In an exemplary embodiment, the unexpected desired effects include, but not limited to, an enhanced affinity to FcRn and a change in effector function. [0008] In one aspect, the 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 said 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. [0009] In one exemplary embodiment, the porcine IgG constant domain comprises one or more of mutations of 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, E293delete, N297G, P307Q, E311A, E311C, E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E311N, E311P, E311Q, E311R, E311S, E311T, E311V, E311W, E311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P329L, A330S, P331S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426H, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, A426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W. [00010] In another aspect, the invention provides a polypeptide comprising: a porcine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.
ZP000432A [00011] In yet another aspect, the invention provides an antibody or a molecule comprising: a porcine IgG constant domain comprising one or more amino acid substitutions of the invention described herein. [00012] In a further aspect, the invention provides a method for producing or manufacturing an antibody or a molecule, the method comprising: providing a vector or a host cell having a nucleic acid sequence that encodes an antibody, wherein said antibody comprises a porcine IgG constant domain comprising one or more amino acid substitutions of the invention described herein. [00013] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS [00014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [00015] FIGURE 1 shows the alignment of the amino acid sequences of human IgG1 and porcine IgG1a, IgG2, IgG3, IgG4a, IgG5a, and IgG6a. 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. [00016] FIGURE 2 shows cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc subclass CTLA4 fusion proteins. [00017] FIGURES 3A and 3B show cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) activity of porcine wild type Fc subclass CTLA4 fusion proteins. [00018] FIGURE 4 shows cell-based antibody-dependent cell-mediated phagocytosis (ADCP) of porcine wild type Fc subclass CTLA4 fusion proteins. [00019] FIGURES 5A, 5B, and 5C show cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc IgG6 subclass CTLA4 fusion proteins and Fc mutants of that subclass.
ZP000432A [00020] FIGURES 6A, 6B, and 6C show cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) activity of porcine wild type Fc IgG6 subclass CTLA4 fusion protein and mutations of that Fc subclass. [00021] FIGURES 7A and 7B show cell-based antibody-dependent cell-mediated phagocytosis (ADCP) activity of porcine wild type Fc IgG6 subclass CTLA4 fusion protein and mutations of that Fc subclass. [00022] FIGURES 8A and 8B show cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc IgG4a and 4b subclasses CTLA4 fusion proteins and Fc mutants of those subclasses. [00023] FIGURE 9 shows cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) activity of porcine wild type Fc IgG4a and IgG4b subclass CTLA4 fusion proteins and SAP mutations of those Fc subclasses. [00024] FIGURE 10 shows cell-based antibody-dependent cellular phagocytosis (ADCP) activity of porcine wild type Fc IgG4a subclass CTLA4 fusion protein, as well as SAP and WinPG mutations of that Fc subclass. [00025] FIGURE 11 shows cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc IgG2 subclass CTLA4 fusion proteins and Fc mutants of that subclass. [00026] FIGURE 12 shows cell-based antibody-dependent cytotoxicity (ADCC) activity of porcine wild type IgG2 subclass CTLA4 fusion protein and Fc mutants of that subclass. [00027] FIGURES 13A and 13B show cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc IgG1a and 1b subclasses CTLA4 fusion proteins and Fc mutants of those subclasses. [00028] FIGURE 14 shows cell-based antibody-dependent cytotoxicity activity (ADCC) of porcine wild type IgG1a and 1b subclass CTLA4 fusion proteins and Fc mutants of these subclasses. [00029] FIGURES 15A and 15B. Cell-based antibody-dependent cell-mediated phagocytosis (ADCP) activity of porcine wild type and corresponding mutations of IgG6 subclass. [00030] FIGURES 16A 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 those subclasses.
ZP000432A [00031] FIGURES 17A and 17B. Cell-based antibody-dependent cellular phagocytosis (ADCP) activity of porcine wild type Fc IgG4a subclass mutations of that Fc subclass. [00032] FIGURES 18A and 18B. Cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc IgG2 subclass CTLA4 fusion proteins and Fc mutants of that subclass. [00033] FIGURES 19A and 19B. Cell-based antibody-dependent phagocytosis of porcine wild type IgG2 subclass and Fc mutants of that subclass. [00034] FIGURES 20A and 20B. Cell-based complement-dependent cytotoxicity activity of porcine wild type Fc IgG1a subclasses CTLA4 fusion proteins and Fc mutants of those subclasses. [00035] FIGURE 21. Cell-based antibody-dependent phagocyosis of porcine wild type IgG1a proteins and Fc mutants of these subclasses. [00036] FIGURE 22. Cell-based antibody-dependent phagocyosis of porcine wild type IgG1a proteins and Fc mutants of these subclasses. [00037] FIGURE 23. A) Overlay of protein models of Porcine IgG1a and IgG1b Fc regions showing amino acid residue positions important for effector function in ball-and-stick form B) Protein model of Porcine IgG2 (2a and 2b Fc are identical) Fc region showing amino acid residue positions important for effector function in ball-and-stick form. [00038] FIGURE 24. A) Overlay of protein models of Porcine IgG4a and IgG4b Fc regions showing amino acid residue positions important for effector function in ball-and-stick form B) Protein model of Porcine IgG6a and IgG6b Fc regions showing amino acid residue positions important for effector function in ball-and-stick form. [00039] FIGURE 25. Root Mean Square Deviation (RMSD) comparisons of wild-type constructs of porcine IgG1a, IgG1b, IgG2, IgG4a, IgG4b, IgG6a and IgG6b. BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS [00040] SEQ ID NO.: 1 refers to the amino acid sequence of porcine IgG1a wildtype constant region. [00041] SEQ ID NO.: 2 refers to the amino acid sequence of porcine IgG1b wildtype constant region.
ZP000432A [00042] SEQ ID NO.: 3 refers to the amino acid sequence of porcine IgG2a wildtype constant region. [00043] SEQ ID NO.: 4 refers to the amino acid sequence of porcine IgG2b wildtype constant region. [00044] SEQ ID NO.: 5 refers to the amino acid sequence of porcine IgG3 wildtype constant region. [00045] SEQ ID NO.: 6 refers to the amino acid sequence of porcine IgG4a wildtype constant region. [00046] SEQ ID NO.: 7 refers to the amino acid sequence of porcine IgG4b wildtype constant region. [00047] SEQ ID NO.: 8 refers to the amino acid sequence of porcine IgG5a wildtype constant region. [00048] SEQ ID NO.: 9 refers to the amino acid sequence of porcine IgG5b wildtype constant region. [00049] SEQ ID NO.: 10 refers to the amino acid sequence of porcine IgG6a wildtype constant region. [00050] SEQ ID NO.: 11 refers to the amino acid sequence of porcine IgG6b wildtype constant region. [00051] SEQ ID NO.: 12 refers to the amino acid sequence of human IgG1 wildtype constant region. DETAILED DESCRIPTION OF THE INVENTION [00052] The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. [00053] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of
ZP000432A ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. [00054] As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings. Definitions [00055] In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a molecule” or "a 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 forth. The term "plurality", as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. [00056] In the specification and claims, the numbering of the amino acid residues in an immunoglobulin heavy chain is that of the Eu index as in Kabat, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The " Eu index as in Kabat " refers to the residue numbering of the IgG antibody and is reflected herein in FIG.1. [00057] The term "isolated" when used in relation to a nucleic acid is a nucleic acid that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is in a form or setting different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide encoded therein where, for example, the nucleic acid molecule is in a plasmid or a chromosomal location different from that of natural cells. The isolated nucleic acid may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand, but may contain both the sense and anti-sense strands (i.e., may be double-stranded). [00058] A nucleic acid molecule is "operably linked" or "operably attached" when it is placed into a functional relationship with another nucleic acid molecule. For example, a promoter or
ZP000432A enhancer is operably linked to a coding sequence of nucleic acid if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence of nucleic acid if it is positioned so as to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof which does not, as it exists in nature, comprise an Fc region) if it is positioned such that the expressed fusion protein comprises the heterologous protein or functional fragment thereof adjoined either upstream or downstream to the variant Fc region polypeptide; the heterologous protein may by immediately adjacent to the variant Fc region polypeptide or may be separated therefrom by a linker sequence of any length and composition. Likewise, a polypeptide (used synonymously herein with "protein") molecule is "operably linked" or "operably attached" when it is placed into a functional relationship with another polypeptide. [00059] As used herein the term "functional fragment" when in reference to a polypeptide or protein (e.g., a variant Fc region, or a monoclonal antibody) refers to fragments of that protein which retain at least one function of the full-length polypeptide. The fragments may range in size from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. A functional fragment of a variant Fc region polypeptide of the present invention retains at least one "amino acid substitution" as herein defined. A functional fragment of a variant Fc region polypeptide retains at least one function known in the art to be associated with the Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, down regulation of cell surface receptors or may, e.g., increase the in vivo or in vitro half-life of a polypeptide to which it is operably attached). [00060] The term "purified" or "purify" refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody may 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 protein; it may also be purified by the removal of immunoglobulin protein that does not bind to the same antigen. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind a particular antigen results in an increase in the percent of antigen-specific immunoglobulins in the sample. In another example, a polypeptide (e.g., an immunoglobulin) expressed in bacterial host cells is purified by the complete or substantial removal of host cell proteins; the percent of the polypeptide is thereby increased in the sample.
ZP000432A [00061] The term "native" as it refers to a polypeptide (e.g., Fc region) is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of the polypeptide as it commonly occurs in nature or a naturally occurring polymorphism thereof. A native polypeptide (e.g., native Fc region) may be produced by recombinant means or may be isolated from a naturally occurring source. [00062] The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. [00063] 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 or in situ, or in vivo [00064] As used herein, the term "Fc region" refers to a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of the Fc region of an immunoglobulin heavy chain might vary, the porcine IgG heavy chain Fc region is usually defined to stretch, for example, from residue 231 to the c-terminus in Figure 1. In some embodiments, variants comprise only portions of the Fc region and can include or not include the carboxy-terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants without such constant domains (or with only portions of such constant domains) are contemplated. [00065] The "CH2 domain" of a porcine IgG Fc region refers to, for example, the residues starting at residue 231 and extending to residue 340 in Figure 1. The CH2 domain is unique in that it is not closely paired with another domain. [00066] The "CH3 domain" of a porcine IgG Fc region generally is the stretch of residues C- terminal to a CH2 domain in an Fc region, for example, residue 341 to the c-terminus in Figure 1. [00067] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions
ZP000432A may require the Fc region to be operably linked to a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assay, ADCC assays, CDC assays, ADCP assays, target cell depletion from whole or fractionated blood samples, etc.). [00068] 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. Exemplary native sequence porcine Fc regions are from residue 231 to the c-terminus in Figure 1. [00069] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region (or fragment thereof) by virtue of at least one "amino acid substitution" as defined herein. In preferred embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or in the Fc region of a parent polypeptide, preferably 1, 2, 3, 4 or 5 amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. In an alternative embodiment, a variant Fc region may be generated according to the methods herein disclosed and this variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a non- antibody polypeptide, e.g., binding domain of a receptor or ligand. [00070] As used herein, the term "derivative" in the context of polypeptides refers to a polypeptide that comprises and amino acid sequence which has been altered by introduction of an amino acid residue substitution. The term "derivative" as used herein also refers to a polypeptide which has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative polypeptide may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide possesses a similar or identical function as the polypeptide from which it was derived. It is understood that a polypeptide comprising a variant Fc region of the present invention may be a derivative as defined herein, preferably the derivatization occurs within the Fc region. [00071] "Substantially of porcine origin" as used herein in reference to a polypeptide (e.g., an Fc region or a monoclonal antibody), indicates the polypeptide has an amino acid sequence at
ZP000432A least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94% or even more preferably at least 95%, 96%, 97%, 98% or 99% homologous to that of a native porcine amino polypeptide. [00072] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). The preferred FcR is a native sequence FcR. Moreover, a preferred FcR is one which binds an IgG antibody Fc region, an Fc gamma receptor or “FcgR”, and includes receptors of the Fc gamma RI (FcgR1), Fc gamma RII (FcgR2), Fc gamma RIII (FcgR3) subclasses, including allelic variants and alternatively spliced forms of these receptors as well as the novel porcine Fc gamma 2R. Another preferred FcR includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. [00073] The phrase "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) that express FcgRs (e.g., Natural Killer ("NK") cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cells. The primary cells for mediating ADCC in humans, NK cells, express FcgR3 only, whereas monocytes express FcgR1, FcgR2 and FcgR3. [00074] 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) that express FcgRs (e.g., FcgR1, FcgR2a and FcgR3) recognize bound IgG antibody Fc region on a target cell and subsequently trigger a signaling cascade leading to the engulfment of the IgG-opsonized particle (e.g., bacteria, dead tissue cells). [00075] As used herein, the phrase "effector cells" refers to leukocytes (preferably porcine) which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcgR3 and perform ADCC effector function. Examples of leukocytes which mediate ADCC include PBMC, NK cells, monocytes, macrophage, cytotoxic T cells and neutrophils. The effector cells may be isolated from a native source (e.g., from blood or PBMCs). In one example, the leukocytes express FcgR1, or other relevant Fc gamma receptor, and trigger ADCP function.
ZP000432A [00076] A variant polypeptide with "altered" Fc receptor binding affinity is one which has either enhanced (i.e., increased, greater or higher) or diminished (i.e., reduced, decreased or lesser) Fc receptor binding affinity compared to the variant's parent polypeptide or to a polypeptide comprising a native Fc. A variant polypeptide which displays increased binding or increased binding affinity to an Fc receptor binds Fc receptor with greater affinity than the parent polypeptide. A variant polypeptide which displays decreased binding or decreased binding affinity to an Fc receptor, binds Fc receptor with lower affinity than its parent polypeptide. Such variants which display decreased binding to an Fc receptor may possess little or no appreciable binding to an Fc receptor, e.g., 0-20% binding to Fc receptor the Fc receptor compared to a parent polypeptide. A variant polypeptide which binds an Fc receptor with "enhanced affinity" as compared to its parent polypeptide, is one which binds Fc receptor with higher binding affinity than the parent polypeptide, when the amounts of variant polypeptide and parent polypeptide in a binding assay are essentially the same, and all other conditions are identical. For example, a variant polypeptide with enhanced Fc receptor binding affinity may display from about 1.10 fold to about 100 fold (more typically from about 1.2 fold to about 50 fold) increase in Fc receptor binding affinity compared to the parent polypeptide, where Fc receptor binding affinity is determined, for example, in an ELISA assay or other method available to one of ordinary skill in the art. [00077] As used herein, an "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with another different "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and 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). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein. A "non-naturally occurring amino acid residue" refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues (s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym.202: 301-336 (1991).
ZP000432A [00078] The term "assay signal" refers to the output from any method of detecting protein- protein interactions, including but not limited to, absorbance measurements from colorimetric assays, fluorescent intensity, or disintegrations per minute. Assay formats could include ELISA, FACS, or other methods. A change in the "assay signal" may reflect a change in cell viability and/or a change in the kinetic off-rate, the kinetic on-rate, or both. A "higher assay signal" refers to the measured output number being larger than another number (e.g., a variant may have a higher (larger) measured number in an ELISA assay as compared to the parent polypeptide). A "lower" assay signal refers to the measured output number being smaller than another number (e.g., a variant may have a lower (smaller) measured number in an ELISA assay as compared to the parent polypeptide). [00079] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. The binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g., seconds-1) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g., molar/second). In general, it is not possible to unequivocally state whether changes in equilibrium dissociation constants (KD or KD) are due to differences in on-rates, off-rates or both unless each of these parameters are experimentally determined (e.g., by BIACORE or SAPIDYNE measurements). [00080] As used herein, the term "hinge region" refers to the stretch of amino acids that links the Fab antigen binding region to the Fc region of an antibody. Hinge regions of IgG subclasses may be aligned by placing the first and last cysteine residues forming inter-heavy chain disulfide (S—S) bonds in the same positions. As shown in Figure 1, the hinge region, for example, in porcine IgG constant region starts at residue 216 and extends to residue 230. [00081] "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, the first component of the CDC pathway. [00082] 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 so long as they exhibit the desired biological activity or functional activity. Single chain antibodies, and chimeric, porcine, or porcinized antibodies, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising
ZP000432A portions derived from different species, are also encompassed by the present invention and the term "antibody". The various portions of these antibodies can be joined together chemically by conventional techniques, synthetically, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or porcinized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. No.4,816,567; U.S. Pat. No. 4,816,397; WO 86/01533; U.S. Pat. No. 5,225,539; and U.S. Pat. Nos. 5,585,089 and 5,698,762. See also, Newman, R. et al. BioTechnology, 10: 1455-1460, 1993, regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242:423-426, 1988, regarding single chain antibodies. It is understood that all forms of the antibodies comprising an Fc region (or portion thereof) are encompassed herein within the term "antibody." Furthermore, the antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art. [00083] As used herein, the term "antibody fragments" 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 fragments preferably retain at least part of the hinge and optionally the CH1 region of an IgG heavy chain. In other preferred embodiments, the antibody fragments comprise at least a portion of the CH2 region or the entire CH2 region. [00084] As used herein, the term "functional fragment", when used in reference to a monoclonal antibody, is intended to refer to a portion of the monoclonal antibody that still retains a functional activity. A functional activity can be, for example, antigen binding activity or specificity, receptor binding activity or specificity, effector function activity and the like. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single chain Fv (scFv); and Fc fragments. Such terms are described in, for example, Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497- 515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The term functional fragment is intended to include, for example, fragments
ZP000432A produced by protease digestion or reduction of a monoclonal antibody and by recombinant DNA methods known to those skilled in the art. [00085] 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 contiguous amino acid residues of the amino acid sequence of another polypeptide. In a preferred embodiment, a fragment of a polypeptide retains at least one function of the full-length polypeptide. [00086] As used herein, the term "chimeric antibody" includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through disulfide bridges with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy chain-light chain dimers associated through at least one disulfide bridge. A chimeric heavy chain of an antibody for use in porcine comprises an antigen-binding region derived from the heavy chain of a non-porcine antibody, which is linked to at least a portion of a porcine heavy chain constant region, such as CH1 or CH2. A chimeric light chain of an antibody for use in porcine comprises an antigen binding region derived from the light chain of a non-porcine antibody, linked to at least a portion of a porcine light chain constant region (CL). Antibodies, fragments or derivatives having chimeric heavy chains and light chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps. With this approach, hosts expressing chimeric heavy chains are separately cultured from hosts expressing chimeric light chains, and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin or fragment or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Pat. Nos. 6,284,471; 5,807,715; 4,816,567; and 4,816,397). [00087] As used herein, "porcinized" forms of non-porcine (e.g., murine) antibodies (i.e., porcinized antibodies) are antibodies that contain minimal sequence, or no sequence, derived from non-porcine immunoglobulin. For the most part, porcinized antibodies are porcine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-porcine species (donor antibody) such as mouse, rat, rabbit, human or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the
ZP000432A porcine immunoglobulin are replaced by corresponding non-porcine residues. Furthermore, porcinized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further refine antibody performance. In general, the porcinized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-porcine immunoglobulin and all or substantially all of the 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. [00088] As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding domain of a heterologous "adhesin" protein (e.g., a receptor, ligand or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e., is "heterologous") with an immunoglobulin constant domain sequence. [00089] As used herein, the term "ligand binding domain" refers to any native receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability of a corresponding native receptor. In certain embodiments, the receptor is from a cell-surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergene family. Other receptors, which are not members of the immunoglobulin supergene family but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L-, and P-selectins). [00090] As used herein, the term "receptor binding domain" refers to any native ligand for a receptor, including, e.g., cell adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability of a corresponding native ligand. [00091] As used herein, an "isolated" polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In certain embodiments, the isolated polypeptide is purified (1) to
ZP000432A greater than 95% by weight of polypeptides as determined by the Lowry method, and preferably, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-page under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by a least one purification step. [00092] As used herein, the term "disorder" and "disease" are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder). [00093] As used herein, the term "receptor" refers to a polypeptide capable of binding at least one ligand. The preferred receptor is a cell-surface or soluble receptor having an extracellular ligand-binding domain and, optionally, other domains (e.g., transmembrane domain, intracellular domain and/or membrane anchor). A receptor to be evaluated in an assay described herein may be an intact receptor or a fragment or derivative thereof (e.g. a fusion protein comprising the binding domain of the receptor fused to one or more heterologous polypeptides). Moreover, the receptor to be evaluated for its binding properties may be present in a cell or isolated and optionally coated on an assay plate or some other solid phase or labeled directly and used as a probe. [00094] 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 parent antibody is one which in vitro or in vivo is substantially less active at mediating ADCC, ADCP and/or CDC, when the amounts of variant polypeptide and parent antibody used in the assay are essentially 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 an identical ADCC assay. Such variants may be identified, for example, using an ADCC, ADCP or CDC assay, but other assays or methods for determining ADCC, ADCP or CDC activity may also be employed (e.g., animal models). In preferred embodiments, the variant polypeptide is about 100, 75, 50, or 25 percent less active at mediating ADCC, ADCP and CDC than the parent polypeptide.
ZP000432A Porcine Wildtype IgG [00095] Porcine IgGs are well known in the art and fully described in, for example, Butler et al., 2009, Immunogenetics, vol.61(3): pages 209-30; and Paudyal et al., 2022, Front Immunol, vol 13, pages 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. [00096] Allotypes of porcine IgG subclasses, shown in Table 1 below, are also well known in the art. IgG1 described herein can be, for example, IgG1a or 1b; IgG2 described herein can be, for example, IgG2a or 2b; IgG4 described herein can be, for example, IgG4a or 4b; IgG5 described herein can be, for example, IgG5a or 5b; and IgG6 described herein can be, for example, IgG6a or 6b. In a particular example, porcine IgG is IgG6a. [00097] The amino acid and nucleic acid sequences of IgGs are also well known in the art. [00098] In one example, IgG of the invention comprises a constant domain, for example, CH1, CH2, or CH3 domains, or a combination thereof. In another example, the constant domain of the invention comprises Fc region, including, for example, CH2 or CH3 domains or a combination thereof. [00099] In a particular example, the wild-type constant domain comprises any one of the amino acid sequences set forth in SEQ ID NOs.: 1-11. In a particular embodiment, the wild-type constant domain of IgG1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, and 6b comprises the amino acid sequence set forth 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 homologue, a variant, an isomer, or a functional fragment of any one of SEQ ID NOs.: 1-11, but without any mutation described herein. Each possibility represents a separate embodiment of the present invention. [000100] IgG contant domains also include polypeptides with amino acid sequences substantially similar to the amino acid sequence of the heavy and/or light chain. Substantially the same amino acid sequence is defined herein as a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a compared amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988). [000101] The present invention also includes nucleic acid molecules that encode IgGs or portion thereof, described herein. In one embodiment, the nucleic acids may encode an
ZP000432A antibody heavy chain comprising, for example, CH1, CH2, CH3 regions, or a combination thereof. In another embodiment, the nucleic acids may encode an antibody heavy chain comprising, for example, any one of the VH regions or a portion thereof, or any one of the VH CDRs, including any variants thereof. The invention also includes nucleic acid molecules that encode an antibody light chain comprising, for example, any one of the CL regions or a portion thereof, any one of the VL regions or a portion thereof or any one of the VL CDRs, including any variants thereof. In certain embodiments, the nucleic acid encodes both a heavy and light chain, or portions thereof. [000102] The amino acid sequence of the wild-type constant domain set forth in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 is encoded by its corresponding nucleic acid sequence. Modified Porcine IgG [000103] The inventors of the instant application have found that substituting 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 with another amino acid surprisingly and unexpectedly exhibited a desired effect. The term, position, as used herein, refers to a position numbered according to the Eu index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In one embodiment, the desired effect is a higher affinity to FcRn, relative to an IgG having the wild-type porcine IgG constant domain. In another embodiment, the desired effect is eliminating or reducing a complement-dependent cytotoxicity (CDC), relative to an IgG having the wild-type porcine IgG constant domain. In another embodiment, the desired effect is eliminating or reducing an antibody-dependent cell-mediated cytotoxicity (ADCC), relative to an IgG having the wild-type porcine IgG constant domain. In another embodiment, the desired effect is eliminating or reducing an antibody-dependent cellular phagocytosis (ADCP), relative to an IgG having the wild-type porcine IgG constant domain. In yet another embodiment, the desired effect is eliminating or reducing the binding of the IgG to Fc gamma receptor (pFcgR). [000104] In one embodiment, the 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, numbered according to the Eu index as in Kabat. The amino acid at these positions can
ZP000432A be substituted with any other amino acid. Examples of substitution amino acid includes, 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 substitution amino acid is a non-natural amino acid. [000105] The modified porcine IgG of the invention can be any suitable porcine IgG, known to one of skilled in the art. Examples of the modified porcine IgG include a modified variant 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). [000106] In another exemplary embodiment, the porcine IgG constant domain comprises one or more of 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, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, T289A, R290A, K292A, E293delete, N297G, P307Q, E311A, E311C, E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E311N, E311P, E311Q, E311R, E311S, E311T, E311V, E311W, E311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P329L, A330S, P331S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426H, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, A426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W. [000107] In one embodiment, the modified porcine IgG is IgG1a constant domain comprising one or more of 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.
ZP000432A [000108] In another embodiment, the modified porcine IgG is IgG1b constant domain comprising one or more of 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. [000109] In another embodiment, the modified porcine IgG is IgG2a constant domain comprising one or more of 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. [000110] In another embodiment, the modified porcine IgG is IgG2b constant domain comprising one or more of 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. [000111] In another embodiment, the modified porcine IgG is IgG4a constant domain comprising one or more of 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. [000112] In another embodiment, the modified porcine IgG is IgG4b constant domain comprising one or more of 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. [000113] In another embodiment, the modified porcine IgG is IgG6a constant domain comprising one or more of 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. [000114] In another embodiment, the modified porcine IgG is IgG6b constant domain comprising one or more of 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. [000115] In another example, the mutant IgG constant domain of the invention comprises one or more mutations described herein. In some embodiments, the mutant IgG constant domain
ZP000432A is a homologue, a variant, an isomer, or a functional fragment, but with mutation of the invention described herein. Each possibility represents a separate embodiment of the present invention. [000116] The amino acid sequence of the mutant constant domain is encoded by its corresponding mutant nucleic acid sequence. Methods for Making Antibody Molecules of the Invention [000117] Methods for making antibody molecules are well known in the art and fully described in U.S. Patents 8,394,925; 8,088,376; 8,546,543; 10,336,818; and 9,803,023 and U.S. Patent Application Publication 20060067930, which are incorporated by reference herein in their entirety. Any suitable method, process, or technique, known to one of skilled in the art, can be used. An antibody molecule having a variant Fc region of the invention may be generated according to the methods well known in the art. In some embodiments, the variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or binding domain of a receptor or ligand. [000118] With the advent of methods of molecular biology and recombinant technology, a person of skilled in the art can produce antibody and antibody-like molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of the antibodies. Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with assembly of the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This has permitted the ready production of antibodies having sequences characteristic of neutralizing antibodies from different species and sources. [000119] Regardless of the source of the antibodies, or how they are recombinantly constructed, or how they are synthesized, in vitro or in vivo, using transgenic animals, large cell cultures of laboratory or commercial size, using transgenic plants, or by direct chemical synthesis employing no living organisms at any stage of the process, all antibodies have a similar overall 3 dimensional structure. This structure is often given as H2L2 and refers to the fact that antibodies commonly comprise two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as "variable" or `V" regions and are characterized by differences in amino acid sequence from antibodies of
ZP000432A different antigenic specificity. The variable regions of either H or L chains contain the amino acid sequences capable of specifically binding to antigenic targets. [000120] As used herein, the term "antigen binding region" refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody binding region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the H or L chains that provide for the antigen binding regions are smaller sequences dubbed "hypervariable" because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. [000121] The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called "framework regions" made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs. [000122] The present invention further provides a vector including 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 particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which would be capable of encoding the amino acid. The probability that a particular oligonucleotide will, in fact, constitute the actual encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an antibody or portion. Such "codon usage rules" are disclosed by Lathe, et al., 183 J. Molec. Biol.1-12 (1985). Using the "codon usage rules" of Lathe, a single nucleotide sequence, or a set of nucleotide sequences that contains a theoretical "most probable" nucleotide sequence capable of encoding porcine IgG sequences can be identified. It is also intended that the antibody coding regions for use in the present invention could also be provided by altering existing antibody genes using standard molecular
ZP000432A biological techniques that result in variants of the antibodies and peptides described herein. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the antibodies or peptides. [000123] For example, one class of substitutions is conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a porcine antibody peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg, replacements among the aromatic residues Phe, Tyr, and the like. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., 247 Science 1306-10 (1990). [000124] Variant porcine antibodies or peptides may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative variations or variations in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non- conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region. [000125] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as epitope binding or in vitro ADCC activity. Sites that are 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). [000126] Moreover, polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known
ZP000432A modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP ribosylation, for instance, are described in most basic texts, such as Proteins-Structure and Molecular Properties (2nd ed., T. E. Creighton, W. H. Freeman & Co., N.Y., 1993). Many detailed reviews are available on this subject, such as by Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, N.Y., 1983); Seifter et al.182 Meth. Enzymol.626-46 (1990); and Rattan et al.663 Ann. NY Acad. Sci.48-62 (1992). [000127] In another aspect, the invention provides antibody derivatives. A "derivative" of an antibody contains additional chemical moieties not normally a part of the protein. Covalent modifications of the protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting 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 useful for cross-linking the antibody or fragment to a water-insoluble support matrix or to other macromolecular carriers. [000128] Derivatives also include radioactively labeled monoclonal antibodies that are labeled. For example, with radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, tritium (H3) or the like; conjugates of monoclonal antibodies with biotin or avidin, with enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholine esterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemoluminescent agents (such as acridine esters) or fluorescent agents (such as phycobiliproteins).
ZP000432A [000129] Another derivative bifunctional antibody of the invention is a bispecific antibody, generated by combining parts of two separate antibodies that recognize two different antigenic groups. This may be achieved by crosslinking or recombinant techniques. Additionally, moieties may be added to the antibody or a portion thereof to increase half-life in vivo (e.g., by lengthening the time to clearance from the blood stream. Such techniques include, for example, adding PEG moieties (also termed pegylation), and are well-known in the art. See U.S. Patent. Appl. Pub. No.20030031671. [000130] In some embodiments, the nucleic acids encoding a subject antibody are introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. After the subject nucleic acids have been introduced into a cell, the cell is typically incubated, normally at 37° C., sometimes under selection, for a period of about 1-24 hours in order to allow for the expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the media in which the cell is growing. Traditionally, monoclonal antibodies have been produced as native molecules in murine hybridoma lines. In addition to that technology, the present invention provides for recombinant DNA expression of the antibodies. This allows the production of antibodies, as well as a spectrum of antibody derivatives and fusion proteins in a host species of choice. [000131] A nucleic acid sequence encoding at least one antibody, portion or polypeptide of the invention may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 supra, may be used to construct nucleic acid sequences which encode an antibody molecule or antigen binding region thereof. [000132] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as peptides or antibody portions in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to
ZP000432A organism, as is well known in the analogous art. See, e.g., Sambrook et al., 2001 supra; Ausubel et al., 1993 supra. [000133] The present invention accordingly encompasses the expression of an antibody or peptide, in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts including bacteria, yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. The mammalian cell or tissue may be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin. Any other suitable mammalian cell, known in the art, may also be used. [000134] In one embodiment, the nucleotide sequence of the invention will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. See, e.g., Ausubel et al., 1993 supra. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. [000135] Example prokaryotic vectors known in the art include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, CoIE1, pSC101, pACYC 184, .pi.vX). Such plasmids are, for example, disclosed by Maniatis et aI., 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 bacteriophages such as phLC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John et aI., 8 Rev. Infect. Dis.693-704 (1986); lzaki, 33 Jpn. J. Bacteriol.729-42 (1978); and Ausubel et aI., 1993 supra. [000136] Alternatively, gene expression elements useful for the expression of cDNA encoding antibodies or peptides include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et aI., 3 Mol. Cell. Biol.280 (1983), Rous sarcoma virus LTR (Gorman et aI., 79 Proc. Natl. Acad. Sci., USA 6777 (1982), and Moloney murine leukemia virus LTR (Grosschedl et aI., 41 Cell 885 (1985); (b) splice
ZP000432A regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et aI., 1983), and (c) polyadenylation sites such as in SV40 (Okayama et aI., 1983). [000137] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin genes comprised of part cDNA, part genomic DNA (Whittle et aI., 1 Protein Engin.499 (1987)), the transcriptional promoter can be human cytomegalovirus, the promoter enhancers can be cytomegalovirus and mouse/human immunoglobulin, and mRNA splicing and polyadenylation regions can be the native chromosomal immunoglobulin sequences. [000138] In one embodiment, for expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells. [000139] Each fused gene can be assembled in, or inserted into, an expression vector. Recipient cells capable of expressing the immunoglobulin chain gene product are then transfected singly with a peptide or H or L chain-encoding gene, or are co-transfected with H and L chain gene. The transfected recipient cells are cultured under conditions that permit expression of the incorporated genes and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture. [000140] In one embodiment, the fused genes encoding the peptide or H and L chains, or portions thereof are assembled in separate expression vectors that are then used to cotransfect a recipient cell. Alternatively the fused genes encoding the H and L chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the antibody, the recipient cell line may be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. Myeloma cells can be grown
ZP000432A in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as B lymphocytes of porcine or non-porcine origin, hybridoma cells of porcine or non-porcine origin, or interspecies heterohybridoma cells. [000141] The expression vector carrying an antibody construct or polypeptide of the invention can be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988). [000142] Yeast may provide substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982). [000143] Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of peptides, antibodies, fragments and regions thereof. Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A number of approaches can be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985). [000144] Bacterial strains can also be utilized as hosts for the production of antibody molecules or peptides described by this invention. Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches
ZP000432A can be taken for evaluating the expression plasmids for the production of antibodies, fragments and regions or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001 supra; Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, N.Y. (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2001). [000145] Host mammalian cells may be grown in vitro or in vivo. Mammalian cells provide posttranslational modifications to immunoglobulin protein molecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein. Mammalian cells which can be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems are available for the expression of cloned peptides Hand L chain genes in mammalian cells (see Glover, 1985 supra). Different approaches can be followed to obtain complete H2L2 antibodies. It is possible to co-express Hand L chains in the same cells to achieve intracellular association and linkage of Hand L chains into complete tetrameric H2L2 antibodies and/or peptides. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both Hand L chains and/or peptides can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker. cell lines producing peptides and/or H2L2 molecules via either route could be transfected with plasmids encoding additional copies of peptides, H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines. [000146] For long-term, high-yield production of recombinant antibodies, stable expression may be used. For example, cell lines, which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and grow to form foci which in turn can be cloned
ZP000432A and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluation of compounds/components that interact directly or indirectly with the antibody molecule. [000147] Once an antibody of the invention has been produced, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In many embodiments, antibodies are secreted from the cell into culture medium and harvested from the culture medium. Pharmaceutical and Veterinary Applications [000148] The invention also provides a pharmaceutical composition comprising molecules of the invention and one or more pharmaceutically acceptable carriers. More specifically, the invention provides for a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody or peptide according to the invention. [000149] “Pharmaceutically acceptable carriers” include any excipient which is nontoxic to the cell or animal being exposed thereto at the dosages and concentrations employed. The pharmaceutical composition may include one or additional therapeutic agents. [000150] "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 excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. [000151] 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. [000152] Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphate, citrate and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; EDTA; salt forming counterions such as sodium; and/or nonionic surfactants such as
ZP000432A TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride; as well as combinations thereof. [000153] The pharmaceutical compositions of the invention may be formulated in a variety of ways, including for example, liquid, semi-solid, or solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the compositions are in the form of injectable or infusible solutions. The composition can be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The composition may be formulated as an immediate, controlled, extended or delayed release composition. [000154] The compositions of the invention can be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of the antibodies (typically carried in a pharmaceutical formulation) to an airway surface. Topical administration to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler). Topical administration of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. [000155] In some desired embodiments, the antibodies are administered by parenteral injection. For parenteral administration, antibodies or molecules can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier such vehicles are water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine and the like. Liposomes and nonaqueous vehicles such as fixed oils
ZP000432A can also be used. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjustment agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 1% to as much as 15% or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980). [000156] The antibodies or molecules of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins. Any suitable lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate. The compositions containing the present antibodies or a cocktail thereof can be administered for prevention of recurrence and/or therapeutic treatments for existing disease. Suitable pharmaceutical carriers are described in the most recent edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference text in this field of art. In therapeutic application, compositions are administered to a subject already suffering from a disease, in an amount sufficient to cure or at least partially arrest or alleviate the disease and its complications. [000157] Effective doses of the compositions of the present invention, for treatment of conditions or diseases as described herein vary depending upon many different factors, including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; target site; physiological state of the animal; other medications administered; whether treatment is prophylactic or therapeutic; age,
ZP000432A health, and weight of the recipient; nature and extent of symptoms kind of concurrent treatment, frequency of treatment, and the effect desired. [000158] Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating veterinarian. In any event, the pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the subject. [000159] Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. [000160] The pharmaceutical compositions of the invention may include a “therapeutically effective amount.” A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary according to 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 one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects. [000161] In another aspect, the compositions of the invention can be used, for example, in the treatment of various diseases and disorders in porcine. As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented. [000162] All patents and literature references cited in the present specification are hereby incorporated by reference in their entirety. [000163] The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should
ZP000432A not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention. EXAMPLES EXAMPLE 1 Porcine IgG and FcRn [000164] Ex vivo porcine olfactory tissue, primary cells from porcine olfactory epithelium (OEPC) and the human cell line RPMI 2650 were used for evaluation. IgGs Comparable permeation rates for human and porcine IgG were observed in OEPC, which display the highest expression of FcRn. Only traces of porcine IgGs could be recovered at the basolateral compartment in ex vivo olfactory tissue, while human IgGs reached far higher levels. [000165] The porcine FcRn cDNA is 1,577 bp in length (GenBank Accession Number: AAP49846.1) and contains a 1,077 bp open reading frame (ORF) encoding a 356-amino acid polypeptide. The 3′ end of the sequence contains a poly(A) stretch, 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 identity with the corresponding gene in human, mouse and cattle respectively. The complete porcine FcRn genomic DNA sequence spans 8,900 bp (GenBank accession number: HQ026019) and it consists of 5 introns separating 6 exons. The intron/exon organization of porcine (5 introns and 6 exons) is identical with that of the human and mouse FcRn gene. [000166] Porcine IgG used to evaluate the in vitro FcRn binding are provided in Table1. Table 1. Porcine IgG Subtypes and Allotypes
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[000167] The porcine FcRn was generated recombinantly. The porcine beta-2-microglobulin (B2M) small subunit that associates with FcRn to form a functional complex was also generated recombinantly and used in surface plasmon resonance (SPR) binding affinity experiments. [000168] DNA for porcine FcRn/B2M (Genbank accession: AAP49846.1/NP_999143.1) and all porcine mAb genes was codon-optimized for mammalian expression, and constructs were transiently expressed either in HEK 293 cells using a standard lipofectamine transfection protocol (Invitrogen Life Technologies, Carlsbad, CA, USA) or into CHO cells using the ExpiCHO transient system (ThermoFisher Scientific) kit protocols. ExpiCHO expression followed protocols outlined by ThermoFisher for either mAb or FcRn/B2M transfection. For mAbs, plasmid containing gene sequence encoding for an IgG kappa light chain was co- transfected with a plasmid encoding for IgG heavy chain. For HEK293 expression, equal amounts by weight of heavy chain plasmid and kappa chain plasmid were co-transfected. For FcRn/B2M, the two plasmids encoding each were transfected. Cells were allowed to grow for 7 days (HEK293) or 12 days (CHO) after which supernatants were collected for protein purification. mAbs were screened for binding to protein A or protein G sensors via Octet QKe quantitation (Pall ForteBio Corp, Menlo Park, CA, USA). Expression was quantified on Octet with protein A or protein G sensors using standard curves, and mAbs were purified with protein G or protein A/G affinity chromatography. For all protein constructs, Sodium Acetate pH 5.5 was used as binding and wash buffer, and elution was performed at pH 3.4. The purified proteins were neutralized and dialyzed into 20 mM Na acetate, pH 5.5, 140 mM NaCl for further analysis. The FcRn plasmid contained a c-terminal His tag, thus the FcRn/B2M complex was purified by IMAC affinity purification. The concentration of the mAbs and FcRn/B2M proteins was measured via NanoDrop at 280 nm. Protein quality was assessed via analytical SEC and standard coomassie protein gels. [000169] The purified FcRn/B2M was biotinylated as follows. The purified FcRn/B2M protein was dialyzed into 10 mM Tris–HCl, pH 8.0 and concentrated using AmiconUltra,10KMWCO (EMD Millipore, Billerica, MA). The Biotin Acceptor Peptide (BAP) AGLNDIFEAQKIEWHE which was expressed at the c-terminus of the receptor allowed for
ZP000432A transfer of biotin to this stretch of amino acids using the biotin ligase BirA. Biotinylation reactions were carried out as described in the manufacturer protocol (Avidity, LLC,Aurora, CO). The FcRn/B2M receptor was then dialyzed into PBS to remove residual biotin. EXAMPLE 2 Construction of Porcine IgG Fc Mutants [000170] Plasmids containing sequence encoding for porcine constant regions for the IgG6a were utilized and VH/VL sequences for each mAb investigated herein were inserted upstream and in frame with the nucleotides encoding for the constant domains. Mutations were incorporated into either CH2 or CH3 domain positions of each plasmid by direct DNA synthesis of the constant region as gene fragment and were subsequently sub-cloned into respective variable region of interest. Expression and Purification [000171] The monoclonal antibody (mAbs) mutants were expressed in mammalian suspension cell systems, EXPICHO-S (Chinese Hamster Ovary) cells, obtained from Thermo Fisher. Suspension EXPICHO-S cells were maintained in EXPICHO expression medium (Gibco) between 0.14 and 8.0x10e6 cells/ml. Cells were diluted following the ExpiCHO Protocol user manual on Day -1 and transfection day. Diluted cells were transfected as described in the protocol using reagents sourced from ExpiFectamine CHO Transfection Kit (Gibco) following Max Titer conditions. Following 12-14 days of incubation, the cultures were harvested and clarified. Antibodies were purified from the clarified supernatant via Protein A chromatography over MabSelect Sure LX (GE Healthcare) which had been pre-equilibrated with PBS. Following sample load, the resin was washed with PBS and then with 20 mM sodium acetate, pH 5.5. Samples were eluted from the column with 20 mM acetic acid, pH 3.5. Following elution, pools were made and neutralized with the addition of 1 M sodium acetate to 4%. Depending on available volume and intended use, samples were sometimes exchanged into a final buffer (e.g. PBS, other). Concentration was measured by absorbance at 280 nm. SDS-PAGE [000172] Non-reduced (nr) and reduced sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) was performed using 4-12% Bis-Tris NuPAGE gels in MES- SDS running buffer, and SeeBlue Plus 2 standards, all from Invitrogen. For non-reduced samples, 1mM of alkylating agent N-ethylmaleimide (NEM) was added, for reduced samples
ZP000432A reducing agent dithiothreitol (DTT) was added. Gels were stained with Coomassie Blue to detect the protein bands. EXAMPLE 3 FcRn Binding assay Biacore method for pFcRn: [000173] Porcine Fc-based antibodies or fusion protein binding affinities to porcine FcRn were determined by surface plasmon resonance (SPR). All reported KD's were measured in Biacore T200 (Cytiva, Marlborough, MA, USA) or Biacore 8K (Cytiva, Marlborough, MA, USA) using SA sensor. Porcine FcRn was captured on the surface of the sensor for a desired surface density. Running buffer of 20 mM MES, 150 mM NaCl, 0.005% Tween 20, 0.5 mg/mL BSA, pH 6 and/or PBS, 0.0005% Tween 20, pH7.4 were used. Various concentrations of porcine mAbs were titrated in proper running buffer and flowed over the receptor surface. Regeneration was performed with 50 mM Tris-HCl, pH8. Kinetic binding affinity was analyzed using Biacore T200 Evaluation software (Cytiva, Marlborough, MA, USA) or Biacore 8K Insight Evaluation Software with method of double referencing: the reference flow cell was subtracted from the flow cell containing immobilized porcine FcRn and blank runs containing buffer only were subtracted out from all runs. The resulting curve was fitted with the 1:1 binding model. Runs were performed at 25°C. [000174] Mutations made at respective positions have a marked effect on the affinity of the IgG to FcRn at pH6. [000175] Binding of wild-type (WTs) and mutant IgGs to porcine FcRn were measured by surface plasmon resonance (Biacore). The results are shown in Table 2 below. Table 2. Effect of mutants on pFcRn binding affinity.
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Mutants are numbered according to the EU Index as in Kabat. LS=Low Signal. [000176] The results clearly show that mutations made at various positions have a marked effect on the affinity of the IgG to porcine FcRn.
ZP000432A EXAMPLE 4 Effector function and its modulation [000177] Antibodies exhibit their therapeutic function either by blocking the antigen by “neutralization” or by mediating effector functions. Antibody effector functions are an important part of the humoral immune response and are induced via the constant (Fc) region of the antibody, which can interact with complement proteins and specialized Fc-receptors. The most well-known Fc-mediated antibody effector functions are antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). [000178] All three effector functions described above require two main components to neutralize the target in a specific and effective manner – the right Fc region and its corresponding Fc receptor. The isotype and the subclasses/allotypes of the Fc region determine the binding capability of an IgG to mediate an effector function. [000179] The IgG subclasses in swine are poorly understood since the subclasses had not been defined until recently. This lack of progress has been largely due to the lack of IgG subclass reagents for use in immunoassays and due to the lack of subclass knockout animals. [000180] In pigs, to date, six pig IgG subclasses are known - IgG1, G2, G3, G4, G5 and G6. These are further divided into several allotypes based on relative occurrence, differences in intron sequences, and sequence similarity of their CH2-CH3 domain complex - IgG1a, IgG1b, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a and IgG6b. [000181] 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 identification numbers of publicly available NCBI database. Generation of Fcgamma receptors [000182] Recombinant porcine FcgR1, FcgR2b, FcgR3a, and Fcg3b DNA were codon- optimized for mammalian expression and synthesized based on sequences from NCBI database as in Table 3.
ZP000432A Table 3: NCBI accession numbers of Fc gamma receptors
[000183] DNA was cloned into pcDNA3.1(+) vectors, 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. [000184] The purified FcRs were biotinylated as follows. The purified Fc receptor proteins were dialyzed into10 mM Tris–HCl, pH 8.0 and concentrated using AmiconUltra,10KMWCO (EMD Millipore, Billerica, MA). The Biotin Acceptor Peptide (BAP) AGLNDIFEAQKIEWHE which was expressed at the c-terminus of the receptors allowed for transfer of biotin to this stretch of amino acids using the biotin ligase BirA. Biotinylation reactions were carried out as described in the manufacturer protocol (Avidity, LLC,Aurora, CO). The receptors were then dialyzed into PBS to remove residual biotin. [000185] A Biacore SPR binding assay was designed to test the affinity of porcine IgG subclasses and mutants to pFcgR1, pFcgR2b, pFcgR3a, pFcg3b. Generation of Fc fusion proteins and mAbs [000186] Recombinant CTLA4-Fc fusions were constructed via insertion of the canine CTLA4 gene (NCBI NM_001003106.1) into pcDNA3.1(+) mammalian expression vector containing the pIgG1a, pIgG2a, pIgG3, pIgG4a, pIgG5a or pIgG6a. Fc starting just upstream of the heavy chain hinge region. No additional linkers were required. [000187] Recombinant mAbs with pIgG1a, pIgG2a, pIgG3, pIgG4a, pIgG5a or pIgG6a Fc regions were constructed via insertion of VH sequences upstream and in frame with the nucleotides encoding for the constant domains in pcDNA3.1(+) mammalian expression vector. Similarly, light chains were constructed via insertion of VL sequences upstream and in frame with the porcine kappa allele 1 constant region (NCBI AAA03520.1). [000188] Mutations were introduced into the three wildtype subclasses in both the CTLA4 Fc fusion and full mAb formats to knock out binding to FcgRs, knock out CDC and/or ADCP. Mutations were incorporated into various positions on each wildtype plasmid using Agilent’s
ZP000432A QuikChange II Mutagenesis and associated Agilent primer design tools for single site directed mutagenesis (www.agilent.com/store/primerDesignProgram.jsp). [000189] DNA for all CTLA4 fusion and mAb genes was codon-optimized for mammalian expression, and constructs were transiently expressed either in HEK 293 cells using a standard lipofectamine transfection protocol (Invitrogen Life Technologies, Carlsbad, CA, USA) or into CHO cells using the ExpiCHO transient system (ThermoFisher Scientific) kit protocols. ExpiCHO expression followed protocols outlined by ThermoFisher for either mAb or CTLA4 Fc fusion transfection. For mAbs, plasmid containing gene sequence encoding for an IgG kappa light chain was co-transfected with a plasmid encoding for IgG heavy chain. For HEK293 expression, equal amounts by weight of heavy chain plasmid and kappa chain plasmid were co-transfected. For the Fc fusions, the single plasmid was transfected. Cells were allowed to grow for 7 days (HEK293) or 12 days (CHO) after which supernatants were collected for protein purification. CTLA4 Fc fusions and mAbs were screened for binding to protein A or protein G sensors via Octet QKe quantitation (Pall ForteBio Corp, Menlo Park, CA, USA). Expression was quantified on Octet with protein A or protein G sensors using standard curves, and mAbs/fusion proteins were purified with protein G or protein A/G affinity chromatography. For all protein constructs, Sodium Acetate pH 5.5 was used as binding and wash buffer, and elution was performed at pH 3.4. The purified proteins were neutralized and dialyzed into 20 mM Na acetate, pH 5.5, 140 mM NaCl for further analysis. The concentration of the mAbs and fusion proteins was measured via NanoDrop at 280 nm. Protein quality was assessed via analytical SEC and standard coomassie protein gels. SPR Method for biotinylated pFcgR1, pFcgR2 and pFcgR3 [000190] A Biacore SPR binding assay was designed to test the affinity of bovine IgG subclasses to pFcgR1, pFcgR2 and pFcgR3. All reported KD's were measured by Biacore (cytiva, Marlborough, MA, USA) using series S SA sensor. Biotinylated-bovine FcgR1, R2 and R3 were captured on the sensor surface using a modified SA capture method to reach the desired surface density. 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% v/v surfactant P20, pH7.4 buffer was used as the running and titration buffer. Various concentrations of porcine CTLA4-Fc fusions or mAbs were titrated and flowed over the receptor surface and affinities were determined using Biacore T200 Evaluation software (cytiva, Marlborough, MA, USA) with 1:1 binding model. The method of double referencing has been applied where the reference flow cell was subtracted from the flow cell containing immobilized receptors and
ZP000432A blank runs containing buffer only were subtracted out from all runs. Flow cells were regenerated using 10 mM Glycine pH1.5. Runs were performed at 15°C. CDC assay [000191] The CDC cell-based assay was developed and employed to characterize the effectiveness of the nine CTLA4 porcine IgG subclass Fc fusion proteins in mediating CDC and to investigate Fc region mutations in the subclasses. This will help define key residues in the Fc region that determine CDC activity of the porcine IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 which binds to CTLA4 on the Fc fusion proteins. These target cells have been used in past canine ADCC assays and were utilized in the CDC assay due to their dependability. [000192] Incubation of the fusion protein-bound target cells with complement-preserved serum can result in Fc binding on the fusion proteins to complement component C1q initiating the complement cascade, ultimately forming membrane attack complexes. The pore-forming complexes mediate cell lysis of the target cells measured by loss of cell viability. If there is no Fc binding to C1q there is no resultant cell lysis/death. [000193] Briefly, CD80-expressing CHO cells (target cells) were plated at 40,000 cells/well in CD CHO media in round-bottomed 96-well plates. Titrated fusion proteins in CD CHO media were added to the target cells and allowed to bind for 60 minutes at 37°C. Porcine complement preserved serum (20% in CD CHO media) was added to the plates 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 no fusion protein + complement preserved serum controls. [000194] As shown in Figure 2 and Table 4, all of the porcine wild type Fc subclasses showed robust and potent CDC activity with the exception of IgG3 and IgG5a. EC50 values range from 0.010 to 0.044 µg/mL.
ZP000432A Table 4. CDC effects induced by porcine IgG Fc wildtype constructs.
[000195] 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 diminished CDC activity and IgG3 showed little CDC activity. ADCC assay [000196] The ADCC cell-based assay was developed and employed to characterize the effectiveness of the nine CTLA4 porcine IgG subclass Fc fusion proteins in mediating ADCC and to investigate Fc region mutations in the subclasses. This will help define key residues in the Fc region that determine ADCC activity of the porcine IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 which binds to CTLA4 on the Fc fusion proteins. These target cells have been used in past canine ADCC assays and were utilized in the CDC assay due to their dependability. [000197] Incubation of the fusion protein-bound target cells with cultured activated porcine PBMCs can result in Fc binding on the fusion proteins to FcγRIII mediating Granzyme and perforin release form the NK cells within the PBMC population. The actions of these proteins will result in cytotoxicity of the target cells bound by the fusion proteins measured by quantification of dead target cells by flow cytometry. If there is no Fc binding to FcγRIII there is no resultant target cell death. [000198] Briefly, CD80-expressing CHO cells (target cells) were plated at 20,000 cells/well in CD CHO media in round-bottomed 96-well plates. Titrated fusion proteins in CD CHO media were added to the target cells and allowed to bind for 60 minutes at 37°C. Single donor porcine PBMCs (effector cells), cultured overnight in RPMI 1640 medium + IL-2 and IL-15,
ZP000432A were added to the plates for 18-20 hours at 37°C using an effector:target cell ratio (E:F Ratio) of 40-50:1. Cells were then stained fixed and analyzed via flow cytometry quantifying the live/dead stain of the target cells. Data were expressed as “% Dead Target cells” normalized to fusion protein alone (minus effector PBMCs). [000199] Figures 3A and 3B show the results of cell-based antibody-dependent cell-mediated cytotoxicity activity of porcine wild type Fc subclass CTLA4 fusion proteins. As shown in Figures 3A and 3B and Table 5, all the porcine wild type Fc subclasses showed some ADCC activity with the exception of IgG3. Degree of ADCC and potency varied with EC50 values range from 0.031 to 0.477 µg/mL. [000200] Our results showed 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. Table 5. ADCC effects induced by porcine IgG Fc wildtype CTLA4 fusion proteins
ADCP assay [000201] The antibody dependent cellular phagocytosis (ADCP) assay utilizes CHO target cells engineered to express canine CD80, which binds to canine CTLA4 on the Fc fusion proteins. The Fc region of the fusion protein may then bridge this complex to Fc gamma receptors on alveolar macrophage effector cells, which have the capacity to phagocytose the target cell. ADCP is measured by signal intensity and frequency of a pH-sensitive fluorescent dye within the population of effector macrophages in the co-culture, wherein fluorescent cells are indicative of an effector cell that has successfully internalized a target cell into the acidic lysosome.
ZP000432A [000202] Briefly, canine CD80-expressing CHO cells (CD80 target cells) or wild-type CHO cells which do not express CD80 (parental target cells) were stained with pHrodo red dye for 30 minutes at 37 degrees C. Stained cells were subsequently incubated with CTLA4-Fc fusion proteins for 20 minutes to mediate CTLA4:CD80 binding. 60,000 target cells were then added to 30,000 pre-plated porcine alveolar macrophages which had been previously stained with a cell marker (CellTrace Violet, CTV) to aid in later identification. Cocultures were maintained for 5-6 hours at 37 C, and subsequently harvested and analyzed by flow cytometry to identify effector cells (CTV+) that have successfully performed ADCP (pHrodo+). [000203] Figure 4 shows the results of cell-based antibody-dependent cell-mediated phagocytosis of porcine wild type Fc subclass CTLA4 fusion proteins. Of the 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 showed no ADCP activity. Of those with activity, IgG4b and IgG6b showed the lowest (most effective) EC50s at 2.6ng/mL and 5.9 ng/mL respectively. When each construct was incubated with parental target cells that did not express canine CD80, and thus did not bind the CTLA4:Fc constructs, no increase in phagocytosis was observed (range of observed phagocytosis in WT ChoK1 target cells is displayed as horizontal grey bar on the graph, with the average displayed as a dotted line). pIgG6 Mutations Knock Out Effector Function CDC assay [000204] As shown in Figure 5, wild type IgG6 subclass showed robust and potent CDC activity. The Fc mutations investigated all showed varying effects on CDC activity ranging from no effect to completely knocking our CDC effector function. [000205] Both A and B allotypes of porcine IgG6 Fc CTLA4 fusion protein showed CDC activity. Mutations in the Fc showed a range of effect on CDC activity from no effect to modest effect to complete knockout of effector function. Mutations SSP, WIN-LSP, WIN-PG, PG, EP-PSS, KAPA, KA, PA, and PG appear to most effective at knocking out CDC effector function for the pIgG6a allotype. Additionally, alanine substitutions T289A, R290A and K292A knocked out CDC effector function for IgG6a. Mutations DANG-SAP, WIN-PG and KAPA appear to most effective at knocking out CDC effector function of the pIgG6b allotype. Mutation SAP showed partial knockout of CDC effector function for both the 6a and 6b allotype.
ZP000432A ADCC assay [000206] As shown in Figure 6, wild type IgG6a or IgG6b subclasses showed robust and potent ADCC activity. IgG6a_WIN, IgG6a_WIN-LSP, IgG6a_WIN-PG, IgG6a_PG, IgG6a_DANG, IgG6a_EP, IgG6a_EP-PSS, IgG6a_SAP and IgG6a_SSP dramatically knockdown ADCC. The SAP Fc mutation showed dramatic knock down of ADCC effector function activity for IgG6b. ADCP assay [000207] Of the 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 showed no ADCP activity. Of those with activity, IgG4b and IgG6b showed the lowest (most effective) EC50s at 2.6ng/mL and 5.9 ng/mL respectively. When each construct was incubated with parental target cells that did not express canine CD80, and thus did not bind the CTLA4:Fc constructs, no increase in phagocytosis was observed (range of observed phagocytosis in WT ChoK1 target cells is displayed as horizontal grey bar on the graph, with the average displayed as a dotted line) [000208] Of the IgG6a mutants tested, the PG, WIN2, WIN-PG, DANG, DANG-GSP, EP, EP- PSS, KA, SAP and PA mutants all showed no significant amount of ADCP. WIN-LSP, SSP, KAPA and T289A mutations had ADCP knocked down. [000209] Table 6A below lists various constructs, their mutations, and their corresponding codon usage. Table 6B below summarizes the results of effector function of pIgG6 WT and mutations. Table 6A. Constructs, Mutations, and Their Codons
ZP000432A
ZP000432A
Table 6B. Summary of effector function of pIgG6 WT and mutations )
ZP000432A )
PA = Partial Activity; KO = Knock Out; NT = Not Tested, NC= No Change, EE= Effector Enhancement. pIgG4 Mutations Knock Out Effector Function CDC assay [000210] Figures 8 and 16 show the results of cell-based CDC activity of porcine wild type Fc IgG4a and 4b subclasses CTLA4 fusion proteins and Fc mutants of those subclasses. [000211] Porcine IgG4a and 4b Fc CTLA4 fusion proteins showed robust CDC activity. Mutations in the Fc showed modest knockdown of CDC activity, but no mutation appeared to dramatically knock down CDC activity for either of these two subclasses. ADCC assay [000212] Figure 9 shows the results of cell-based ADCC activity of porcine wild type Fc IgG4a and IgG4b subclass CTLA4 fusion proteins and SAP mutations of those Fc subclasses. [000213] The wild type pIgG4a and pIgG4b constructs show ADCC activity that is knocked out with SAP mutations in each of the two allotypes.
ZP000432A ADCP assay [000214] Figures 10, 17A, and 17B show the results of cell-based ADCP activity of porcine wild type Fc IgG4a subclass CTLA4 fusion protein, as well as SAP and WinPG mutations of that Fc subclass. [000215] The wild type pIgG4a construct shows robust activity that is fully knocked out with SAP, KAPA, GSP, EP-PAS, DANG, SSP, WIN2, and WIN-PG mutations. Mutations EP, PA, PG, R290A, T289A and DANG-SAP showed partial knock down of ADCP. [000216] Table 7 below summarizes the results of effector function of pIgG4 WT and mutations. Table 7. Summary of effector function of pIgG4 WT and mutations )
PA = Partial Activity; KO = Knock Out; NT = Not Tested, NC= No Change, EE= Effector Enhancement.
ZP000432A pIgG2 Mutations Knock Out Effector Function CDC assay [000217] Figure 11 shows the results of cell-based CDC activity of porcine wild type Fc IgG2 subclass CTLA4 fusion proteins and Fc mutants of that subclass. As shown in Figure 11, wild type IgG2 Fc subclass showed robust and potent CDC activity. The Fc mutations investigated all showed diminished CDC activity to varying degrees. [000218] Porcine IgG2 Fc CTLA4 fusion protein showed CDC activity. Mutations in the Fc showed knockdown of CDC activity with the GSP mutation appearing to be the mutation that most effectively knocks down CDC activity. ADCC assay [000219] Figure 12 shows the results of cell-based antibody-dependent cytotoxicity activity of porcine wild type IgG2 subclass CTLA4 fusion protein and Fc mutants of that subclass. [000220] The wild type pIgG2 construct showed ADCC activity that is knocked out with SAP mutation. ADCP assay [000221] The wild type pIgG2 construct showed ADCP activity that is knocked out with SAP, KAPA, GSP, EP-PAS, DANG, PG, SSP and WIN-PG mutations. Mutations EP, KA, PA, R290A, T289A and WIN2 showed partial knock down of ADCP. See Figures 19A and 19B. [000222] Table 8 below summarizes the results of effector function of pIgG2 WT and mutations. The results support for both pIgG2a and 2b allotypes because both 2a and 2b are identical in the constant region domains CH2 and CH3. Table 8. Summary of effector function of pIgG2 WT and mutations
ZP000432A
PA = Partial Activity; KO = Knock Out; NT = Not Tested, NC= No Change, EE= Effector Enhancement. pIgG1 Mutations Knock Out Effector Function CDC assay [000223] Figure 13 shows the results of cell-based complement-dependent cytotoxicity activity of porcine wild type Fc IgG1a and 1b subclasses CTLA4 fusion proteins and Fc mutants of those subclasses. As shown in Figure 13, wild type IgG1a and IgG1b Fc subclasses showed robust and potent CDC activity. The Fc mutations investigated all showed diminished CDC activity to varying degrees. [000224] Porcine IgG1a and 1b Fc CTLA4 fusion proteins both showed CDC activity. Mutations in the Fc showed knockdown of CDC activity with the KAPA mutation appearing to be the mutation that most effectively knocks down CDC activity. ADCC assay [000225] Figure 14 shows the results of cell-based antibody-dependent cytotoxicity activity of porcine wild type IgG1a and 1b subclass CTLA4 fusion proteins and Fc mutants of these subclasses. [000226] Porcine IgG1a and 1b Fc CTLA4 fusion proteins both showed ADCC activity. As shown in figure 12, SAP mutations significantly knock out ADCC function.
ZP000432A ADCP assay [000227] Porcine IgG1a showed ADCP activity. As shown in figure 15, SAP, DANG, PG, SSP, WIN2 and WIN-PG mutations significantly knock out ADCP function. Mutations EP, KA, PA, R290A and T289A showed partial knock down of ADCP. [000228] Table 9 below summarizes the results of effector function of pIgG1 WT and mutations. Table 9. Summary of effector function of pIgG1 WT and mutations )
PA = Partial Activity; KO = Knock Out; NT = Not Tested, NC= No Change, EE= Effector Enhancement.
ZP000432A EXAMPLE 5 FcRn Binding Validations Methods [000229] The Fc regions of the four porcine subclasses and their allotypes, IgG1 (IgG1a, IgG1b), IgG2, IgG4 (IgG4a and IgG4b), IgG6 (IgG6a and IgG6b) were first 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. [000230] Molecular Operating Environment (MOE) developed by Chemical Computing Group (MOE2019.0102) provides a flexible and automated graphical user interface for protein modeling. To analyze the difference in structures, the sequence-to-profile alignment algorithm uses a scoring algorithm to rank the sequence templates and scores higher than 85% ensure the selection of protein templates with physically realistic structures. The model was then optimized using the same pipeline and the structural stability of the models was verified using Ramachandran Plots, which checks the stereochemical quality of a protein structure. Results [000231] The method described above was performed on the Porcine wild-type (WT) constructs and the following mutants in 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. The positions for the mutational library are represented in a ball-and-stick form in Fig.23 and Fig 24. [000232] An RMSD plot was generated to calculate the root mean square deviation of the WT structures, Porcine IgG1a, Procine-IgG1b, Porcine IgG2, Porcine-IgG4a, Porcine-IgG4b, Porcine-IgG6a and Porcine IgG6b relative to each other (Fig 25). An RMSD value of 2.0Å or lower is considered the standard for considering two structures to be alike. Results indicated that the protein fold of porcine IgG4a construct was identical to the porcine IgG4b allotype with average RMSD value for the structure being 0.11Å (individual position RMSD in Table 10). Porcine IgG6a construct was identical to the porcine IgG6b allotype with an average RMSD value of 0.66 Å (individual position RMSD in Table 11). Allotypes IgG1a, IgG1b and IgG2 also showed RMSDs of 0.2 and 0.78 Å. RMSDs at the positions where mutational scanning was performed is noted in Tables 10 through 13.
ZP000432A Table 10. Root Mean Square Deviation (RMSD) comparisons of residues in the protein models of WT Porcine IgG4a and IgG4b.
Table 11. Root Mean Square Deviation (RMSD) comparisons of residues in the protein models of WT Porcine IgG6a and IgG6b.
ZP000432A
Table 12. Root Mean Square Deviation (RMSD) comparisons of residues in the protein models of WT Porcine IgG1a and IgG1b.
Table 13. Root Mean Square Deviation (RMSD) comparisons of residues in the protein models of WT Porcine IgG1a and IgG2.
ZP000432A
Conclusion [000233] Molecular modeling of all mutations across the two allotypes, IgG6a and IgG6b of porcine backbones were performed and validated using MOE2019.0102 and AlphaFold2.2. Similarly, comparisons of molecular models of all mutations across the two allotypes of each subclass, IgG4a vs IgG4b, IgG1a vs IgG1b and IgG2 of porcine backbones were performed and validated using MOE2019.0102 and AlphaFold2.2. The Fc fold and residue conformation between subclasses were shown to have RMSDs less than 2Å, indicating that the protein structures have very high identity and therefore would function in a similar manner. [000234] Having described preferred embodiments of the invention, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.
Claims
ZP000432A ABSTRACT [000216] The invention relates generally to porcine antibody mutants and uses thereof. Specifically, the invention relates to mutations in the constant region of porcine antibody for improving various characteristics.
4. The method of any of the claims above, wherein the modified IgG is a porcine or porcinized IgG.
5. The method of any of the claims above, wherein the IgG is IgGl a, IgGlb, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a, or IgG6b.
6. The method of any of the claims above, wherein the IgG constant domain comprises CHI domain or a hinge region.
7. The method of any of the claims above, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domain.
8. The method of any of the claims above, wherein the wild-type porcine IgG constant domain comprises one of the amino acid sequences set forth in SEQ ID NOs.: 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 said 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, numbered according to the Eu index as in Kabat.
10. The modified IgG of claim 9, wherein said constant domain comprises one or more of 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, E293delete, N297G, P307Q, E311A, E311C, E311D, E311F, E311G, E311H, E311I, E311K, E311L, E311M, E311N, E311P, E311Q, E311R,
E311S, E311T, E311V, E311W, E311Y, D312A, D312C, D312E, D312F, D312G,
D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S,
D312T, D312V, D312W, D312Y, K322A, P329G, P329S, P329L, A330S, P331S, P331A, D378V, A426C, A426D, A426E, A426F, A426G, A426H, A426I, A426K, A426L, A426M, A426N, A426P, A426Q, A426R, A426S, A426T, A426V, A426W, A426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y,
N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W. The modified IgG of any of claims 9-10 above, wherein the modified IgG is a porcine or porcinized IgG. The modified IgG of any of claims 9-11 above, wherein the IgG is IgGla, IgGlb, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a, or IgG6b. The modified IgG of any of claims 9-12 above, wherein the IgG is IgGla and wherein said constant domain comprising one or more of 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. The modified IgG of any of claims 9-12 above, wherein the IgG is IgGlb and wherein said constant domain comprising one or more of 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. The modified IgG of any of claims 9-12 above, wherein the IgG is IgG2 and wherein said constant domain comprising one or more of 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 P331 S; (v) K322A and P331 A; and (vi) P329S. The modified IgG of any of claims 9-12 above, wherein the IgG is IgG4a and wherein said constant domain comprising one or more of 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 P331 S; (v) K322A and P331 A; and (vi) P329S. The modified IgG of any of claims 9-12 above, wherein the IgG is IgG4b and wherein said constant domain comprising one or more of 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 P331 S; (v) K322A and P331 A; and (vi) P329S.
18. The modified IgG of any of claims 9-12 above, wherein the IgG is IgG6a and wherein said constant domain comprising one or more of 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) P331 A; (viii) K322A; (ix) E233P; (x) P329S and A330S; (xi) G234A, P235L, G236A, P329L, and A330S; (xii) K322A and P331A; and (xiii) P329S.
19. The modified IgG of any of claims 9-12 above, wherein the IgG is IgG6b and wherein said constant domain comprising one or more of 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 P331 S; (v) K322A and P331 A; and (vi) P329S.
20. The modified IgG of any of claims 9-19 above, wherein the modified IgG has a higher affinity for FcRn than the IgG having the wild-type porcine IgG constant domain.
21. The modified IgG of any of claims 9-20 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 of any of claims 9-21 above, wherein the IgG constant domain comprises CHI domain or a hinge region.
23. The modified IgG of any of claims 9-22 above, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domain.
24. The modified IgG of any of claims 9-23 above, wherein the wild-type porcine IgG constant domain comprises one of the amino acid sequences set forth in SEQ ID NOs.: 1-11.
25. A pharmaceutical composition comprising the modified IgG of any of the claims above and a pharmaceutically acceptable carrier.
26. A kit comprising the modified IgG of any of the claims 9-24, in a container, and instructions for use.
27. A polypeptide comprising the modified IgG of any of the claims 9-24.
28. An antibody comprising the modified IgG of any of the claims 9-24.
29. A vector comprising the nucleic acid sequence encoding the amino acid sequence of the modified IgG of any of the claims 9-24.
30. An isolated cell comprising the vector of claim 29.
31. A method of manufacturing an antibody or a molecule, the method comprising: providing the cell of claim 30; and culturing said cell.
32. A method of manufacturing an antibody, the method comprising: providing an antibody of claim 28.
33. A fusion molecule comprising: a porcine IgG constant domain fused to an agent, said porcine IgG constant domain comprising the modified IgG of any of the claims 9-24.
34. A method for enhancing the binding affinity of a porcine IgG to FcRn, the method comprising: providing the modified IgG of any of the claims 9-24.
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