Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/524—CH2 domain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/526—CH3 domain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/71—Decreased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/72—Increased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

• 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.
• the unexpected desired effects include, but not limited to, an enhanced affinity to FcRn and a change in effector function.
• 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, T
• 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.
• FIGURE 2 shows cell-based complement-dependent cytotoxicity (CDC) activity of porcine wild type Fc subclass CTLA4 fusion proteins.
• CDC complement-dependent cytotoxicity
• SEQ ID NO.: 4 refers to the amino acid sequence of porcine IgG2b wildtype constant region.
• SEQ ID NO.: 5 refers to the amino acid sequence of porcine IgG3 wildtype constant region.
• SEQ ID NO.: 6 refers to the amino acid sequence of porcine IgG4a wildtype constant region.
• SEQ ID NO.: 7 refers to the amino acid sequence of porcine IgG4b wildtype constant region.
• SEQ ID NO.: 8 refers to the amino acid sequence of porcine IgG5a wildtype constant region.
• SEQ ID NO.: 9 refers to the amino acid sequence of porcine IgG5b wildtype constant region.
• 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.
• 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.
• effector cells refers to leukocytes (preferably porcine) which express one or more FcRs and perform effector functions.
• the cells express at least FcgR3 and perform ADCC effector function.
• 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).
• the leukocytes express FcgR1, or other relevant Fc gamma receptor, and trigger ADCP function.
• 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.
• 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.
• 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).
• amino acid residues i.e., encoded by the genetic code
• 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.
• 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).
• test 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).
• 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).
• 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.
• 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.
• 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.
• 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.
• antibody fragments refers to a portion of an intact antibody.
• a fragment of a polypeptide retains at least one function of the full-length polypeptide.
• 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.
• 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.
• 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.
• porcine IgG is IgG6a.
• the amino acid and nucleic acid sequences of IgGs are also well known in the art.
• IgG of the invention comprises a constant domain, for example, CH1, CH2, or CH3 domains, or a combination thereof.
• the constant domain of the invention comprises Fc region, including, for example, CH2 or CH3 domains or a combination thereof.
• the wild-type constant domain comprises any one of the amino acid sequences set forth in SEQ ID NOs.: 1-11.
• 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.
• 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.
• 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.
• the nucleic acid encodes both a heavy and light chain, or portions thereof.
• 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.
• position 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)).
• the desired effect is a higher affinity to FcRn, relative to an IgG having the wild-type porcine IgG constant domain.
• the desired effect is eliminating or reducing a complement-dependent cytotoxicity (CDC), relative to an IgG having the wild-type porcine IgG constant domain.
• CDC complement-dependent cytotoxicity
• 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.
• 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, T
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the mutant IgG constant domain of the invention comprises one or more mutations described herein.
• the antibody binding region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues.
• 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.
• hypervariable regions are also referred to as “complementarity determining regions” or “CDR” regions.
• CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure.
• 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.
• variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others.
• 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.
• 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.
• 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.
• variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the antibodies or peptides.
• 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.
• 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.
• 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).
• polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids.
• 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.
• 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.
• 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.
• derivatization with bifunctional agents is useful for cross-linking the antibody or fragment to a water-insoluble support matrix or to other macromolecular carriers.
• Derivatives also include radioactively labeled monoclonal antibodies that are labeled.
• radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, tritium (H 3 ) 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
• bioluminescent agents such as luciferase
• chemoluminescent agents such as acridine esters
• fluorescent agents such as phycobiliproteins
• 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.
• 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.
• the antibody is secreted into the supernatant of the media in which the cell is growing.
• 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.
• 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.
• 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.
• 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
• the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized.
• cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells.
• 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.
• 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.
• the fused genes encoding the H and L chains can be assembled on the same expression vector.
• 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.
• 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.
• biochemical means such 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.
• DEAE diethylaminoethyl
• 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.
• 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.
• the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized.
• PGK phosphoglycerate kinase
• 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.
• 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 include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells.
• 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.
• stable expression may be used.
• cell lines which stably express the antibody molecule may be engineered.
• host cells can be transformed with immunoglobulin expression cassettes and a selectable marker.
• 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.
• an antibody of the invention 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.
• 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.
• the invention provides for a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody or peptide according to the invention.
• “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.
• “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.
• 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.
• 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 chlor
• 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.
• 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.
• 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.
• respirable particles of a pharmaceutical formulation including both solid and liquid particles
• Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.
• the antibodies are administered by parenteral injection.
• antibodies or molecules can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating veterinarian.
• the pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the subject.
• Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
• 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.
• compositions of the invention can be used, for example, in the treatment of various diseases and disorders in porcine.
• 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.
• 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.
• ORF open reading frame
• 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.
• Porcine IgG used to evaluate the in vitro FcRn binding are provided in Table1. Table 1.
• Porcine IgG Subtypes and Allotypes ZP000432A [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.
• SPR surface plasmon resonance
• plasmid containing gene sequence encoding for an IgG kappa light chain was co- transfected with a plasmid encoding for IgG heavy chain.
• HEK293 expression equal amounts by weight of heavy chain plasmid and kappa chain plasmid were co-transfected.
• 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).
• 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.
• 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).
• 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.
• 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).
• 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.
• 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.
• BAP Biotin Acceptor Peptide
• 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.
• 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.
• 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.
• light chains were constructed via insertion of VL sequences upstream and in frame with the porcine kappa allele 1 constant region (NCBI AAA03520.1).
• 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.
• HEK293 expression 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.
• 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.
• 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.
• 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.
• 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.
• EC50 values range from 0.010 to 0.044 ⁇ g/mL.
• ZP000432A Table 4 CDC effects induced by porcine IgG Fc wildtype constructs.
• 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 Fc ⁇ RIII 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 Fc ⁇ RIII there is no resultant target cell death.
• CD80-expressing CHO 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
• 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.
• E:F Ratio effector:target cell ratio
• FIGS 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 ⁇ g/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.
• ADCC effects induced by porcine IgG Fc wildtype CTLA4 fusion proteins ADCP assay
• 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.
• FIG. 4 shows the results of cell-based antibody-dependent cell-mediated phagocytosis of porcine wild type Fc subclass CTLA4 fusion proteins.
• 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.
• 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.
• 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.
• Table 6A lists various constructs, their mutations, and their corresponding codon usage.
• Table 6B 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.
• FIG. 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.
• 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.
• 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.
• FIG. 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.
• FIG. 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.
• 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.
• FIG 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.
• Porcine IgG1a and 1b Fc CTLA4 fusion proteins both showed ADCC activity.
• SAP mutations significantly knock out ADCC function.
• ZP000432A ADCP assay [000227] Porcine IgG1a showed ADCP activity.
• 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.
• Molecular Operating Environment developed by Chemical Computing Group (MOE2019.0102) provides a flexible and automated graphical user interface for protein modeling.
• 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.
• the positions for the mutational library are represented in a ball-and-stick form in Fig.23 and Fig 24.
• 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 2.0 ⁇ or lower is considered the standard for considering two structures to be alike.
• Porcine IgG6a construct was identical to the porcine IgG6b allotype with an average RMSD value of 0.66 ⁇ (individual position RMSD in Table 11). Allotypes IgG1a, IgG1b and IgG2 also showed RMSDs of 0.2 and 0.78 ⁇ . 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.

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