Canine animal antibody variant

Abstract

The present invention relates generally to canine antibody variants and their uses. Specifically, the invention relates to mutations in the constant regions of canine antibodies to improve their half-life and other characteristics.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 011453, filed on 17 April 2020, which is incorporated herein by reference in its entirety.

[0002] This invention generally relates to canine antibody variants and their uses. Specifically, it relates to mutations in the Fc constant region of canine antibodies to improve half-life. [Background technology]

[0003] Canine IgG monoclonal antibodies (mAbs) are being developed as effective therapeutic agents in veterinary medicine. Several years ago, four canine IgG subclasses were identified and characterized (Bergeron et al., 2014, Vet Immunol Immunopathol., vol.157(1-2), pages 31-41). However, the extension of the half-life of canine IgG has not been extensively studied.

[0004] Neonatal Fc receptors (FcRn) extend the half-life of IgG through a recycling mechanism and pH-dependent interaction with their fragment crystallizable (Fc) domains. Specifically, the Fc domains extending across the interface of the CH2 and CH3 domains interact with FcRn on the cell surface to regulate IgG homeostasis. This interaction is favorable for acidic interactions after IgG pinocytosis, thus protecting IgG from degradation. Endocytized IgG is then recycled to the cell surface and released into the bloodstream at alkaline pH, thereby maintaining sufficient serum IgG for proper function. Therefore, the pharmacokinetic profile of IgG depends on the structural and functional properties of their Fc domains.

[0005] Three canine IgG subclasses have been compared to human IgG analogs for their binding to canine FcRn. The half-life of canine IgG needs to be well studied because without any experimental evidence, it is not possible to anticipate or predict whether they will align in a state similar to human IgG.

[0006] Prolonging the half-life of IgG can enable reduction in the dosing frequency of antibody drugs and / or a decrease in dosage, which in turn reduces veterinary visits, improves patient compliance, and decreases concentration-dependent cytotoxicity / adverse events.

[0007] Therefore, there is a need to identify mutations in the Fc constant region to improve the half-life.

Summary of the Invention

[0008] The present invention relates to a mutant canine IgG that provides higher FcRn affinity and a higher half-life compared to wild-type canine IgG. Specifically, the inventors of the present application have surprisingly and unexpectedly found that substituting the amino acid residue asparagine (Asn or N) at position 434 with another amino acid improved the affinity for FcRn, thereby increasing the half-life of IgG.

[0009] In one aspect, the present invention provides a modified IgG comprising a canine IgG constant domain with at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 434 numbered according to the EU index in Kabat. In an exemplary embodiment, the substitution is the substitution of asparagine with histidine at position 434.

[0010] In another aspect, the present invention provides a polypeptide comprising a canine IgG constant domain that contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 434 numbered according to the EU index in Kabat.

[0011] In yet another aspect, the present invention provides an antibody or molecule comprising a canine IgG constant domain that contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 434 numbered according to the EU index in Kabat.

[0012] In a further aspect, the present invention provides a method for generating or manufacturing an antibody or molecule, the method comprising providing a vector or host cell having an antibody comprising a canine IgG constant domain, wherein the canine IgG constant domain contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, and the substitution is at amino acid residue 434 numbered according to the EU index in Kabat.

[0013] In another aspect, the present invention provides a method for increasing the antibody serum half-life in dogs, the method comprising administering to the dog a therapeutically effective amount of an antibody comprising a canine IgG constant domain, wherein the canine IgG constant domain contains at least one amino acid substitution compared to the wild-type canine IgG constant domain, and the substitution is at amino acid residue 434 numbered according to the EU index in Kabat. In an exemplary embodiment, the antibody increases the half-life by about 30 days.

[0014] In another embodiment, the present invention provides a method for maintaining therapeutic serum levels of an antibody in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody comprising a canine IgG constant domain, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to a wild-type canine IgG constant domain, the substitution being located at amino acid residue 434, numbered according to the EU index in Kabat. In an exemplary embodiment, the antibody maintains therapeutic serum levels of the antibody in the dog over a period ranging from about one month to about seven months.

[0015] Other features and advantages of the present invention will become apparent from the examples and drawings in the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific embodiments are given only as examples, while illustrating preferred embodiments of the present invention. [Brief explanation of the drawing]

[0016] This patent or application file includes at least one drawing in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Office upon request and payment of the necessary fees.

[0017] [Figure 1] The domain structure of IgG is shown. The Fc mutation N434H was created within the CH3 domain, and the affinity for FcRn was increased at pH 6, thereby increasing the half-life of IgG. [Figure 2A] The amino acid sequences of canid IgGB containing N434H and wild-type (WT) canid IgGB are shown. [Figure 2B-1]This shows the amino acid sequence alignments of wild-type (WT) human IgG1, WT canid 1gGA, WT canid IgGB, WT canid IgGC, and WT canid IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are shown in red, purple, blue, and green, respectively. [Figure 2B-2] This shows the amino acid sequence alignments of wild-type (WT) human IgG1, WT canid 1gGA, WT canid IgGB, WT canid IgGC, and WT canid IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are shown in red, purple, blue, and green, respectively. [Figure 2B-3] This shows the amino acid sequence alignments of wild-type (WT) human IgG1, WT canid 1gGA, WT canid IgGB, WT canid IgGC, and WT canid IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are shown in red, purple, blue, and green, respectively. [Figure 2B-4] This shows the amino acid sequence alignments of wild-type (WT) human IgG1, WT canid 1gGA, WT canid IgGB, WT canid IgGC, and WT canid IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are shown in red, purple, blue, and green, respectively. [Figure 2B-5] This shows the amino acid sequence alignments of wild-type (WT) human IgG1, WT canid 1gGA, WT canid IgGB, WT canid IgGC, and WT canid IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are shown in red, purple, blue, and green, respectively. [Figure 2B-6]This shows the amino acid sequence alignments of wild-type (WT) human IgG1, WT canid 1gGA, WT canid IgGB, WT canid IgGC, and WT canid IgGD. Amino acid residues are numbered according to the Eu index in Kabat. CH1, hinge, CH2, and CH3 amino acid residues are shown in red, purple, blue, and green, respectively. [Figure 2C] The Fc nucleotide sequence of WT IgGB 65 is shown. [Figure 3] The individual serum concentrations of WT mAb1 IgG in four dogs (two males, 01M and 02M) and two females (03F and 04F) after a single 2 mg / kg injection, measured over a 56-day period, are shown. [Figure 4] The individual serum concentrations of N434H mAb1 IgG in four dogs (two males, 17M and 18M) and two females (19F and 20F) after a single 2 mg / kg injection, measured over a 56-day period, are shown. [Figure 5] Individual serum concentrations of WT mAb2 IgG in eight dogs (four males: H03433, H03434, H03435, H03436) and four females (H03453, H03454, H03455, H03456) after three injections of 2 mg / kg (SC / SC / IV) over a 98-day period are shown. [Figure 6] Individual serum concentrations of N434H mAb2 IgG are shown in eight dogs (four males: H03433, H03434, H03435, H03436) and four females (H03453, H03454, H03455, and H03456) after three injections of 2 mg / kg (SC / SC / IV) over a 98-day period. [Figure 7] This shows the serum profile of ZTS-00008183 in dogs after a single subcutaneous administration of 4 mg / kg. Colors represent different animal identification numbers. [Figure 8] The mean serum profile of ZTS-00008183 in dogs after a single subcutaneous administration of 4 mg / kg is shown. [Figure 9]Plot of least squares mean values ​​for treatment at different time points (3-5 months). Alpha levels: Day 84 = 0.07085, Day 112 = 0.04575, Day 140 = 0.04352. [Figure 10] Plot of least squares mean and percentage change due to treatment at time points (3-5 months). Change % in mean = 100 × [mean(T01) - mean(T02) / mean(T01)]. [Figure 11] Box plot of pruritus scores at all time points. T01 = placebo 0 mg / kg, T02 = ZTS-00008183 4 mg / kg. [Figure 12] Plot of pruritus scores by arithmetic means based on treatment at all time points. Error bars represent the standard error. [Figure 13] Plot of pruritus score as an arithmetic mean and percentage change due to treatment at all time points. Change % in mean = 100 × [mean(T01) - mean(T0X) / mean(T01)], X = 2, 3.

[0018] A brief explanation of sequence listings Sequence ID 1 is the amino acid sequence of the constant domain of the IgGB of a mutant canid with the N434H mutation. Sequence ID 2 is the amino acid sequence of the constant domain of wild-type canid IgGB. Sequence ID 3 is the nucleic acid sequence for wild-type canid IgG constant domain codon optimization (IgGB_65_WT), Sequence ID 4 is the nucleic acid sequence of the constant domain of wild-type canid IgGB. Sequence ID 5 is the amino acid sequence at positions 118-215 of the IgGB CH1 domain. Sequence ID 6 is the amino acid sequence at positions 217-230 of the IgGB hinge domain. Sequence ID 7 is the amino acid sequence of the wild-type IgGB CH2 domain from positions 231 to 340. Sequence ID 8 is the amino acid sequence at positions 341-447 of the wild-type IgGB CH3 domain. Sequence ID 9 is the nucleic acid sequence of the IgGB CH1 domain. Sequence ID 10 is the nucleic acid sequence of the IgGB hinge domain, Sequence ID 11 is the nucleic acid sequence of the wild-type IgGB CH2 domain. Sequence ID 12 is the nucleic acid sequence of the wild-type IgGB CH3 domain. Sequence ID 13 is a variable heavy chain CDR1 of an anti-IL31 antibody referred to herein as 11E12-VH-CDR1, Sequence ID No. 14 is a variable heavy chain CDR1 of an anti-IL31 antibody referred to herein as 34D03-VH-CDR1, Sequence ID 15 is a variable heavy chain CDR2 of an anti-IL31 antibody referred to herein as 11E12-VH-CDR2, Sequence ID 16 is a variable heavy chain CDR2 of an anti-IL31 antibody referred to herein as 34D03-VH-CDR2, Sequence ID 17 is a variable heavy chain CDR3 of an anti-IL31 antibody referred to herein as 11E12-VH-CDR3. Sequence ID No. 18 is a variable heavy chain CDR3 of an anti-IL31 antibody referred to herein as 34D03-VH-CDR3, Sequence ID 19 is a variable light chain CDR1 of an anti-IL31 antibody referred to herein as 11E12-VL-CDR1, Sequence ID No. 20 is a variable light chain CDR1 of an anti-IL31 antibody referred to herein as 34D03-VL-CDR1, Sequence ID 21 is a variable light chain CDR2 of an anti-IL31 antibody referred to herein as 11E12-VL-CDR2, Sequence ID No. 22 is a variable light chain CDR2 of an anti-IL31 antibody referred to herein as 34D03-VL-CDR2, Sequence ID 23 is a variable light chain CDR3 of an anti-IL31 antibody referred to herein as 11E12-VL-CDR3, Sequence ID No. 24 is a variable light chain CDR3 of an anti-IL31 antibody referred to herein as 34D03-VL-CDR3. Sequence ID 25 is a variable light chain sequence of the anti-IL31 antibody referred to herein as MU-11E12-VL. Sequence ID 26 is a variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VL-cUn-FW2, Sequence ID 27 is a variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VL-cUn-13, Sequence ID 28 is the variable light chain sequence of the anti-IL31 antibody referred to herein as MU-34D03-VL. Sequence ID 29 is a variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-34D03-VL-998-1, Sequence ID 30 is the variable heavy chain sequence of the anti-IL31 antibody referred to herein as MU-11E12-VH, Sequence ID 31 is a variable heavy chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VH-415-1, Sequence ID 32 is the variable heavy chain sequence of the anti-IL31 antibody referred to herein as MU-34D03-VH, Sequence ID 33 is the variable heavy chain sequence of the anti-IL31 antibody referred to herein as CAN-34D03-VH-568-1, Sequence ID 34 is the amino acid sequence corresponding to GenBank accession number C7G0W1, and corresponds to the full-length IL-31 protein of canids. Sequence ID 35 is a nucleotide sequence corresponding to GenBank accession number C7G0W1, and corresponds to the nucleotide sequence encoding the canine IL-31 full-length protein. Sequence ID 36 is a nucleotide sequence encoding the variable light chain sequence of the anti-IL31 antibody referred to herein as MU-11E12-VL. Sequence ID 37 is a nucleotide sequence encoding the variable heavy chain sequence of the anti-IL31 antibody referred to herein as MU-11E12-VH. Sequence ID 38 is a nucleotide sequence encoding the variable light chain sequence of the anti-IL31 antibody referred to herein as MU-34D03-VL. Sequence ID 39 is a nucleotide sequence encoding the variable heavy chain sequence of the anti-IL31 antibody referred to herein as MU-34D03-VH, Sequence ID 40 is the amino acid sequence of the heavy chain constant region of wild-type canids, referred to herein as HC-64 (GenBank accession number AF354264). Sequence ID 41 is a nucleotide sequence (GenBank accession number AF354264) that encodes the canid wild-type heavy chain constant region referred to herein as HC-64. Sequence ID 42 is the amino acid sequence of the canid wild-type heavy chain constant region referred to herein as HC-65 (GenBank accession number AF354265), Sequence ID 43 is a nucleotide sequence (GenBank accession number AF354265) that encodes the canid wild-type heavy chain constant region referred to herein as HC-65. Sequence ID 44 is the amino acid sequence of the constant region of the light chain of a canid animal referred to herein as kappa (GenBank accession number XP_532962), Sequence ID 45 is a nucleotide sequence (GenBank accession number XP_532962) that codes for the constant region of the light chain of a canid animal known as kappa. Sequence ID 46 is a nucleotide sequence encoding the variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-34D03-VL-998-1, Sequence ID 47 is a nucleotide sequence encoding the variable heavy chain sequence of the anti-IL31 antibody referred to herein as CAN-34D03-VH-568-1, Sequence ID 48 is a nucleotide sequence encoding the variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VL-cUn-FW2, Sequence ID 49 is a nucleotide sequence encoding the variable heavy chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VH-415-1, Sequence ID 50 is a nucleotide sequence encoding the variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VL-cUn-13. Sequence ID 51 is a variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12_VL_cUn_1, Sequence ID 52 is a nucleotide sequence encoding the variable light chain sequence of the anti-IL31 antibody referred to herein as CAN-11E12-VL-cUn-1, Sequence ID 53 corresponds to the amino acid sequence of the canine IL-31 full-length construct for E. coli expression. Sequence ID 54 is a nucleotide sequence corresponding to the canine IL-31 full-length construct for E. coli expression. Sequence ID 55 is a nucleotide sequence encoding the variable heavy chain sequence of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 56 is an amino acid sequence that encodes the variable heavy chain sequence of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 57 is a variable heavy chain CDR1 of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 58 is a variable heavy chain CDR2 of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 59 is a variable heavy chain CDR3 of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 60 is a nucleotide sequence encoding the variable light chain sequence of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 61 is an amino acid sequence that encodes the variable light chain sequence of the anti-NGF antibody referred to herein as ZTS-841. Sequence ID 62 is a variable light chain CDR1 of the anti-NGF antibody referred to herein as ZTS-841, Sequence ID 63 is a variable light chain CDR2 of an anti-NGF antibody referred to herein as ZTS-841. Sequence ID 64 is a variable light chain CDR3 of an anti-NGF antibody referred to herein as ZTS-841. Sequence ID 65 is the amino acid sequence of the mutant CH3 domain at positions 341-447 of IgGB. Sequence ID 66 is the amino acid sequence of a variant region within the CH3 domain of IgGB. Sequence ID 67 is the nucleic acid sequence of the light chain of the anti-NGF antibody referred to herein as ZTS-00008183. Sequence ID 68 is the amino acid sequence of the light chain of the anti-NGF antibody referred to herein as ZTS-00008183. Sequence ID 69 is the nucleic acid sequence of the heavy chain of the anti-NGF antibody referred to herein as ZTS-00008183. Sequence ID 70 is the amino acid sequence of the heavy chain of the anti-NGF antibody referred to herein as ZTS-00008183. [Modes for carrying out the invention]

[0019] The subject matter of the present invention can be more readily understood by referring to the following detailed description, which forms part of this disclosure. It should be understood that the present invention is not limited to any specific products, methods, conditions, or parameters described and / or shown herein, and that the terms used herein are for illustrative purposes only to illustrate specific embodiments and are not intended to limit the claimed invention.

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms.

[0021] Where used above and throughout this disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise indicated.

[0022] definition In this disclosure, the singular forms “a,” “an,” and “the” include plural references, and references to specific numerical values ​​include at least that specific value unless the context clearly indicates otherwise. Thus, for example, a reference to “molecule” or “compound” refers to one or more such molecules or compounds and their equivalents, etc., known to those skilled in the art. The term “plural” as used herein means more than one. Where a range of values ​​is expressed, another embodiment includes one specific value and / or other specific values. Similarly, where a value is expressed as an approximation, it is understood that by using the preceding “about,” a specific value forms another embodiment. All ranges are comprehensive and combinable.

[0023] In this specification and in the claims, the numbering of amino acid residues in immunoglobulin heavy chains is based on the Eu index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "Kabat's Eu index" refers to the residue numbering of IgG antibodies, which is reflected in Figure 2 in this specification.

[0024] The term "isolated," when used in reference to nucleic acids, refers to nucleic acids that are identified and separated from nucleic acids that are typically associated with at least one contaminant in their natural source. Isolated nucleic acids are in a different form or configuration than those found in nature. Therefore, isolated nucleic acid molecules are distinguished from nucleic acid molecules present in natural cells. Isolated nucleic acid molecules include nucleic acid molecules contained within cells that normally express the polypeptide they encode, and for example, the nucleic acid molecules are located in plasmid or chromosomal positions different from those in natural cells. Isolated nucleic acids can exist in single-stranded or double-stranded form. When using isolated nucleic acid molecules to express proteins, oligonucleotides or polynucleotides contain minimal sense or coding strands, but may contain both sense and antisense strands (i.e., they can be double-stranded).

[0025] A nucleic acid molecule is "operably linked" or "operably bound" when it is positioned in a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to the coding sequence of a nucleic acid when it affects the transcription of the sequence, or a ribosome binding site is operably linked to the coding sequence of a nucleic acid when it is positioned to facilitate translation. If the fusion protein to be expressed is positioned to contain a heterologous protein or a functional fragment thereof adjacent either upstream or downstream of the variant Fc region polypeptide, the nucleic acid molecule encoding the variant Fc region is operably linked to the nucleic acid molecule encoding the heterologous protein (i.e., a protein or a functional fragment thereof that does not contain the Fc region as it would naturally exist), and the heterologous protein may be adjacent to the variant Fc region polypeptide in the immediate vicinity or separated from it by a linker sequence of any length and composition. Similarly, a polypeptide molecule (used herein synonymously with "protein") is "operably linked" or "operably bound" when it is positioned in a functional relationship with another polypeptide.

[0026] As used herein, the term “functional fragment” refers, when referring to a polypeptide or protein (e.g., a variant Fc region or a monoclonal antibody), to a fragment of that protein that retains at least one function of the full-length polypeptide. The fragment may be in size ranging from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. The functional fragments of the variant Fc region polypeptide of the present invention retain at least one “amino acid substitution” as defined herein. The functional fragments of the variant Fc region polypeptide retain at least one function known in the art to be associated with the Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, downregulation of cell surface receptors, or, for example, increasing the in vivo or in vitro half-life of the polypeptide to which it is operably bound).

[0027] The terms “purified” or “to purify” refer to the substantial removal of at least one contaminant from a sample. For example, antigen-specific antibodies can be purified by the 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 contaminant, non-immunoglobulin protein, and also by the removal of immunoglobulin proteins that do not bind to the same antigen. The removal of non-immunoglobulin proteins and / or immunoglobulins that do not bind to a particular antigen results in an increase in the percentage of antigen-specific immunoglobulins in the sample. In another example, polypeptides expressed in bacterial host cells (e.g., immunoglobulins) are purified by the complete or substantial removal of host cell proteins, thereby increasing the percentage of polypeptides in the sample.

[0028] The term “natural” is used herein to indicate that a polypeptide (e.g., an Fc region) has an amino acid sequence that is naturally occurring and commonly found, or an amino acid sequence consisting of a naturally occurring polymorphism thereof. Natural polypeptides (e.g., natural Fc regions) can be produced by recombinant means or isolated from natural sources.

[0029] As used herein, the term “expression vector” refers to a recombinant DNA molecule containing a desired coding sequence and a suitable nucleic acid sequence necessary for the expression of an operablely linked coding sequence in a particular host organism.

[0030] As used herein, the term “host cell” means any eukaryotic or prokaryotic cell, whether arranged in vitro, in situ, or in vivo (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).

[0031] As used herein, the term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. While the generally accepted boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a canine IgG heavy chain is typically defined as extending, for example, from the amino acid residue at position 231 to its carboxyl terminus. In some embodiments, a variant may consist only of a portion of the Fc region, with or without the carboxyl terminus. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. In some embodiments, a variant having one or more of these constant domains is intended. In other embodiments, a variant without such constant domains (or having only a portion of such constant domains) is intended.

[0032] The "CH2 domain" in the Fc region of canid IgG typically extends for approximately 231 to 340 amino acids (see Figure 2B). The CH2 domain is unique in that it does not closely pair with other domains. Two N-linked branched carbohydrate chains interpose between the two CH2 domains of the natural IgG molecule.

[0033] The "CH3 domain" of the Fc region in canid IgG generally extends from the C-terminus of the Fc region to the CH2 domain, for example, from approximately 341 to 447 amino acid residues (see Figure 2B).

[0034] A "functional Fc region" has the "effector function" of a native sequence Fc region. At least one effector function of a polypeptide containing the variant Fc region of the present invention may be enhanced or reduced compared to a polypeptide containing a native Fc region or the parent Fc region of the variant. Examples of effector functions, but not limited to, include Clq binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions may require the Fc region to be operably linked to a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, target cell depletion from whole blood samples or fractionated blood samples).

[0035] The term "natural sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of a naturally occurring and commonly found Fc region. An example of a natural sequence canid Fc region is shown in Figure 2, which includes the natural sequence of the canid IgGB_65Fc region.

[0036] A “variant Fc region” contains an amino acid sequence that differs from that of the natural sequence Fc region (or a fragment thereof) due to at least one “amino acid substitution” as defined herein. In a preferred embodiment, the variant Fc region has at least one amino acid substitution, preferably one, two, three, four, or five amino acid substitutions, compared to the natural sequence Fc region or the Fc region of the parent polypeptide. In an alternative embodiment, the variant Fc region may be generated according to the methods disclosed herein, and this variant Fc region may be fused to an antibody variable domain or a non-antibody polypeptide, such as a selected heterologous polypeptide, including a receptor or ligand binding domain.

[0037] As used herein, the term “derivative” in the context of polypeptides refers to a polypeptide comprising an amino acid sequence modified by the introduction of amino acid residue substitutions. As used herein, the term “derivative” also refers to a polypeptide modified by the covalent bonding of any type of molecule to the polypeptide. For example, antibodies may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Derivative polypeptides may be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tunicamycin. Furthermore, derivative polypeptides have the same or identical function as the polypeptide from which they were derived. Polypeptides comprising the variant Fc region of the present invention may be derivatives as defined herein, and preferably, derivatization is understood to occur within the Fc region.

[0038] When used herein in relation to polypeptides (e.g., Fc regions or monoclonal antibodies), “substantially of canine origin” means that the polypeptide has an amino acid sequence that is at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, or even more preferably at least 95%, 95%, 97%, 98%, or 99% homologous to that of a natural canine aminopolypeptide.

[0039] The terms “Fc receptor” or “FcR” are used to describe receptors that bind to the Fc region (e.g., the Fc region of an antibody). Preferred FcRs are natural sequence FcRs. Furthermore, preferred FcRs include receptors of the Fc-gamma-RI, Fc-gamma-RII, and Fc-gamma-RIII subclasses, which bind to IgG antibodies (gamma receptors) and include allelic variants and, alternatively, spliced ​​forms of these receptors. Another preferred FcR is FcRn, the neonatal receptor responsible for the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)). Other FcRs, including those to be identified in the future, are encompassed herein by the term “FcR”.

[0040] The terms "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing FcR (e.g., nonspecific cells such as natural killer ("NK") cells, neutrophils, and macrophages) recognize antibodies bound to target cells, subsequently causing lysis of those target cells. NK cells, the primary cells mediating ADCC, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

[0041] As used herein, the term “effector cell” refers to a leukocyte (preferably from a canid) that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least Fc gamma RIII and performs ADCC effector function. Examples of leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources (e.g., blood or PBMCs).

[0042] A variant polypeptide with "modified" FcRn binding affinity, when measured at pH 6.0, will have either improved (i.e., increased, greater, or higher) or decreased (i.e., reduced, decreased, or lower) FcRn binding affinity compared to the variant's parent polypeptide or a polypeptide containing the natural Fc region. A variant polypeptide exhibiting increased binding to FcRn or increased binding affinity will bind to FcRn with higher affinity than the parent polypeptide. A variant polypeptide exhibiting decreased binding to FcRn or decreased binding affinity will bind to FcRn with lower affinity than its parent polypeptide. Such a variant exhibiting decreased binding to FcRn may have little to no binding to FcRn, for example, 0-20% binding to FcRn compared to the parent polypeptide. When the amounts of the variant polypeptide and parent polypeptide in the binding assay are essentially the same and all other conditions are identical, a variant polypeptide that binds to FcRn with "improved affinity" compared to its parent polypeptide will bind to FcRn with higher binding affinity than the parent polypeptide. For example, when FcRn binding affinity is determined by an ELISA assay or other method available to those skilled in the art, a variant polypeptide with improved FcRn binding affinity may show an increase of approximately 1.10 to 100 times (more typically, approximately 1.2 to 50 times) in FcRn binding affinity compared to the parent polypeptide.

[0043] As used herein, “amino acid substitution” means replacing at least one existing amino acid residue in a given amino acid sequence with another different “substitution” amino acid residue. The substitution residue or the substitution residues may be “naturally occurring amino acid residues” (i.e., encoded by the genetic code) and are selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile):leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Substitution with one or more non-naturally occurring amino acid residues is also included in the definition of amino acid substitution herein. "Naturally unexisting amino acid residues" refer to residues other than the naturally occurring amino acid residues listed above, and can be covalently bonded to adjacent amino acid residues within a polypeptide chain. Examples of naturally unexisting amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991).

[0044] The term “assay signal” refers to the output from any method of detecting protein-protein interactions, including but not limited to colorimetric assays, fluorescence intensity, or absorbance measurements from decay per minute. Assay formats may include ELISA, FACS, or other methods. Changes in the “assay signal” may reflect changes in cell viability, and / or changes in kinetic off-rate, kinetic on-rate, or both. A “higher assay signal” refers to a number of measured outputs greater than another (e.g., a variant may have a higher (greater) count in an ELISA assay compared to its parent polypeptide). A “lower” assay signal refers to a number of measured outputs less than another (e.g., a variant may have a lower (smaller) count in an ELISA assay compared to its parent polypeptide).

[0045] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. Binding affinity is measured in units of kinetic off-rate (generally the reciprocal of time, e.g., seconds). -1 It is directly related to the ratio obtained by dividing the reported (as reported) by the dynamic on-rate (generally reported in units of concentration per unit time, e.g., moles / second). Generally, unless each of these parameters is experimentally determined (e.g., by BIACORE or SAPIDYNE measurements), it is impossible to definitively state whether the change in the equilibrium dissociation constant is due to the difference in the on-rate, the off-rate, or both.

[0046] As used herein, the term “hinge region” refers to the range of amino acids extending to canine IgG, for example, from position 216 to 230 in canine IgG. Hinge regions of other IgG isotypes can be aligned with the IgG sequence by placing cysteine ​​residues that form interchain disulfide (SS) bonds at the same positions.

[0047] Clq is a polypeptide that contains a binding site for the Fc region of immunoglobulins. Clq, together with two serine proteases, Clr and Cls, forms the complex Cl, which is the first component of the CDC pathway.

[0048] As used herein, the term “antibody” is used synonymously with “immunoglobulin” or “Ig” and is used in its broadest sense to specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological or functional activity. Single-chain antibodies containing portions derived from different species, and chimeric, canid, or caninized antibodies, as well as single-chain antibodies transplanted with chimeric or CDRs, are also included in the present invention and the term “antibody.” These various portions of antibodies can be chemically and synthetically joined together by conventional techniques or prepared as a continuous protein using genetic engineering techniques. For example, nucleic acids encoding chimeric or caninized chains can be expressed to produce a continuous protein. For example, see U.S. Patent Nos. 4,816,567, 4,816,397, WO86 / 01533, 5,225,539, 5,585,089, and 5,698,762. For primate antibodies, see Newman, R. et al., BioTechnology, 10:1455-1460, 1993, and for single-chain antibodies, see Ladner et al., U.S. Patent No. 4,946,778 and Bird, R. et al., Science, 242:423-426, 1988. In this specification, all forms of antibodies containing an Fc region (or a portion thereof) are understood to be encompassed within the term “antibody.” Furthermore, antibodies can be labeled with detectable labels, immobilized on a solid phase, and / or conjugated with heterologous compounds (e.g., enzymes or toxins) according to methods known in the art.

[0049] As used herein, the term “antibody fragment” refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments; and multispecific antibodies formed from antibody fragments. The antibody fragment preferably retains at least a portion of the hinge and, optionally, the CH1 region of the IgG heavy chain. In other preferred embodiments, the antibody fragment includes at least a portion of the CH2 region or the entire CH2 region.

[0050] As used herein, the term “functional fragment” is intended to refer to a portion of a monoclonal antibody that still retains functional activity, when used in reference to a monoclonal antibody. Functional activity may be, for example, antigen-binding activity or specificity, receptor-binding activity or specificity, effector functional activity, etc. Examples of monoclonal antibody functional fragments include individual heavy or light chains such as VL, VH, and Fd, and their fragments; monovalent fragments such as Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single-chain Fv(scFv); and Fc fragments. Such terminology is described, for example, in Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989), Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.), Huston et al., Cell Biophysics, 22:189-224 (1993), Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The term functional fragment is intended to include, for example, fragments produced by protease digestion or reduction of monoclonal antibodies and by recombinant DNA methods known to those skilled in the art.

[0051] As used herein, the term “fragment” refers to a polypeptide comprising an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more consecutive amino acid residues from the amino acid sequence of another polypeptide. In preferred embodiments, the polypeptide fragment retains at least one function of the full-length polypeptide.

[0052] As used herein, the term “chimeric antibody” includes monovalent, bivalent, or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated with a chimeric light chain via a disulfide crosslink. A bivalent chimeric antibody is a tetramer formed by two heavy-light chain dimers associated with at least one disulfide crosslink. The chimeric heavy chain of an antibody for use in canids includes an antigen-binding region derived from a non-canine antibody heavy chain, which is linked to at least a portion of the constant region (CL) of the canine heavy chain. The chimeric light chain of an antibody for use in canids includes an antigen-binding region derived from a non-canine antibody light chain, which is linked to at least a portion of the constant region (CL) of the canine heavy chain. Antibodies, fragments, or derivatives having the same or different chimeric heavy and light chains of variable region binding specificity can also be prepared by appropriate association of individual polypeptide chains according to known method steps. Using this approach, a host expressing a chimeric light chain and a host expressing a chimeric heavy chain are cultured separately, the immunoglobulin chains are recovered separately, and then they are assembled. Alternatively, the hosts may be co-cultured, the chains may be spontaneously assembled in the culture medium, and then the assembled immunoglobulin or fragments may be recovered, or both the heavy and light chains may be expressed in the same host cells. Methods for generating chimeric antibodies are well known in the art (see, for example, U.S. Patents 6,284,471, 5,807,715, 4,816,567, and 4,816,397).

[0053] As used herein, a “canine” form of a non-canine (e.g., mouse) antibody (i.e., a canine antibody) is an antibody that contains minimal or no sequences derived from non-canine immunoglobulins. In most cases, a canine antibody is a canine immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-canine species (donor antibody), such as mouse, rat, rabbit, human, or non-human primate, having the desired specificity, affinity, and capability. In some cases, framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, a canine antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are generally made to further improve antibody performance. Generally, canine antibodies contain substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable loop (CDR) corresponding to that of a non-canine immunoglobulin, and all or substantially all of the FR residues being from a canine immunoglobulin sequence. Canine antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically that of a canine immunoglobulin.

[0054] As used herein, the term “immunoadhesin” refers to an antibody-like molecule that combines the binding domain of a heterogeneous “adhesin” protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an immunoadhesin amino acid sequence having desired binding specificity, which is other than the antigen recognition and binding site (antigen binding site) of an antibody (i.e., “heterogeneous”), and an immunoglobulin constant domain sequence.

[0055] As used herein, the term “ligand-binding domain” refers to any intrinsic receptor, or any region or derivative thereof, that retains at least the qualitative ligand-binding ability of the corresponding intrinsic receptor. In certain embodiments, the receptor is derived from a cell surface polypeptide having an extracellular domain homologous to a member of the immunoglobulin supergene family. Other receptors that are not members of the immunoglobulin supergene family but are nevertheless specifically covered by this definition are cytokine receptors, 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).

[0056] As used herein, the term “receptor-binding domain” refers to any natural ligand of a receptor, for example, a cell adhesion molecule, or any region or derivative of such a natural ligand that retains at least the qualitative receptor-binding ability of the corresponding natural ligand.

[0057] As used herein, “isolated” polypeptide is a lipeptide that has been identified and separated from its natural environment components and / or recovered. Contaminating components from its natural environment are substances that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the isolated polypeptide is purified to (1) more than 95% by weight, preferably more than 99% by weight, of the polypeptide by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequencer, or (3) to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining. Since the isolated polypeptide will not contain at least one component of the polypeptide’s natural environment, it will also contain the polypeptide in situ within recombinant cells. However, typically, the isolated polypeptide will be prepared by at least one purification step.

[0058] As used herein, the terms “disorder” and “disease” are used synonymously to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising the variant Fc region of the present invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder).

[0059] As used herein, the term "receptor" refers to a polypeptide capable of binding to at least one ligand. Preferred receptors are cell surface or soluble receptors having an extracellular ligand-binding domain and optionally other domains (e.g., transmembrane domains, intracellular domains, and / or membrane anchors). The receptors evaluated in the assays described herein can be the native 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). Further, the receptors whose binding properties are evaluated can be present intracellularly, or can be isolated and optionally coated onto an assay plate or some other solid phase, or can be directly labeled and used as a probe.

[0060] Wild-type IgG of Canidae Canidae IgG is well-known in the art and is fully described, for example, in Bergeron et al., 2014, Vet Immunol Immunopathol., vol. 157(1-2), pages 31-41. In one embodiment, Canidae IgG is IgG A In another embodiment, Canidae IgG is IgG B In yet another embodiment, Canidae IgG is IgG C In a further embodiment, Canidae IgG is IgG D In a still further embodiment, Canidae IgG is IgG B _65.

[0061] IgG A IgG B IgG C and IgG D The amino acid and nucleic acid sequences of are also well-known in the art.

[0062] In one example, the IgG of the present invention includes a constant domain, for example, a CH1, CH2, or CH3 domain, or a combination thereof. In another example, the constant domain of the present invention includes an Fc region, for example, a CH2 or CH3 domain, or a combination thereof.

[0063] In certain examples, the wild-type constant domain contains the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the wild-type IgG constant domain is a homolog, variant, isomer, or functional fragment of SEQ ID NO: 2, but without any mutation at position 434. Each possibility represents a distinct embodiment of the present invention.

[0064] The IgG contain domain also contains polypeptides having an amino acid sequence substantially similar to that of the heavy and / or light chain. A substantially identical amino acid sequence is defined herein as a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the compared amino acid sequence, as determined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988).

[0065] The present invention also includes nucleic acid molecules encoding IgG or a portion thereof as described herein. In one embodiment, the nucleic acid may encode an antibody heavy chain comprising, for example, a CH1, CH2, CH3 region, or a combination thereof. In another embodiment, the nucleic acid may encode an antibody heavy chain comprising, for example, one of the VH region or a portion thereof, or one of the VH CDRs, comprising any variant thereof. The present invention also includes nucleic acid molecules encoding an antibody light chain comprising, for example, one of the CL region or a portion thereof, one of the VL region or a portion thereof, or one of the VL CDRs, comprising any variant thereof. In certain embodiments, the nucleic acid encodes both the heavy chain and the light chain, or both portions thereof.

[0066] The amino acid sequence of the wild-type constant domain described in SEQ ID NO: 2 is encoded by the nucleic acid sequence described in SEQ ID NO: 4.

[0067] Modified canid IgG The inventors of this application have surprisingly and unexpectedly found that substituting the amino acid residue asparagine (Asn or N) at position 434 with another amino acid improved affinity for FcRn and increased the half-life of IgG. As used herein, the term position 434 refers to the position numbered according to the EU index in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0068] Accordingly, in one embodiment, the present invention provides a modified IgG domain comprising a canine IgG constant domain comprising at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is located at amino acid residue 434, numbered according to the EU index in Kabat. The asparagine at position 434 may be substituted with any other amino acid. For example, the asparagine at position 434 may be histidine (i.e., N434H), serine (i.e., N434S), alanine (i.e., N434A), phenylalanine (i.e., N434F), glycine (i.e., N434G), isoleucine (i.e., N434I), lysine (i.e., N434K), leucine (i.e., N434L), methionine (i.e., N434M), glutamine ( It can be substituted with N434Q, arginine (i.e., N434R), threonine (i.e., N434T), valine (i.e., N434V), tryptophan (i.e., N434W), tyrosine (i.e., N434Y), cysteine ​​(i.e., N434C), aspartic acid (i.e., N434D), glutamic acid (i.e., N434E), or proline (i.e., N434P). In certain embodiments, the substitution is a substitution with histidine (i.e., N434H).

[0069] In certain examples, the mutant constant domain of the present invention comprises the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the mutant IgG constant domain is a homolog, variant, isomer, or functional fragment of SEQ ID NO: 1, but with a mutation at position 434. Each possibility represents a distinct embodiment of the present invention.

[0070] The amino acid sequence of the mutant constant domain described in Sequence ID No. 1 is encoded by the variant form of the corresponding mutant nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 4.

[0071] In some embodiments, the mutant constant domain of the present invention comprises the amino acid sequence described in SEQ ID NO: 65 or 66. In some embodiments, the mutant IgG constant domain is a homolog, variant, isomer, or functional fragment of SEQ ID NO: 65 or 66, but having a mutation at position 434. Each possibility represents a distinct embodiment of the present invention.

[0072] The amino acid sequence of the mutant constant domain described in SEQ ID NO: 65 or 66 is encoded by its corresponding mutant nucleic acid sequence.

[0073] In one embodiment, the modified IgG of the present invention provides a half-life in the range of about 10 to about 35 days. In one embodiment, the modified IgG of the present invention provides a half-life of about 10, 12, 15, 17, 19, 20, 23, 26, 28, 30, 33, or 35 days. In a particular embodiment, the modified IgG of the present invention provides a half-life of more than 30 days.

[0074] In one embodiment, the modified IgG of the present invention maintains therapeutic serum levels for a period ranging from about 1 month to about 7 months. In one embodiment, the modified IgG of the present invention maintains therapeutic serum levels for about 7, 14, 28, 56, 84, 112, 140, 168, or 210 days. In a particular embodiment, the modified IgG of the present invention maintains therapeutic serum levels for more than 3 months.

[0075] Method for producing antibody molecules of the present invention Methods for producing antibody molecules are well known in the art and are 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 2006 / 0067930, which are incorporated herein by reference in their entirety. Any preferred method, process, or technique known to those skilled in the art may be used. Antibody molecules having the variant Fc region of the present invention can be produced according to methods well known in the art. In some embodiments, the variant Fc region can be fused to a selected heterologous polypeptide, such as the antibody variable domain or binding domain of a receptor or ligand.

[0076] With the advent of molecular biology methods and recombination techniques, those skilled in the art can generate antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences encoding specific amino acid sequences found in the polypeptide structure of antibodies. Such antibodies can be produced by either cloning the gene sequence encoding the polypeptide chain of the antibody, or by directly synthesizing the polypeptide chain and assembling the synthesized chain to form an active tetramer (H2L2) structure having affinity for a specific epitope and antigenic determinant. This has made it possible to immediately generate antibodies having sequences characterized by neutralizing antibodies from different species and sources.

[0077] Regardless of how they are recombinantly constructed or synthesized—whether from an antibody source, or using transgenic animals, which are large cell cultures of laboratory or commercial size, in vitro or in vivo, using transgenic plants, or by direct chemical synthesis without the use of living organisms at any stage of the process—all antibodies have a generally similar three-dimensional structure. This structure is often provided as H2L2, referring to the fact that antibodies generally contain two light-chain (L) amino acids and two heavy-chain (H) amino acids. Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting region is referred to as the "variable" or "V" region and is characterized by differences in the amino acid sequence from antibodies with different antigen specificities. The variable region of either the H or L chain contains an amino acid sequence capable of specifically binding to the antigen target.

[0078] As used herein, the term “antigen-binding region” refers to a portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer its specificity and affinity to the antigen to the antibody. The antibody-binding region contains “framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable region of the H or L chain that provides the antigen-binding region, there is a smaller sequence called “hypervariable,” which is the reason for the extreme variability between antibodies of different specificities. Such hypervariable regions are also called “complementarity-determining regions” or “CDR regions.” These CDR regions are responsible for the fundamental specificity of the antibody to a particular antigenic determinant structure.

[0079] CDRs represent discontinuous ranges of amino acids within the variable region, and it has been found that, regardless of species, the positions of these important amino acid sequences within the variable heavy and light chain regions are similar within the amino acid sequence of the variable chain. All antibody variable heavy and light chains each have three CDR regions, each discontinuous with the others. In all mammalian species, antibody peptides contain both constant (i.e., highly conserved) regions and variable regions, the latter of which include CDRs and so-called "framework regions" composed of amino acid sequences that are within the variable region of the heavy or light chain but outside the CDRs.

[0080] The present invention further provides vectors comprising at least one of the nucleic acids described above. Since the genetic code degrades, more than one codon may be used to code for a particular amino acid. Using the genetic code, one or more different nucleotide sequences may be identified, each of which may be capable of coding for an amino acid. The probability that a particular oligonucleotide will actually constitute an actual coding sequence can be estimated by considering unusual base pairing relationships in eukaryotic or prokaryotic cells expressing the antibody or portion, and the frequency with which a particular codon is actually used (to code for a particular amino acid). Such “codon usage rules” are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe’s “codon usage rules,” a single nucleotide sequence or set of nucleotide sequences containing the theoretically “most likely” nucleotide sequence capable of coding a canine IgG sequence can be identified. Furthermore, antibody coding regions for use in the present invention are also intended to be provided by modifying existing antibody genes using standard molecular biological techniques that produce 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 an antibody or peptide.

[0081] For example, one category of substitutions is conserved amino acid substitution. Such substitutions replace a given amino acid in a canine antibody peptide with another amino acid that has similar characteristics. Typical examples of conserved substitutions include the exchange of aliphatic amino acids Ala, Val, Leu, and lie; the exchange of hydroxyl residues Ser and Thr; the exchange of acidic residues Asp and Glu; the exchange of amide residues Asn and Gin; the exchange of basic residues Lys and Arg; and the exchange of aromatic residues Phe and Tyr. Guidance on which amino acid changes are likely to be phenotypic silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0082] Variant canine antibodies or peptides may be fully functional or lack function in one or more activities. Fully functional variants typically contain only conserved mutations or mutations in non-essential residues or regions. Functional variants may also contain similar amino acid substitutions that do not alter function or alter it only slightly. Alternatively, such substitutions may have a positive or negative effect to some extent. Non-functional variants typically contain one or more non-conserved amino acid substitutions, deletions, insertions, inversions, or cleavages, or substitutions, insertions, inversions, or deletions in essential residues or regions.

[0083] The amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces a single alanine mutation into all residues in the molecule. The resulting mutant molecule is then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites crucial for ligand-receptor binding can also be determined by structural analysis, such as 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).

[0084] Furthermore, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Moreover, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and other transfer-RNA mediated additions of amino acids to proteins, as well as ubiquitination. Such modifications are well known to those skilled in the art and are described in detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfated proteins, gamma-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues, are described in the most basic textbooks, for example, *Proteins - Structure and Molecular Properties* (2nd ed., TECreighton, WH Freeman & Co., NY, 1993). Numerous detailed reviews on this subject are available, including by Wold, *Posttranslational Covalent Modification of Proteins*, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. 182 *Meth. Enzymol.* 626-46 (1990), and Rattan et al. 663 *Ann. NY Acad. Sci.* 48-62 (1992).

[0085] In another aspect, the present invention provides antibody derivatives. “Derivatives” of antibodies typically contain additional chemical portions that are not part of the protein. Covalent modifications of proteins are within the scope of the present invention. Such modifications can be introduced into a molecule by reacting target amino acid residues of an antibody with an organic derivatizing agent that can react with selected side-chain or terminal residues. For example, derivatization using bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to a water-insoluble support matrix or other macromolecule carrier.

[0086] The derivatives also include radiolabeled monoclonal antibodies, such as radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, and tritium (H). 3 ) etc; conjugates of monoclonal antibodies containing biotin or avidin with enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters), or fluorescent agents (such as phycovir protein).

[0087] Another derivative bifunctional antibody of the present invention is a bispecific antibody produced by combining two separate antibody portions that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques. In addition, the portion can be added to the antibody or a portion thereof to increase the in vivo half-life (for example, by extending the time to clearance from the bloodstream). Such a technique is, for example, the addition of a PEG portion (also called pegylation), which is well known in the art. See U.S. Patent Application Publication 2003 / 0031671.

[0088] In some embodiments, the nucleic acid encoding the subject antibody is directly introduced into host cells, and the cells are incubated under conditions sufficient to induce the expression of the encoded antibody. After the subject nucleic acid is introduced into the cells, the cells are typically incubated for a period of about 1 to 24 hours, usually at 37°C, sometimes under selective conditions, to allow antibody expression. In one embodiment, the antibody is secreted into the supernatant of the culture medium in which the cells are growing. Conventionally, monoclonal antibodies have been produced as innate molecules in mouse hybridoma strains. In addition to that technique, the present invention provides recombinant DNA expression of antibodies. This enables the production of antibodies in selected host species, as well as the generation of spectra of antibody derivatives and fusion proteins.

[0089] Nucleic acid sequences encoding at least one antibody, moiety, or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques including blunt or protruding ends for ligation, restriction enzyme digestion to provide suitable ends, proper packing of sticky ends, alkaline phosphatase treatment to avoid undesirable binding, and ligation with a suitable ligase. Techniques for such operations are disclosed, for example, by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989) and Ausubel et al. 1993 (above), and these can be used to construct nucleic acid sequences encoding an antibody molecule or its antigen-binding region.

[0090] Nucleic acid molecules such as DNA contain nucleotide sequences that contain transcriptional and translational regulatory information, and if such sequences are "operably linked" to nucleotide sequences that encode polypeptides, then the polypeptide is said to be "expressible." An operable linkage is a linkage in which a regulatory DNA sequence and a DNA sequence to be expressed are connected in a manner that enables gene expression as a peptide or antibody portion in a recoverable amount. The exact properties of the regulatory region required for gene expression can vary from organism to organism, as is well known in similar techniques. See, for example, Sambrook et al., 2001 (above) and Ausubel et al., 1993 (above).

[0091] Accordingly, the present invention encompasses the expression of antibodies or peptides in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds, and mammalian cells, in vivo or in situ, or in host cells of mammalian, insect, bird, or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog, or cat origin. Any other suitable mammalian cells known in the art may also be used.

[0092] In one embodiment, the nucleotide sequence of the present invention would be incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a wide variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993 (above). Important factors in the selection of a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the desired number of copies of the vector in a particular host; and whether it is desirable that the vector can be "shuttle" between host cells of different species.

[0093] Examples of known prokaryotic vectors in the art include plasmids that can replicate within E. coli (e.g., pBR322, CoIE1, pSC101, pACYC184, .pi.vX, etc.). Such plasmids are disclosed, for example, by Maniatis et al., 1989 (above) and Ausubel et al., 1993 (above). Examples of Bacillus plasmids include pC194, pC221, and pT127. Such plasmids are disclosed by Gryczan in THE MOLEC.BIO.OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include Streptomyces bacteriophages such as p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)) and phLC31 (Chater et al., SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986)). Pseudomonas plasmids have been reviewed by John et al., 8 Rev. Infect. Dis. 693-704 (1986), lzaki, 33 Jpn. J. Bacteriol. 729-42 (1978), and Ausubel et al., 1993 (above).

[0094] Alternatively, useful gene expression elements for the expression of antibodies or peptides encoding cDNA 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 mouse leukemia virus LTR (Grosschedl et aI., 41 Cell 885 (1985)), (b) splice regions and polyadenylation sites derived from the late region of SV40 (Okayarea et aI., 1983), and (c) polyadenylation sites such as SV40 (Okayama et aI., 1983).

[0095] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., Gene 21 (1987), using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin intervention sequence, immunoglobulin, the rabbit S-globin polyadenylation site, and the SV40 polyadenylation element as expression elements. In immunoglobulin genes consisting of a cDNA portion and a genomic DNA portion (Whittle et al., Protein Engine 499 (1987)), the transcription promoter may be human cytomegalovirus, the promoter enhancer may be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation region may be native chromosomal immunoglobulin sequences.

[0096] In one embodiment, in the expression of a cDNA gene in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosome sequence corresponding to the synthesized immunoglobulin chain. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements listed above to achieve protein expression in mammalian cells.

[0097] Each fusion gene can be assembled into an expression vector or inserted into an expression vector. Recipient cells capable of expressing immunoglobulin chain gene products are then transfected with a single peptide or H or L chain coding gene, or co-transfected with both H and L chain genes. The transfected recipient cells are cultured under conditions that enable the expression of the incorporated genes, and the expressed immunoglobulin chains or the antibody or fragments are recovered from the culture.

[0098] In one embodiment, a peptide or a fusion gene encoding H and L chains, or portions thereof, is then assembled into a separate expression vector used for co-transfection of recipient cells. Alternatively, fusion genes encoding H and L chains may be assembled on the same expression vector. The recipient cell line for transfection of the expression vector and antibody production may be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin gene and have a mechanism for immunoglobulin glycosylation. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, where secreted immunoglobulins may be obtained from ascites fluid. Other suitable recipient cells include lymphocytes such as B lymphocytes of canid or non-canid origin, hybridoma cells of canid or non-canid origin, or interspecies heterohybridoma cells.

[0099] Expression vectors carrying antibody constructs or polypeptides of the present invention can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical means such as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[0100] Yeast can offer a substantial advantage over bacteria in the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies currently exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes a peptide (i.e., a pre-peptide) that carries the leader sequence. Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0101] Yeast gene expression systems can be routinely evaluated for the levels of production, secretion, and stability of peptides, antibodies, fragments, and their regions. One of a series of yeast gene expression systems incorporating promoters and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich media, may be utilized. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, promoter and terminator signals from the phosphoglycerate kinase (PGK) gene may be utilized. Several approaches can be selected to evaluate the optimal expression plasmid for the expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed., IRL Press, Oxford, UK 1985).

[0102] Bacterial strains may also be used as hosts for the production of antibody molecules or peptides described in this invention. Plasmid vectors containing replicons and control sequences derived from species compatible with the host cells are used in conjunction with these bacterial hosts. The vectors carry replication sites and specific genes capable of providing phenotypic selection in transformed cells. Several approaches may be selected for evaluating expression plasmids for the production of antibodies, fragments, and regions, or antibody chains encoded by cloned immunoglobulin cDNA in bacteria (see Glover, 1985 (above), Ausubel, 1993 (above), Sambrook, 2001 (above), Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001), Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001)).

[0103] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, hand L chain folding and assembly, antibody molecule glycosylation, and secretion of functional antibody proteins. Mammalian cells that may be useful as hosts for antibody protein production include, in addition to the lymphoid cells mentioned above, fibroblast-derived cells such as Vero (ATCC CRL81) or CHO-K1 (ATCC CRL61) cells. Many vector systems are available for the expression of cloned peptide hand L chain genes in mammalian cells (see Glover, 1985 (above)). Different approaches may be followed to obtain complete H2L2 antibodies. Co-expression of the hand L chain within the same cell is possible to achieve intracellular association and linkage of the hand L chain to the complete tetrameric H2L2 antibody and / or peptide. Co-expression can be performed using either the same or different plasmids within the same host. By placing the genes for both the H2L2 L chain and / or the peptide in the same plasmid and then transfecting cells with it, cells expressing both chains can be directly selected. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the L chain, and then the resulting cell line can be transfected with an H chain plasmid containing a second selectable marker. To generate cell lines with improved properties, such as higher production of assembled H2L2 antibody molecules or improved stability of the transfected cell line, cell lines that produce the peptide and / or H2L2 molecules via either pathway can be transfected with plasmids encoding additional copies of the peptide, H, L, or H-plus L chain, along with additional selectable markers.

[0104] Stable expression can be used for the long-term high-yield production of recombinant antibodies. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing viral replications, host cells may be transformed with immunoglobulin expression cassettes and selectable markers. Following the introduction of exogenous DNA, engineered cells may be grown in concentrated medium for 1-2 days, then switched to selective medium. Selectable markers in recombinant plasmids confer resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes and grow, thereby forming a focus that can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluating compounds / components that directly or indirectly interact with antibody molecules.

[0105] Once the antibody of the present invention is generated, it can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. In many embodiments, the antibody is secreted from cells into a culture medium and then collected from the culture medium.

[0106] In another embodiment, the present invention provides an antibody comprising a canine IgG constant domain comprising at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is located at amino acid residue 434. In one embodiment, the substitution is a substitution of asparagine with histidine at position 434 (N434H).

[0107] The antibody having substitution may be any suitable antibody known to those skilled in the art. In one example, the antibody is an anti-IL31 antibody. In another example, the antibody is an anti-NGF antibody.

[0108] Anti-IL31 antibodies without the substitutions described herein are well known in the art and are fully described, for example, in U.S. Patents 10,526,405, 10,421,807, 9,206,253, and 8,790,651. Similarly, anti-NGF antibodies without the substitutions described herein are well known in the art and are fully described, for example, in U.S. Patents 10,125,192, 10,093,725, 9,951,128, 9,617,334, and 9,505,829.

[0109] In one embodiment, the anti-IL31 antibody of the present invention (i.e., an antibody having a substitution) reduces, inhibits, or neutralizes IL-31-mediated pruritus or allergic conditions. In another embodiment, the anti-IL31 antibody of the present invention reduces, inhibits, or neutralizes IL-31 activity in dogs.

[0110] For example, the anti-IL-31 antibody of the present invention binds to IL-31 in the region of approximately 95 to 125 amino acids in the canine IL-31 amino acid sequence of SEQ ID NO: 44, preferably in the region of approximately 102 to 122 amino acids in the canine IL-31 sequence of SEQ ID NO: 44.

[0111] The VL, VH, and CDR sequences of anti-IL31 antibodies are well known in the art and are fully described, for example, in U.S. Patents 10,526,405, 10,421,807, 9,206,253, and 8,790,651. For example, the anti-IL31 antibody of the present invention may comprise at least one of the following combinations of complementary determination region (CDR) sequences: (1) 11E12: variable weight (VH)-CDR1 of SEQ ID NO: 13, VH-CDR2 of SEQ ID NO: 15, VH-CDR3 of SEQ ID NO: 17, variable light (VL)-CDR1 of SEQ ID NO: 19, VL-CDR2 of SEQ ID NO: 21, and VL-CDR3 of SEQ ID NO: 23; or (2) 34D03: VH-CDR1 of SEQ ID NO: 14, VH-CDR2 of SEQ ID NO: 16, VH-CDR3 of SEQ ID NO: 18, VL-CDR1 of SEQ ID NO: 20, VL-CDR2 of SEQ ID NO: 22, and VL-CDR3 of SEQ ID NO: 24. In some embodiments, the anti-IL31 antibody of the present invention may comprise at least one of the CDRs described herein.

[0112] In one embodiment, the anti-IL31 antibody of the present invention may include a variable light chain containing the amino acid sequence described in SEQ ID NO: 25 (MU-11E12-VL), SEQ ID NO: 26 (CAN-11E12-VL-cUn-FW2), SEQ ID NO: 27 (CAN-11E12-VL-cUn-13), SEQ ID NO: 28 (MU-34D03-VL), or SEQ ID NO: 29 (CAN-34D03-VL-998-1).

[0113] In another embodiment, the anti-IL31 antibody of the present invention may include a variable heavy chain comprising the amino acid sequence described in SEQ ID NO: 30 (MU-11E12-VH), SEQ ID NO: 31 (CAN-11E12-VH-415-1), SEQ ID NO: 32 (MU-34D03-VH), or SEQ ID NO: 33 (CAN-34D03-VH-568-1).

[0114] In one embodiment, the mutant anti-NGF antibody (i.e., an antibody with substitution) of the present invention had an enhanced ability to reduce, inhibit, or neutralize NGF activity in animals and / or inhibit NGF binding to Trk A and p75 in order to treat NGF-mediated pain or conditions.

[0115] The VL, VH, and CDR sequences of anti-NGF antibodies are also well known in the art and are fully described, for example, in U.S. Patents 10,125,192, 10,093,725, 9,951,128, 9,617,334, and 9,505,829. In one example, the anti-NGF antibody of the present invention may comprise at least one of the following complementary determination region (CDR) sequences: ZTS-841: variable weight (VH)-CDR1 of SEQ ID NO: 57, VH-CDR2 of SEQ ID NO: 58, VH-CDR3 of SEQ ID NO: 59, variable light (VL)-CDR1 of SEQ ID NO: 62, VL-CDR2 of SEQ ID NO: 63, and VL-CDR3 of SEQ ID NO: 64. In some embodiments, VL-CDR2 has the GNG residue of SEQ ID NO: 63.

[0116] In one embodiment, the anti-NGF antibody of the present invention may include a variable light chain having the amino acid sequence described in SEQ ID NO: 61 (CAN-ZTS-841-VL).

[0117] In another embodiment, the anti-NGF antibody of the present invention may include a variable heavy chain having the amino acid sequence described in SEQ ID NO: 56 (CAN-ZTS-841-VH). Pharmaceutical and veterinary uses

[0118] The present invention also provides a pharmaceutical composition comprising the molecule of the present invention and one or more pharmaceutically acceptable carriers. More specifically, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and an antibody or peptide according to the present invention as an active ingredient.

[0119] A "pharmaceutically acceptable carrier" includes any excipients that are non-toxic to cells or animals to which the drug is used at the dosage and concentration used. The pharmaceutical composition may include one or additional therapeutic agents.

[0120] "Pharmacologically acceptable" means a compound, material, composition, and / or dosage form that is suitable for contact with animal tissues within the bounds of sound medical judgment, without undue toxicity, irritation, allergic response, or complications of other problems commensurate with a reasonable benefit / risk ratio.

[0121] Pharmaceutically acceptable carriers include solvents, dispersions, buffers, coatings, antimicrobial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents, and absorption retarders.

[0122] Pharmaceutically acceptable carriers include water; physiological saline; phosphate-buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphates, citrates, and other organic acids; ascorbic acid; low molecular weight (less than approximately 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 dextrin; EDTA; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride; and combinations thereof.

[0123] The pharmaceutical compositions of the present invention can be formulated in a variety of ways, including liquid, semi-solid, or solid dosage forms such as liquid solutions (e.g., injectable and injectable solutions), dispersions, or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the composition is in the form of an injectable or injectable solution. The composition may be in a form suitable for intravenous, intra-arterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The composition may be formulated as an immediate-release, controlled-release, sustained-release, or delayed-release composition.

[0124] The compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. They may be administered alone, but generally, they are administered with a pharmaceutical carrier selected based on a chosen route of administration and standard pharmaceutical practice. The antibodies disclosed herein may be administered orally or to the airway surface by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), or by topical administration of the antibody (typically performed in a pharmaceutical formulation). Topical administration to the airway surface may be performed by intranasal administration (e.g., by using a dropper, cotton swab, or inhaler). Topical administration of antibodies to the airway surface may also be performed by inhalation, such as by creating breathable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibody as an aerosol suspension, and then having the subject inhale the breathable particles. Methods and apparatus for administering breathable particles of pharmaceutical formulations are well known, and any conventional techniques may be used.

[0125] In some preferred embodiments, antibodies are administered by parenteral injection. For parenteral administration, the antibody or molecule may be formulated as a solution, suspension, emulsion, or lyophilized powder associated with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or its cocktail dissolved in an acceptable carrier such as an aqueous carrier, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, or 0.3% glycine. Liposomes and non-aqueous vehicles such as non-volatile oils may also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pH adjusters and pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as buffers and toxicity modifiers, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The antibody concentration in these formulations can vary widely, for example, from less than about 0.5% by weight, typically from about 1% by weight or more to a maximum of 15% or 20% by weight, and will be selected according to the specific administration mode chosen, mainly based on fluid volume, viscosity, etc. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulations are sterilized by commonly used techniques. Practical methods for preparing parenterally administered compositions are known or obvious to those skilled in the art and are described in detail, for example, in REMINGTON'S PHARMA.SCI. (15th ed., Mack Pub.Co., Easton, Pa., 1980).

[0126] The antibodies or molecules of the present invention may be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution technique may be used. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of antibody activity loss, which may need to be compensated for by adjusting the usage level. Compositions containing these antibodies or cocktails thereof may be administered for the prevention of recurrence of pre-existing diseases and / or for therapeutic treatment. Suitable pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art. In therapeutic use, the composition is administered to a subject already suffering from the disease in an amount sufficient to cure, or at least partially halt or alleviate, the disease and its complications.

[0127] The effective dose of the compositions of the present invention for the treatment of the conditions or diseases described herein will vary depending on many different factors, including, but not limited to, the pharmacodynamic characteristics of a particular agent and its mode and route of administration; the target site; the physiological state of the animal; other pharmaceuticals administered; whether the treatment is prophylactic or therapeutic; the age, health, and weight of the recipient; the nature and severity of the type of symptoms; the type and frequency of concurrent treatments; and the desired effect.

[0128] The composition may be administered as a single or multiple doses at dose levels and in patterns selected by the treating veterinarian. In any case, the pharmaceutical formulation should provide a sufficient amount of the antibody of the present invention to effectively treat the subject. In exemplary embodiments, the composition of the present invention may be administered every other month, every three months, every four months, every five months, every six months, or every seven months.

[0129] The therapeutic dosage may be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0130] The pharmaceutical compositions of the present invention may contain a "therapeutic dose." The "therapeutic dose" refers to the effective amount in the dosage and duration required to achieve the desired therapeutic outcome. The molecular amount of the therapeutic dose may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the individual's ability to induce the desired response. The therapeutic dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the molecule.

[0131] In another embodiment, the compositions of the present invention may be used, for example, to treat a variety of diseases and disorders in dogs. As used herein, the terms “treat” and “cure” refer to a therapeutic action, including preventive or mitigating measures, in which the subject is prevented or slowed (mitigated) of undesirable physiological changes associated with the disease or condition. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in the severity of the disease or condition, stabilization of the disease or condition (i.e., no worsening of the disease or condition), delay or slowing of the progression of the disease or condition, improvement or mitigation of the disease or condition, and remission (partial or complete) of the disease or condition, whether detectable or undetectable. Subjects requiring treatment include subjects who already have the disease or condition, subjects prone to developing the disease or condition, or subjects in whom the disease or condition is to be prevented.

[0132] The mutant molecules of the present invention may be used to treat any suitable disease or disorder. For example, IL-31-mediated pruritus or allergic conditions may be treated using the mutant anti-IL31 antibody of the present invention. Examples of IL-31-mediated pruritus include, but are not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. Examples of IL-31-mediated allergic conditions include, but are not limited to, allergic dermatitis, summer eczema, urticaria, heaves, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity.

[0133] The mutant anti-NGF antibody of the present invention can be used to treat NGF-mediated pain or conditions. Examples of pain include, but are not limited to, chronic pain, inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, cancer pain, pain resulting from burns, wound-related pain, trauma-related pain, neuropathic pain, pain associated with musculoskeletal disorders, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatic) arthritis, non-rheumatoid arthritis, periarthritis, or peripheral neuropathy. In a particular embodiment, the pain is osteoarthritis pain.

[0134] All patents and references cited herein are incorporated herein by reference in their entirety.

[0135] 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 not be considered limiting to the subject matter described herein. The examples and embodiments described herein are for illustrative purposes only, and it should be understood that various modifications or changes in view thereof will be obvious to those skilled in the art, fall within the true scope of the invention, and can be made without departing from the true scope of the invention. [Examples]

[0136] Example 1 Construction of Canid IgG Fc mutants All canid IgGs (Figure 1) were constructed using plasmids containing sequences encoding the canid constant region of the IgGB(65) subclass, with the VH / VL sequences of each mAb investigated herein inserted upstream and within the frame, along with the nucleotides encoding the constant domain, as described by Bergeron et al. (Bergeron et al., 2014, Vet Immunol Immunopathol., vol.157(1-2), pages 31-41). For single-site targeted mutagenesis, mutations were incorporated at position N434 of the CH3 domain of each plasmid (Figure 2) using Agilent's QuikChange II Mutagenesis and related Agilent primer design tools (https: / / www.agilent.com / store / primerDesignProgram.jsp).

[0137] Antibody constructs were transiently expressed in HEK293 cells using either a standard lipofectamine transfection protocol (Invitrogen Life Technologies, Carlsbad, CA, USA) or in CHO cells using the ExpiCHO transient system (ThermoFisher Scientific) kit protocol. ExpiCHO expression followed the protocol outlined by ThermoFisher for co-transfection with plasmids containing gene sequences encoding IgG light and heavy chains. HEK293 expression involved co-transfection with equiweight heavy and light chain plasmids. Cells were grown for 7 days, after which the supernatant was collected for antibody purification. Antibodies were screened for binding to protein A or protein G sensors via Octet QKe quantification (Pall ForteBio Corp, Menlo Park, CA, USA). Constructs bound to protein A were purified, and protein quality was quantified as described by Bergeron et al.

[0138] Example 2 Target binding affinity and titer assay Affinity for each mAb was evaluated using Biacore, and IC50 was determined by a titer assay based on suitable cells. Surface plasmon resonance was performed using Biacore T200 (GE Healthcare, Pittsburgh, PA) to measure the binding affinity of each antibody to the target. Each target protein was immobilized at a final density of approximately 250 RU (resonance units) at 2.5 μg / ml using CM5 sensor flow cells 2-4 by amine coupling with EDC / NHS.

[0139] Flow cell 1 was used as an internal reference to correct for the effect of the electrophoresis buffer. Antibody binding was measured at a contact time of 250 seconds, a flow rate of 30 μl / min, and 15°C. The dissociation period was 300 seconds. Regeneration was performed for 60 seconds each with regeneration buffer (10 mM glycine pH 1.5 and 10 mM NaOH) and a flow rate of 20 μl / min. The electrophoresis / dilution buffer (1 × HBS-EP, GE Healthcare, 10 × BR-1006-69 containing 100 mM HEPES, 150 mM NaCl, 30 mM EDTA, and 0.5% v / v surfactant P20, pH 7.4, 1:10 in filtered MQ H2O) was used as a negative control in the same assay format.

[0140] Data were analyzed using Biacore T200 Evaluation software with a dual-reference method. The resulting curves were fitted to a 1:1 binding model. No difference in binding affinity or IC50 was observed between wild-type and N434H mutant IgG (Table 1). [Table 1]

[0141] Example 3 In vitro FcRn binding assay Following the Bergeron et al. study discussed above, canine FcRn was isolated and prepared, and mutant Fc IgG was assayed against canine FcRn. Standard PCR was used to amplify the canine FcRn-α subunit and β-microglobulin using degenerate primers designed from sequence alignments from cynomolgus monkeys, humans, mice, and rats. To facilitate subcloning into a pcDNA3.1(+) vector manipulated with a c-terminal 6×His+BAP tag (AGLNDIFEAQKIEWHE), the primers contained HindIII at the 3-prime and BamH1 at the 5-prime end. The FcRn-α subunit and β-microglobulin were co-transfected into HEK293 cells, and the FcRn complex was purified by IMAC affinity purification via the c-terminal His tag. KD levels were measured using a Biacore3000 or BiacoreT200 (GE Healthcare, Pittsburgh, PA, USA) with a CM5 sensor chip.

[0142] FcRn was immobilized on the sensor surface using a standard amine immobilization method to achieve the desired surface density. HBS-EP was used as the electrophoresis buffer, and 10 mM MES, 150 mM NaCl, 0.005% Tween 20, 0.5 mg / mL BSA, pH 6 and pH 7.2, and PBS, 0.005% Tween 20, 0.5 mg / mL BSA, pH 7.4 were used as the electrophoresis buffer and titration method. Fc mutant IgG was flowed onto the receptor surface, and affinity was determined by Scrubber 2 software analysis (BioLogic Software Pty, Ltd., Campbell, Australia) or T200 evaluation software (Table 2). Blank experiments containing only buffer were subtracted from all experiments. Flow cells were regenerated using 50 mM Tris pH 8. Experiments were performed at 15°C.

[0143] Mutations at position 434 of mAb1 and 2 have a significant effect on the affinity of IgG to FcRn at pH 6. The mutant N434H exhibits a significant increase in FcRn affinity at pH 6 while maintaining the weak affinity at pH 7.2 for all three mAbs. Extensive mutagenesis at position 434 reveals that several other mutations also exhibit increased affinity for FcRn at pH 6. This study demonstrates that the increase in FcRn affinity for IgG is independent of the VHVL domain and is universal across all canid IgGB(65). [Table 2]

[0144] Example 4 Fc variant IgG PK study in dogs The objective of this study was to determine the pharmacokinetics of two IgG monoclonal antibodies (mAb1 and mAb2) in dogs, which were elevated against two distinct targets using the Fc mutant N434H incorporated into each IgG.

[0145] mAb1 WT and N434H mutant IgG were administered intravenously as a single dose of 2 mg / kg to a group of four male beagle dogs. mAb2 WT and mutant IgG were administered three times at 28-day intervals to a group of four male and four female beagle dogs, with one of the IgGs administered at a dose of 2 mg / kg. The first two doses were administered subcutaneously, and the final dose was administered intravenously. "Free" IgG in canine serum was assayed using validated ligand-binding assays specific to each IgG, automated on the Gyrolab xP® platform (Figures 3-6). Pharmacokinetic calculations were performed using a non-compartmental approach (linear trapezoidal method for AUC calculation) with Watson® (Table 3). For mAb2 IgG, half-lives were estimated for the first and second doses using time points 7–28 days after administration. The half-life of the final dose was estimated using time points 7–42 days after administration. Additional calculations, including AUC correction for overlapping concentration-time profiles after the second and third injections of the drug, were performed in Excel®. Summarizations of concentration-time and pharmacokinetic data, including simple statistics (mean, standard deviation, coefficient of variation), were calculated using Excel® or Watson®. No other statistical analyses were performed. [Table 3]

[0146] The canid IgGB(65) point mutation N434H has been shown to increase the half-lives of two different canid IgGs in beagle dogs. The half-life of mAb1 increased from 9.7+ / -2.6 days to 17.1+ / -5.1 days, and the half-life of Ma2b2 increased from 9.2+ / -1.7 days to 19.3+ / -3.1 days. The mechanism of action is via improved affinity for canid FcRn at pH 6, which has been demonstrated in three canine IgGs that bind to very different and distinct soluble targets. Thus, the half-life extension of the N434 mutation in IgGB(65) has been demonstrated to be independent of the VHVL domain.

[0147] Example 5 FcRn binding assay Following the Bergeron et al. study discussed above, canine FcRn was isolated and prepared, and mutant Fc IgG was assayed against canine FcRn. Standard PCR was used to amplify the canine FcRn-α subunit and β-microglobulin. HEK293 cells were co-transfected with FcRn-α subunit and β-microglobulin, and the FcRn complex was purified by IMAC affinity purification via a c-terminal His tag. The FcRn complex was biotin-labeled via the BirA enzyme biotinylation reaction. KD was measured using a SA sensor chip with Biacore T200 (GE Healthcare, Pittsburgh, PA, USA) or Biacore 8K (Cytiva, Marlborough, MA, USA).

[0148] FcRn was captured on the sensor surface using a modified SA capture method. Electrophoresis buffer and titration were used, with 10 mM MES, 150 mM NaCl, 0.005% Tween20, 0.5 mg / mL BSA, and pH 6 as the capture material. Alternatively, 1× HBS-P, 0.5 mg / mL BSA, and pH 7.4 were used for electrophoresis buffer and titration. Fc mutant IgG was flowed onto the receptor surface, and affinity was determined using T200 evaluation software or Biacore Insight Evaluation software. Blank experiments containing only buffer were subtracted from all experiments. Flow cells were regenerated using 50 mM Tris pH 8 or pH 9. Experiments were performed at 15°C.

[0149] The mutations created at each location have a significant effect on the affinity of IgG to FcRn at pH 6. Binding of wild-type (WT) and mutant IgG to canid FcRn was measured by surface plasmon resonance (Biacore).

[0150] Significant effects on affinity were observed in completely different and structurally distinct antibodies binding to different targets (i.e., anti-IL31 antibody and anti-NGF antibody), as well as in different versions of antibodies binding to the same target (i.e., different versions of anti-IL31 antibody and anti-NGF antibody) (Tables 1-4). Therefore, the increase in FcRn affinity for IgG is independent of the VHVL domain or CDR region. In addition, significant effects on affinity were observed in multiple IgG subclasses, albeit with slight variability. Overall, the results indicate that the increase in FcRn affinity for IgG is independent of canine IgG subclasses. [Table 4] [Table 5]

[0151] Example 6 Fc variant IgG PK study in dogs The objective of the study was to determine the efficacy of a dose of 4.0 mg / kg of ZTS-00008183 based on efficacy in a canine-induced pruritus model, where efficacy was measured by evaluating the reduction of itching over a maximum of 210 days after administration of the test product on day 0. As used herein, the term "ZTS-00008183" refers to an anti-IL31 antibody having the N434H mutation in its constant region. ZTS-00008183 has the variable regions of 34D03 as described herein (i.e., VL and VH, including CDR).

[0152] A Beagle dog (approximately 4 years old) was administered either ZTS-00008183 or placebo via a single SC injection. The treatment is summarized below. [Table 6]

[0153] Serum samples were collected before drug administration (up to day 7) and on days 7, 14, 28, 56, 84, 112, 140, 168, and 210 after drug administration.

[0154] Specifically, in the IL-31-induced pruritus challenge study, beagle dogs (n=8, approximately 4 years old on the day of administration) were treated with a single subcutaneous administration of 4 mg / kg of ZTS-00008183. Serum samples were collected before administration (-7 days) and at 7, 14, 28, 56, 84, 112, 140, 168, and 210 days after drug administration.

[0155] Test mAbs were quantified using a ligand-binding assay. Anti-drug antibodies (ADAs) were evaluated using a qualified ADA assay method.

[0156] Bioanalysis data was received from BioAgilytix Labs as an Excel® spreadsheet. The data was imported into Watson® v.7.4.1. Toxicology and pharmacokinetic parameters (C max , t max , AUC, AUCextrap, and t 1 / 2 The half-life of ZTS-00008183 in group T02 was estimated using a non-compartment approach with Watson® v.7.4.1. The half-life was estimated using time points from 56 to 210 days after administration. Immunogenicity was assessed.

[0157] Table 6 lists the serum concentrations of ZTS-00008183. [Table 7]

[0158] The mean pharmacokinetic parameters of ZTS-00008183 are shown in Table 7 below. [Table 8]

[0159] No treatment-induced immunogenicity was detected.

[0160] The serum profile of ZTS-00008183 is shown in Figure 7. The mean serum profile of ZTS-00008183 is shown in Figure 8.

[0161] In short, the results demonstrate that the constant domain of canine IgG with the N434H mutation provided a half-life of approximately 30 days. This is more than double the half-life of the wild type (i.e., a 200% increase) compared to 9.2–9.7 days (see Table 2).

[0162] Example 7 Long-term therapeutic effect of Fc-mutated IgG In a canine animal model of IL-31-induced pruritus, a single subcutaneous dose of 4 mg / kg of ZTS-00008183 was evaluated.

[0163] In a parallel design efficacy study, 24 healthy beagle dogs were randomized to receive either 4 mg / kg body weight of ZTS-00008183 or placebo using a randomized complete block design. The animals were divided into eight complete blocks of size 3 based on their past pruritus scores. [Table 9]

[0164] Each animal was administered an IL-31 challenge (2.5 μg / kg) to induce pruritus on day 7 of the study, and a baseline pruritus score was established. Additional IL-31 challenges were administered on days 7, 28, 56, 84, 112, 140, 168, and 210 of the study.

[0165] Animals were observed for itching behavior for 120 minutes after each challenge. Observations were conducted in 1-minute time slots, and the animals responded with "yes" to all itching activity within that time slot. The cumulative number of "yes" responses determined the itching score.

[0166] No adverse events were observed during this study, and all test items, control items, and challenge materials were administered according to the protocol.

[0167] The results demonstrate that in a canine animal model of IL-31-induced pruritus, a single SC dose of 4.0 mg / kg of ZTS-00008183 resulted in significantly lower least squares mean pruritus scores at 3, 4, and 5 months compared to controls (Tables 9, 11, and 13) (P<0.0001).

[0168] As shown in Figures 9-13, in a canine animal model of IL-31-induced pruritus, ZTS-0008183(T02), administered at 4 mg / kg, resulted in significantly lower total pruritus scores compared to controls at days 84, 112, 140, 168, and 210 of the study.

[0169] The results also demonstrate that ZTS-00008183 is therapeutically effective for 84, 112, 140, 168, and 210 days (i.e., approximately 7 months) based on the pruritus score.

[0170] The results further demonstrate that ZTS-00008183 has long-term therapeutic effects and can be administered once every 3, 4, 5, 6, or 7 months. [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0171] In short, the results demonstrate that the constant domain of canine IgG with the N434H mutation maintains therapeutic serum levels for approximately 210 days (i.e., 7 months). This is several times higher than the duration of therapeutic levels reported for wild-type anti-IL31 antibodies in previous studies.

[0172] While preferred embodiments of the present invention have been described, it should be understood that the present invention is not limited to specific embodiments and that various changes and modifications can be made by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims. Claims at the time of international filing [Section 1] Modified IgG comprising a canine IgG constant domain comprising at least one amino acid substitution compared to the wild-type canine IgG constant domain, wherein the substitution is located at amino acid residue 434, numbered according to the EU index in Kabat. [Section 2] The modified IgG according to claim 1, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 3] The modified IgG according to claim 1, wherein the modified IgG has an increased half-life compared to the half-life of the IgG having the wild-type canid IgG constant domain. [Section 4] The modified IgG according to claim 1, wherein the modified IgG has a higher affinity for FcRn than the IgG having the wild-type canid IgG constant domain. [Section 5] The modified IgG according to claim 1, wherein the modified IgG is canine IgG or caninized IgG. [Section 6] The aforementioned IgG, A IgG B IgG C , or IgG D The modified IgG according to claim 1. [Section 7] The aforementioned IgG constant domain, A IgG B IgG C , or IgG D The modified IgG according to claim 1, which is a constant domain. [Section 8] The modified IgG according to claim 1, wherein the IgG constant domain includes an Fc constant region having a CH3 domain. [Section 9] The modified IgG according to claim 1, wherein the IgG constant domain includes an Fc constant region having CH2 and CH3 domains. [Section 10] The modified IgG according to claim 1, wherein the IgG constant domain comprises the amino acid sequence described in SEQ ID NO: 1. [Section 11] A pharmaceutical composition comprising the modified IgG described in claim 1 and a pharmaceutically acceptable carrier. [Section 12] A kit comprising, in a container, the modified IgG described in claim 1, and instructions for use. [Section 13] A polypeptide comprising a canid IgG constant domain, wherein the substitution is located at amino acid residue 434, numbered according to the EU index in Kabat, compared to the wild-type canid IgG constant domain, the polypeptide comprising at least one amino acid substitution compared to the wild-type canid IgG constant domain, the polypeptide wherein the substitution is located at amino acid residue 434. [Section 14] The polypeptide according to claim 13, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 15] The polypeptide according to claim 13, wherein the polypeptide has an increased half-life compared to the half-life of the polypeptide of the wild-type canid IgG constant domain. [Section 16] The polypeptide according to claim 13, wherein the polypeptide has a higher affinity for FcRn than the polypeptide of IgG having the wild-type canid IgG constant domain. [Section 17] The polypeptide according to claim 13, wherein the polypeptide is a polypeptide of canid IgG or canine IgG. [Section 18] The aforementioned IgG, A IgG B IgG C , or IgG D The polypeptide according to claim 17. [Section 19] The aforementioned IgG constant domain, A IgG B IgG C , or IgG D The polypeptide according to claim 13, which is a constant domain. [Section 20] The polypeptide according to claim 13, wherein the IgG constant domain includes an Fc constant region having a CH3 domain. [Section 21] The polypeptide according to claim 13, wherein the IgG constant domain includes an Fc constant region having CH2 and CH3 domains. [Section 22] The polypeptide according to claim 13, wherein the IgG constant domain comprises the amino acid sequence described in SEQ ID NO: 1. [Section 23] A pharmaceutical composition comprising the polypeptide according to claim 13 and a pharmaceutically acceptable carrier. [Section 24] A kit comprising, in a container, the polypeptide according to claim 13 and instructions for use. [Section 25] An antibody comprising a canid IgG constant domain, wherein the constant domain contains at least one amino acid substitution compared to the wild-type canid IgG constant domain, the substitution being located at amino acid residue 434, numbered according to the EU index in Kabat. [Section 26] The antibody according to claim 25, wherein the substitution is a substitution of asparagine with histidine at position 434 (N434H). [Section 27] The antibody according to claim 25, wherein the antibody has an increased half-life compared to the half-life of the antibody having the wild-type canid IgG constant domain. [Section 28] The antibody according to claim 25, wherein the antibody has a higher affinity for FcRn than the antibody having the wild-type canid IgG constant domain. [Section 29] The antibody according to claim 25, wherein the antibody is a canine antibody or a canine antibody. [Section 30] The aforementioned antibody is IgG A IgG B IgG C , or IgG D The antibody according to claim 25. [Section 31] The aforementioned IgG constant domain, A IgG B IgG C , or IgG D The antibody according to claim 25, which is a constant domain. [Section 32] The antibody according to claim 25, wherein the IgG constant domain includes an Fc constant region having a CH3 domain. [Section 33] The antibody according to claim 25, wherein the IgG constant domain includes an Fc constant region having CH2 and CH3 domains. [Section 34] The antibody according to claim 25, wherein the IgG constant domain comprises the amino acid sequence described in SEQ ID NO: 1. [Section 35] A pharmaceutical composition comprising the antibody according to claim 25 and a pharmaceutically acceptable carrier. [Section 36] A kit comprising, in a container, the antibody described in claim 25 and an instruction sheet for use. [Section 37] A vector containing a nucleic acid sequence encoding the amino acid sequence described in SEQ ID NO: 1. [Section 38] Isolated cells comprising the vector according to claim 37. [Section 39] A method for producing an antibody or molecule, the method comprising providing the cells described in claim 38 and culturing the cells. [Section 40] A method for producing an antibody, wherein the method includes providing an antibody according to any one of claims 25 to 34. [Section 41] A method for increasing the serum half-life of an antibody in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody containing a canine IgG constant domain, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to a wild-type canine IgG constant domain, the substitution being located at amino acid residue 434, numbered according to the EU index in Kabat. [Section 42] The method according to claim 41, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 43] The method according to claim 41, wherein the antibody has an increased half-life compared to the half-life of the antibody having the wild-type canid IgG constant domain. [Section 44] The method according to claim 41, wherein the antibody has a higher affinity for FcRn than the antibody having the wild-type canid IgG constant domain. [Section 45] The method according to claim 41, wherein the antibody is a canine antibody or a canine antibody. [Section 46] The aforementioned antibody is IgG A IgG B IgG C , or IgG D The method according to claim 41. [Section 47] The aforementioned IgG constant domain, A IgG B IgG C , or IgG D The method according to claim 41, wherein the constant domain is... [Section 48] The method according to claim 41, wherein the IgG constant domain includes an Fc constant region having a CH3 domain. [Section 49] The method according to claim 41, wherein the IgG constant domain includes an Fc constant region having CH2 and CH3 domains. [Section 50] The method according to claim 41, wherein the IgG constant domain comprises the amino acid sequence described in SEQ ID NO: 1. [Section 51] A fusion molecule comprising a canine IgG constant domain fused to a drug, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to a wild-type canine IgG constant domain, and the substitution is located at amino acid residue 434, numbered according to the EU index in Kabat. [Section 52] The molecule according to claim 51, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 53] The molecule according to claim 51, wherein the molecule has an increased half-life compared to the half-life of the molecule having the wild-type canid IgG constant domain. [Section 54] The molecule according to claim 51, wherein the molecule has a higher affinity for FcRn than the molecule having the wild-type canid IgG constant domain. [Section 55] The molecule according to claim 51, wherein the molecule is a canine antibody or a canine antibody. [Section 56] The aforementioned molecule is IgG A IgG B IgG C , or IgG D The molecule according to claim 51, comprising: [Section 57] The aforementioned IgG constant domain, A IgG B IgG C , or IgG D The molecule according to claim 51, which is a constant domain. [Item 58] The molecule according to claim 51, wherein the IgG constant domain comprises an Fc constant region having a CH3 domain. [Item 59] The molecule according to claim 51, wherein the IgG constant domain comprises an Fc constant region having CH2 and CH3 domains. [Item 60] The molecule according to claim 51, wherein the IgG constant domain comprises the amino acid sequence set forth in SEQ ID NO: 1. [Item 61] A pharmaceutical composition comprising the molecule according to claim 51 and a pharmaceutically acceptable carrier. [Item 62] A kit comprising the molecule according to claim 51 and an instruction manual, within a container. [Item 63] A modified IgG comprising a canine IgG constant domain comprising the amino acid sequence set forth in SEQ ID NO: 1, 65, or 66, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having a wild-type canine IgG constant domain. [Item 64] The modified IgG according to claim 63, wherein the increased half-life is during a period ranging from about 25 days to about 35 days. [Item 65] The modified IgG according to claim 63, wherein the increased half-life is about 30 days. [Item 66] A modified IgG comprising a canine IgG constant domain comprising the amino acid sequence set forth in SEQ ID NO: 1, 65, or 66, wherein the modified IgG maintains its therapeutic level for a long period of time. [Item 67] The modified IgG according to claim 66, wherein the modified IgG maintains its therapeutic level over a period ranging from about 1 month to about 7 months. [Item 68] The modified IgG according to claim 66, wherein when the modified IgG is subcutaneously delivered to a canine subject, it maintains its therapeutic level over the period. [Section 69] An antibody comprising a canine IgG constant domain having the amino acid sequence described in SEQ ID NO: 1, 65, or 66, wherein the antibody has an increased half-life compared to an antibody having a wild-type canine IgG constant domain. [Section 70] The antibody according to claim 69, wherein the increased half-life is in the range of approximately 25 days to approximately 35 days. [Section 71] The antibody according to claim 69, wherein the increased half-life is approximately 30 days. [Section 72] An antibody comprising a canine IgG constant domain having an amino acid sequence described in SEQ ID NO: 1, 65, or 66, wherein the antibody maintains its therapeutic level for a long period of time. [Section 73] The antibody according to claim 66, wherein the antibody maintains its therapeutic level over a period of approximately one to seven months. [Section 74] The antibody according to claim 66, wherein when the antibody is delivered subcutaneously to a canid, it maintains its therapeutic level over the period of time. [Section 75] The antibody according to any one of claims 25 to 34 and 69 to 74, wherein the antibody is an anti-IL31 or anti-NGF antibody. [Section 76] A method for increasing the serum half-life of an antibody in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody containing a canine IgG constant domain, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to a wild-type canine IgG constant domain, the substitution being at amino acid residue 434 numbered according to the EU index in Kabat, and the antibody is an anti-IL31 antibody. [Section 77] The method according to claim 76, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 78] The method according to claim 76, wherein the method increases the half-life during a period ranging from about 25 days to about 35 days. [Section 79] The antibody according to claim 76, wherein the method increases the half-life of approximately 30 days. [Section 80] A method for maintaining therapeutic serum levels of an antibody in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody comprising a canine IgG constant domain, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to a wild-type canine IgG constant domain, the substitution being at amino acid residue 434 numbered according to the EU index in Kabat, and the antibody is an anti-IL31 antibody. [Section 81] The method according to claim 80, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 82] The method according to claim 80, wherein the method maintains the therapeutic serum level of the antibody in the dog over a period of about one month to about seven months. [Section 83] The method according to claim 80, wherein the method, when the antibody is delivered subcutaneously to the dog, maintains the therapeutic serum level over the period of time. [Section 84] A method for increasing the serum half-life of an antibody in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody containing a canine IgG constant domain, wherein the canine IgG constant domain comprises at least one amino acid substitution compared to a wild-type canine IgG constant domain, the substitution being at amino acid residue 434 numbered according to the EU index in Kabat, and the antibody is an anti-NGF antibody. [Section 85] The method according to claim 84, wherein the substitution is the substitution of asparagine with histidine at position 434 (N434H). [Section 86] The method according to claim 84, wherein the method increases the half-life during a period ranging from about 10 days to about 35 days. [Clause 87] The antibody according to claim 84, wherein the method increases the half-life of about 30 days. [Clause 88] A method of treating IL-31-mediated pruritus or allergic conditions in a canine subject, the method comprising administering to the subject a therapeutically effective amount of the anti-IL31 antibody according to claim 75, thereby treating the IL-31-mediated pruritus or allergic condition in the canine subject. [Clause 89] The method according to claim 88, wherein the IL-31-mediated pruritus or allergic condition is a pruritic condition selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. [Clause 90] The method according to claim 88, wherein the IL-31-mediated pruritus or allergic condition is an allergic condition selected from the group consisting of allergic dermatitis, summer eczema, urticaria, heaves, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity. [Clause 91] The method according to claim 88, wherein the antibody is administered once every two months, once every three months, once every four months, once every five months, once every six months, or once every seven months. [Clause 92] The method according to claim 88, wherein the antibody is administered subcutaneously at a dosage of less than 4.0 mg / kg body weight. [Clause 93] A method of treating pain in a canine subject, the method comprising administering to the subject a therapeutically effective amount of the anti-NGF antibody according to claim 75, thereby treating the pain in the canine subject. [Clause 94] The method according to claim 93, wherein the pain is chronic pain, inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, cancer pain, pain resulting from burns, wound-related pain, trauma-related pain, neuropathic pain, pain associated with musculoskeletal disorders, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatic) arthritis, non-rheumatoid arthritis, periarthritis, or peripheral neuropathy. [Section 95] The method according to claim 93, wherein the pain is osteoarthritis pain. [Section 96] The method according to claim 93, wherein the antibody is administered every other month, once every three months, once every four months, once every five months, once every six months, or once every seven months.

Claims

1. (i) a canine IgG constant domain comprising an amino acid substitution compared to a wild-type canine IgG constant domain, wherein the substitution is at amino acid residue 434 numbered according to the EU index in Kabat, and the substitution is a substitution of asparagine with histidine at position 434 (N434H); and (ii) A canid IgG variable domain capable of binding to interleukin-31 (IL-31), comprising a variable heavy chain having the amino acid sequence of SEQ ID NO: 68 and a variable light chain having the amino acid sequence of SEQ ID NO: 70, the variable domain A modified IgG comprising the above substitution, wherein the presence of the above substitution in the modified IgG increases the serum circulation period of the modified IgG in dogs by approximately 90 days. Modified IgG.

2. The modified IgG according to claim 1, wherein the modified IgG has an increased half-life compared to the half-life of IgG having the wild-type canid IgG constant domain.

3. The modified IgG according to claim 1, wherein the modified IgG has a higher affinity for FcRn than the IgG having the wild-type canid IgG constant domain.

4. The modified IgG according to claim 1, wherein the modified IgG is canid IgG or caninized IgG.

5. The modified IgG according to claim 1, wherein the IgG steady-state domain includes an Fc steady-state region having a CH2 or CH3 domain or a combination thereof.

6. The modified IgG according to claim 1, wherein the wild-type canid IgG constant domain comprises the amino acid sequence described in SEQ ID NO:

2.

7. A pharmaceutical composition comprising the modified IgG described in claim 1 and a pharmaceutically acceptable carrier.

8. A kit comprising, in a container, the modified IgG described in claim 1 and an instruction sheet for use.

9. A polypeptide comprising the modified IgG described in claim 1.

10. A fusion molecule comprising the modified IgG described in claim 1.

11. An immunoglobulin molecule comprising the modified IgG described in claim 1.

12. Use of an immunoglobulin molecule for the manufacture of a pharmaceutical product, wherein the immunoglobulin molecule is the molecule described in claim 11.

13. Use of an immunoglobulin molecule to increase the serum half-life of an immunoglobulin molecule in dogs, wherein the immunoglobulin molecule is the molecule described in claim 11.

14. The use according to claim 13, wherein the molecule increases its half-life over a period of about 25 to about 35 days.

15. The use according to claim 13, wherein the molecule increases its half-life over a period of approximately 30 days.

16. The immunoglobulin molecule according to claim 11, wherein the immunoglobulin is an anti-IL31 molecule.

Images