Bispecific canine antibodies for treating atopic dermatitis

Abstract

This invention provides a composition for treating canine atopic dermatitis. [Solution] A composition is provided comprising a canine antiproliferative antibody, a canine antipruritic antibody, and a canine anti-inflammatory antibody. Furthermore, a composition is provided comprising a bispecific antibody comprising a canine antipruritic antibody and a canine antiproliferative antibody, or a bispecific antibody comprising a canine antipruritic antibody and a canine anti-inflammatory antibody.

Description

[Technical Field]

[0001] Cross-reference of related applications This application was filed on October 15, 2020, under Section 119(e) of the United States Patent Act. U.S. Provisional Patent Application No. 63 / 092,294, filed on October 15, 2020, is a U.S. patent application filed on October 15, 2020. Patent Application No. 63 / 092,296, filed on April 24, 2020. U.S. Patent Application No. 63 / 015,209, filed on April 24, 2020. U.S. Patent Application No. 5,220, filed December 20, 2019, U.S. Patent Application No. 62 / 951,77 Patent No. 8, and U.S. Patent Application No. 62 / 951,793 filed on December 20, 2019. Priority is claimed in this document, and all of these contents are incorporated herein by reference in their entirety. It is possible.

[0002] The present invention comprises a canine antiproliferative antibody, a canine antipruritic antibody, and a canine anti-inflammatory antibody. The present invention relates to a composition for treating atopic dermatitis in dogs. The present invention further relates to a composition for treating atopic dermatitis in dogs. A bispecific antibody containing an antipruritic antibody and a canine antiproliferative antibody, or a canine antipruritic antibody and This invention relates to a composition containing a bispecific antibody, including a nu-type anti-inflammatory antibody. [Background technology]

[0003] The immune system works together to protect the host from infectious diseases and cancer. This includes a network of specialized cells that recirculate. The ability of the immune system to perform this function. The power is secreted by white blood cells and is produced by a group of proteins collectively called interleukins. It depends heavily on biological activity. Well-studied interleukins include interleukins. Ikin-4 (IL-4), Interleukin-31 (IL-31), and Interleuk There are three important molecules identified as IL-22: IL-4, IL-3. 1 and IL-22 are extracellular pathogens (e.g., parasites present in tissues or tubules). It is an important cytokine for generating the immune response necessary for protection against However, these cytokines are involved in allergies in humans and animals, including atopic dermatitis. It is also involved in the pathogenesis of gynecomastia.

[0004] Atopic dermatitis (AD) is a recurrent, itchy, and chronic inflammatory skin disease affecting humans. It is characterized by immune system dysregulation and epidermal barrier abnormalities. Pathology of atopic dermatitis The phenotypic and immunological characteristics were the subject of extensive investigation [Rahman et al., I nflammation&allergy-drug target 10:486-4 96 (2011) and Harskamp et al., Seminar in Cu Taneous Medicine and Surgery 32:132-139( It is outlined in 2013. Atopic dermatitis is also common in companion animals, especially dogs. It is a common symptom, and its prevalence is estimated to be about 10-15% of the canine population. Pathophysiology of atopic dermatitis in dogs and cats [Nuttall et al.] ,Veterinary Records 172(8):201-207(2013) This is outlined in [reference]. ] involves skin infiltration by various immune cells, as well as IL-4, IL-13 CD4, which is dominated by IL-31 + Th2-polarized cytokine environment shows a marked similarity to the pathogenesis of atopic dermatitis in humans. Furthermore, IL -22 is involved in the excessive epithelial proliferation that leads to the epidermal hyperplasia characteristic of atopic dermatitis .

[0005] For example, antibodies against canine IL-31 have been shown to have a marked effect on the pruritus associated with atopic dermatitis in dogs [U.S. Patent No. 8,790,651 ; U.S. Patent No. 10,093,731]. Furthermore, antibodies against human IL-31 receptor α (IL-31 RA) have been tested and found to have a marked effect on the pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New Eng land Journal of Medicine, 376(9), 826-835( 2017)]. Therefore, blocking the binding of IL-31 to its receptor IL-31RA reduces the pruritus associated with atopic dermatitis .

[0006] Monoclonal antibodies have been developed against human interleukin-4 receptor α, and some of these antibodies have been widely tested for their therapeutic effects in treating atopic dermatitis in humans [see, for example, U.S. Patent Application Publication No. 2015 / 0 017176]. More recently, canineized antibodies against canine interleukin-4 receptor α (canine IL-4Rα, canine IL-4 R , cIL-4Rα or cIL-4R α that block the binding of canine IL- to canine IL-4R have also been disclosed [U.S. Patent Application Publication No. 2018 / 03465, which is incorporated herein by reference in its entirety for reference . α α ). ​​​[Issue 80]. Type II IL-4 receptors consist of the IL-4 receptor α chain and the IL-13 receptor α1 chain. Therefore, both canine IL-4 and canine IL-13 bind to the type II canine IL-4 receptor. Canine IL-4R can block the mating process. α Antibodies against it have been obtained, and Therefore, it helps to block inflammation associated with atopic dermatitis [U.S. Patent Application Publication No. 20 No. 18 / 0346580].

[0007] Interleuk, also known as IL-10-related T cell-derived inducible factor (IL-TIF), IL-22 belongs to the IL-10 cytokine family. In humans, it is produced by normal T cells in response to anti-CD3 stimulation. Mouse IL-22 Furthermore, IL-22 is induced in various organs upon lipopolysaccharide injection, and is involved in the inflammatory response. This suggests that it is possible. IL-22 is also known as IL-10R2 (IL-10Rβ). It consists of a heterodimer complex of (a) and the interleukin-22 receptor (IL-22R). It specifically binds to the receptor complex and transmits signals via the receptor complex [Lee e t al.,Pharmacology Research&Perspectives See Pages 1-13 (2018:e00434). Interleukin-22 The receptor is interleukin-22R, α1;IL-22RA1;IL-22R1;zc Also known as ytor11 and CRF2-9 [Xu et al., Pro c.Nat.Acad.Sci.98(17)9511-9516(2001);Gel ebart and Lai, Atlas of Genetics and Cyto genetics 14(12):1106-1110(2010)]. IL-22 is Epithelial cell proliferation is induced during wound healing, and its deficiency may promote tumorigenesis [Huber et al.] al., Nature 491:259-263 (2012). How many IL-22s are there? In that hepatocellular carcinoma cell line, STAT-1 and STAT-3 were activated, and acute phase proteins It has been shown to upregulate the production of interleukin-22 and IL-2. Antibodies against 2R block the interaction of IL-22 with IL-22R, thereby improving It acts as an antiproliferative agent by blocking related signaling pathways that cause skin proliferation. .

[0008] It has been proven and / or is likely to be helpful in the treatment of atopic dermatitis. Some pharmaceuticals include Janus kinase (JAK) inhibitors [e.g., U.S. 8,133,8]. [See Publication No. 99; U.S. Publication No. 8,987,283; International Publication No. 2018 / 108969] , spleen tyrosine kinase (SYK) inhibitors [e.g., U.S. Patent No. 8,759,366] [Reference], and antagonists for chemoattractant receptor homologous molecules expressed on TH2 cells Nist [for example, U.S. Patent No. 7,696,222, U.S. Patent No. 8,546,422, [See U.S. Patent Nos. 8,637,541 and U.S. Patent Nos. 8,546,422] It can be done.

[0009] However, despite recent successes in treating atopic dermatitis, All current treatments involve improving the skin barrier function while addressing skin inflammation. It does not produce a significant effect while simultaneously providing a rapid onset of antipruritic action. Therefore, atopic To design a better treatment that can address these three symptoms of genital dermatitis at once. It is necessary.

[0010] Any citation of references in this specification does not imply that such references constitute a "subject to this application". This should not be interpreted as an acknowledgment that the technology is available for use as "future technology." [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] U.S. Patent No. 8,790,651 [Patent Document 2] U.S. Patent No. 10,093,731 [Patent Document 3] U.S. Patent Application Publication No. 2015 / 0017176 [Patent Document 4] U.S. Patent Application Publication No. 2018 / 0346580 [Patent Document 5] U.S. Patent No. 8,133,899 [Patent Document 6] U.S. Patent No. 8,987,283 [Patent Document 7] International Publication No. 2018 / 108969 [Patent Document 8] U.S. Patent No. 8,759,366 [Patent Document 9] U.S. Patent No. 7,696,222 [Patent Document 10] U.S. Patent No. 8,546,422 [Patent Document 11] U.S. Patent No. 8,637,541 [Non-patent literature]

[0012] [Non-Patent Document 1] Rahman et al., Inflammation & Allergy-drug target 10:486-496(2011) [Non-Patent Document 2] Harskamp et al.,Seminar in Cutaneous Medicine and Surgery 32:132-139(2013) [Non-Patent Document 3] Nuttall et al., Veterinary Records 172(8):201-207(2013) [Non-Patent Document 4] Ruzicka,et al.,New England Journal of Medicine,376(9),826-835(2017) [Non-Patent Document 5] Lee et al.,Pharmacology Research & Perspectives,Pages 1-13(2018:e00434) [Non-Patent Document 6] Xu et al.,Proc.Nat.Acad.Sci.98(17)9511-9516(2001) [Non-Patent Document 7] Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12):1106-1110(2010) [Non-Patent Document 8] Huber et al.,Nature 491:259-263(2012) [Overview of the Initiative] [Means for solving the problem]

[0013] This invention relates to a bispecific antibody and a dual antibody for treating atopic dermatitis in dogs. The present invention relates to a composition containing specific antibodies. In a particular embodiment, atopic dermatitis in dogs The composition for treating dermatitis contains an antipruritic antibody, an anti-proliferative antibody, and an anti-inflammatory antibody. In a species-specific manner, one, two, or all of the antibodies are found in chimeric rodents (i.e., It is a mouse or rat-dog antibody. In yet other specific embodiments of this kind, the antibody One, two, or all of these are canine antibodies. In yet another specific embodiment of this kind One, two, or all of the antibodies are canine antibodies. In a specific embodiment, antipruritic antibodies This is part of a bispecific antibody that further includes anti-proliferative antibodies or anti-inflammatory antibodies. In a specific embodiment, the bispecific antibody is a monomer of canine IL-31 antibody and canine IL-31 antibody. Contains a monomer of IL-22 antibody. In an alternative embodiment, the bispecific antibody is canine IL- It contains monomers of 31RA antibody and canine IL-4Rα antibody.

[0014] Therefore, in a particular manner, to treat atopic dermatitis in dogs The composition comprises a canine antipruritic antibody, a canine antiproliferative antibody, and a canine anti-inflammatory antibody. In a more specific embodiment of the species, canine antipruritic antibodies are canine antiproliferative antibodies or canine antipruritic antibodies. It is part of a bispecific antibody that also contains inflammatory antibodies.

[0015] In a particular embodiment, the composition comprises a canine anti-inflammatory antibody and a canine heavy chain and a light chain. A dual ion comprising a monomer of a canine anti-pruritic antibody and a monomer of a canine anti-proliferative antibody containing heavy and light chains. It includes specific antibodies. In a particular embodiment of this type, the canine antipruritic antibody is canine inter - It is a leukin-31 (IL-31) antibody. In a more specific embodiment of this type, canine transformation The IL-31 antibody contains a light chain with the amino acid sequence of SEQ ID NO: 12 and the amino acids of SEQ ID NO: 16. It includes a heavy chain containing the sequence. In this alternative embodiment, the canine IL-31 antibody contains the sequence It contains a light chain containing the amino acid sequence of number 11 and a heavy chain containing the amino acid sequence of sequence number 15. In certain embodiments, the light chain of a canine IL-31 antibody replaces the constant light domain (CL). It was modified to include the heavy chain constant region 1 (CH1) derived from the heavy chain of the canine IL-31 antibody. Furthermore, the heavy chain of the canine IL-31 antibody is different from CH1 in the canine IL-31 antibody. It has been modified to include a constant light domain (CL) derived from the body's light chain. In a certain particular form In this context, the bispecific antibody, the canine antiproliferative antibody, is canine interleukin-22(IL- 22) It is an antibody monomer. In a more specific embodiment of this kind, canine IL-22 antibody is A light chain containing the amino acid sequence of SEQ ID NO: 24, and a heavy chain containing the amino acid sequence of SEQ ID NO: 17. This includes. In a more specific embodiment, the canine anti-inflammatory antibody is a canine interleukin. It is an IL-4 receptor α (IL-4Rα) antibody. In a specific embodiment, it is a canine IL-4R The α antibody consists of a light chain containing the amino acid sequence of SEQ ID NO: 4 and a heavy chain containing the amino acid sequence of SEQ ID NO: 5. The chain includes. In an alternative embodiment, the canine IL-4Rα antibody has the amino acid sequence of SEQ ID NO: 6. It comprises a light chain containing a column and a heavy chain containing the amino acid sequence of SEQ ID NO: 7.

[0016] In an alternative embodiment, the composition comprises a canine antiproliferative antibody and a canine antiproliferative antibody comprising a heavy chain and a light chain. A bispecific antibody comprising a monomer of pruritic antibodies and a monomer of canine-type anti-inflammatory antibodies containing heavy and light chains. It includes antibodies. In a particular embodiment of this type, canine antipruritic antibodies are canine interloyal antibodies. It is a Kin-31 receptor α (IL-31RA) antibody. In a related embodiment, it is an anti-inflammatory drug for canine inflammation. The antibody is canine interleukin-4 receptor α (IL-4Rα) antibody. In this form, the canine antipruritic antibody is a canine IL-31RA antibody, and is a canine anti-inflammatory antibody. The antibody is a canine IL-4Rα antibody. In a more specific embodiment, canine IL- The 4Rα antibody contains a light chain with the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 18. It contains heavy chains. In this alternative embodiment, the canine IL-4Rα antibody is SEQ ID NO: It contains a light chain with the amino acid sequence of 6 and a heavy chain with the amino acid sequence of SEQ ID NO: 19. In this embodiment, the light chain of the canine IL-4Rα antibody is instead of the constant light domain (CL). Modified to include heavy chain constant region 1 (CH1) derived from the heavy chain of the canine IL-4Rα antibody. Furthermore, the heavy chain of the canine IL-4Rα antibody is canine IL-4Rα instead of CH1. Modified to include constant light domains (CLs) derived from the antibody light chain. More specific embodiments In this study, the canine IL-31RA antibody contains a light chain with the amino acid sequence of SEQ ID NO: 43, and It comprises a heavy chain containing the amino acid sequence of column number 44. In a more specific embodiment, canine The anti-proliferative antibody is a canine interleukin-22 (IL-22) antibody. In this case, the canine IL-22 antibody contains a light chain with the amino acid sequence of SEQ ID NO: 20, and the sequence It includes a heavy chain containing amino acid sequence number 22. In an alternative embodiment, canine IL-22 antibody The body consists of a light chain containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain containing the amino acid sequence of SEQ ID NO: 23. The chain includes. In other alternative embodiments, the canine IL-22 antibody is the amino acid of SEQ ID NO: 24. It contains a light chain containing an acid sequence and a heavy chain containing the amino acid sequence of SEQ ID NO: 25.

[0017] The present invention further provides the bispecific antibody of the present invention. In a particular embodiment, anti-scratch Itchy antibodies are a type of bispecific antibody that also includes anti-proliferative antibodies or anti-inflammatory antibodies. In this embodiment, the bispecific antibody is a monomer of a canine antipruritic antibody containing a heavy chain and a light chain. , comprising monomers of canine antiproliferative antibodies containing heavy and light chains. In this context, canine antipruritic antibodies are canine interleukin-31 (IL-31) antibodies. In a more specific embodiment of this type, the canine IL-31 antibody is the same as in SEQ ID NO: 12. It contains a light chain containing a amino acid sequence and a heavy chain containing the amino acid sequence of SEQ ID NO: 16. In an alternative embodiment, the canine IL-31 antibody has a light chain containing the amino acid sequence of SEQ ID NO: 11 and , comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 15. In certain embodiments, canine IL- The light chain of the IL-31 antibody is derived from the heavy chain of the canine IL-31 antibody instead of the constant light domain (CL). It has been modified to include the heavy chain constant region 1 (CH1), and canine IL-31 antibody The body's heavy chain is replaced by a constant light domain (CL) derived from the light chain of the canine IL-31 antibody, instead of CH1. ) has been modified to include. In a particular embodiment, canine antibody amplification of bispecific antibodies The proliferative antibody is a monomer of canine interleukin-22 (IL-22) antibody. In a more specific embodiment, the canine IL-22 antibody comprises the amino acid sequence of SEQ ID NO: 24 It contains a light chain and a heavy chain containing the amino acid sequence of SEQ ID NO: 17.

[0018] In an alternative embodiment, the present invention relates to a monomer of a canine antipruritic antibody comprising a heavy chain and a light chain, This invention provides a bispecific antibody comprising a monomer of a canine-type anti-inflammatory antibody containing heavy and light chains. In a specific manner, canine antipruritic antibodies are canine interleukin-31 receptor α(IL) -31RA) antibody. In a related embodiment, canine anti-inflammatory antibodies are canine inter This is a leukin-4 receptor α (IL-4Rα) antibody. It is a more specific form of a bispecific antibody. In this context, the canine antipruritic antibody is the canine IL-31RA antibody, and also the canine anti-inflammatory antibody. The antibody is a canine IL-4Rα antibody. In a more specific embodiment, canine IL- The 4Rα antibody contains a light chain with the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 18. It contains heavy chains. In this alternative embodiment, the canine IL-4Rα antibody is SEQ ID NO: It contains a light chain with the amino acid sequence of 6 and a heavy chain with the amino acid sequence of SEQ ID NO: 19. In this embodiment, the light chain of the canine IL-4Rα antibody is instead of the constant light domain (CL). Modified to include heavy chain constant region 1 (CH1) derived from the heavy chain of the canine IL-4Rα antibody. Furthermore, the heavy chain of the canine IL-4Rα antibody is canine IL-4Rα instead of CH1. Modified to include constant light domains (CLs) derived from the antibody light chain. More specific embodiments In this study, the canine IL-31RA antibody contains a light chain with the amino acid sequence of SEQ ID NO: 43, and It contains a heavy chain containing the amino acid sequence of column number 44.

[0019] In yet another embodiment, compositions comprising the bispecific antibody and / or antibody of the present invention , and may further contain one or more additional therapeutic components. In one such embodiment The therapeutic component is a Janus kinase (JAK) inhibitor. In a particular embodiment of this type, JAK inhibitors are oclacitinib and its pharmaceutically acceptable salts. In another embodiment... In this context, JAK inhibitors are 1-[(3R,4S)-4-cyanotetrahydropyran-3- [Iyl]-3-[(2-fluoro-6-methoxy-4-pyridyl)amino]pyrazole-4 -Carboxamide and its pharmaceutically acceptable salts. In another embodiment, therapeutic The component is a spleen tyrosine kinase (SYK) inhibitor. In a specific embodiment of this type, S YK inhibitors include (1S,4R)-4-hydroxy-2,2-dimethyl-4-{5-[3- Methyl-5-(4-methylpyrimidine-2-ylamino)phenyl]-1,3-thia sol-2-yl-cyclohexanecarboxylic acid or a pharmaceutically acceptable salt thereof. In yet another embodiment, the therapeutic component is a chemoattractant receptor expressed on TH2 cells. It acts as an antagonist to homologous molecules.

[0020] The present invention further relates to a method for treating atopic dermatitis, wherein a person has atopic dermatitis A method comprising administering the composition and / or one of the bispecific antibodies to a dog. provide.

[0021] These and other aspects of the present invention are described below with reference to the brief and detailed descriptions of the drawings. It will be better understood by doing so. [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1 is a graph showing the binding of canine IL-22 to the anti-IL-22 antibody. [Figure 2] Figure 2 is a graph showing the inhibition of IL-4-mediated STAT-6 phosphorylation by IL-4Rα (IL-4Rα) antibodies. Three different canine monoclonal anti-canine IL-4Rα antibodies, called c4H3, c146E2-H3L3, and c152H11-H3L3, were evaluated for their ability to inhibit αSTAT-6 phosphorylation. The data show that all three antibodies result in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4. The IL-4 control in the absence of IL-4Rα (IL-4Rα) antibodies is shown in the upper left portion of the graph. [Figure 3] Figure 3 is a graph showing the inhibition of IL-13-mediated STAT-6 phosphorylation by IL-4Rα antibodies. Three different canine monoclonal anti-canine IL-4Rα antibodies, called c4H3, c146E2-H3L3, and c152H11-H3L3, were evaluated for their ability to inhibit STAT-6 phosphorylation. The data show that all three antibodies result in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13. The IL-13 control in the absence of IL-4Rα (IL-4Rα) antibodies is shown in the upper left portion of the graph. [Figure 4] Figure 4 is a graph showing the binding of IL-31 to the extracellular domain of IL-31R (IL-31RA). The extracellular domain (ECD) of canine IL-31RA was tested for its ability to bind to canine IL-31. The results show that IL-31RA ECD binds to biotinylated canine IL-31 in a dose-dependent manner at an EC50 of 0.3679 μg / ml. [Modes for carrying out the invention]

[0023] In response to the need for better treatments for atopic dermatitis, the present invention Simultaneous regulation of IL-4, IL-31, and IL-22 was achieved, resulting in remarkable efficacy against skin inflammation. It can improve the fruit and skin barrier function while simultaneously initiating a rapid onset of antipruritic effects. To provide formulations and methodologies.

[0024] Abbreviation The following abbreviations are used throughout the detailed description and examples of this invention. ADCC antibody-dependent cytotoxicity CDC complement-dependent cell injury Immunoglobulin variable, defined using the CDR Kabat numbering system. Complementarity determination region within the domain CHO Chinese hamster ovaries CL stationary light domain Concentration that yields 50% efficacy or binding in EC50 ELISA (Enzyme-linked immunosorbent assay) FR Antibody Framework Region: Immunoglobulin variable region excluding the CDR region. HRP (Horseradiol Peroxidase) IFN (Interferon) Concentration that produces 50% inhibition of IC50 IgG (Immunoglobulin G) Immunoglobulins pioneered by Elvin A. Kabat Alignment and numbering system [Sequences of Prote ins of Immunological Interest,5th Ed.Pub lic Health Service, National Institutes o f Health, Bethesda, Md. (1991)] mAb monoclonal antibody (also known as Mab or MAb) MES 2-(N-morpholino)ethanesulfonic acid MOA mechanism of action NHS Normal Human Serum PCR (polymerase chain reaction) PK (Pharmacokinetics) SEB (Staphylococcus enterotoxin B) TT Tetanus Toxoid VH Immunoglobulin Heavy Chain Variable Region VL (Variable Region of Immunoglobulin Light Chain) VK immunoglobulin κ light chain variable region

[0025] definition To make the present invention easier to understand, specific technical and scientific terms are used below. Defined as follows. Unless otherwise specifically defined elsewhere in this Spec. All other technical and scientific terms have meanings that are generally understood by those skilled in the art. do.

[0026] As used herein, including in the attached claims, "a", "an", and "t" The singular form of a word like "he" is used unless the context clearly indicates otherwise. This includes multiple corresponding references.

[0027] "Activation" when applied to cells or receptors, depending on the context or explicitly stated otherwise. Unless otherwise specified, this refers to the activation or processing of cells or receptors by a ligand. "Ligands" are natural and synthetic ligands, such as cytokines, cytokine variants, etc. It includes conjugated compounds derived from analogs, mutains, and antibodies. "Ligands" are small molecules. For example, this includes peptide mimes of cytokines and peptide mimes of antibodies. "Cell activation" refers to cell activation that is regulated not only by internal mechanisms but also by external or environmental factors. It is possible.

[0028] Molecular "activity" includes binding of the molecule to a ligand or receptor, catalytic activity, and gene expression. or the ability to stimulate cell signaling, differentiation, or maturation; antigenic activity, activity of other molecules Regulations, etc., may be described or expressed. The "activity" of a molecule also refers to cell-cell interactions, for example. If so, activity in regulating or maintaining adhesion, or in the structure of cells, for example, the cell membrane or This can also represent activity in maintaining the cytoskeleton. "Activity" can also refer to specific activity, for example, [catalysis]. [Activity] / [mg protein], or [Immune activity] / [mg protein], biological compartment It can refer to the concentration or other properties within a substance. "Activity" refers to the components of the innate or adaptive immune system. This can represent the adjustment of [something].

[0029] "Administration" and "processing" refer to animals, such as dogs, cells, tissues, organs, or biological subjects. When applied to fluids, exogenous pharmaceuticals, therapeutic agents, diagnostic agents, or compositions in animals, such as dogs. This refers to contact with an object, cell, tissue, organ, or biological fluid. Processing cells involves contact with cells. This includes not only drug contact but also the contact of reagents with fluids that are in contact with cells.

[0030] "Administration" and "processing" also refer to, for example, by reagents, diagnostic agents, conjugates, or other This refers to in vitro and ex vivo treatment of cells by cells. The term "subject" means Any living organism, preferably an animal, more preferably a mammal (e.g., a dog, a cat, or a human). ), most preferably including dogs.

[0031] "To treat" or "is treating" means, for example, that the therapeutic agent has therapeutic activity. For dogs or patients exhibiting or suspected to exhibit the above symptoms, internal or Externally, a composition containing either the antibody of the present invention and / or a bispecific antibody is used. It means administering a therapeutic drug.

[0032] Typically, the treatment only reduces such symptoms(s) to any clinically measurable degree. One or more contractures in the treated subject or population by inducing or inhibiting the progression of contracture. To be administered in a dose effective in reducing and / or improving any specific disease / symptom. The amount of a therapeutic agent that is effective in alleviating a disease / symptom (also called the "therapeutic dose") is , the patient's (e.g., dog's) medical condition, age, and weight, as well as the desired response in the subject This can vary depending on factors such as the ability of the inducing pharmaceutical composition to alleviate the disease / symptoms. To determine whether the condition has improved, a veterinarian may assess the severity or progression of the symptom. This is assessed by any clinical measurement that is normally used by other skilled healthcare professionals. This is possible. Embodiments of the present invention (for example, treatment methods or products) can be applied to any subject. It may not be effective in alleviating the target disease / symptom(s) or symptoms. Student's t-test, chi-squared test, Mann-Whitney U test, Claska Le Wallis Test (H Test), Yonkie Terpstra Test, and Wilcoxon Test If determined by any statistical test known in the art, is it statistically significant? It should alleviate target diseases / symptoms / signs in a number of subjects.

[0033] "Procedure" is used when applied to humans, veterinary subjects (e.g., dogs), or research subjects. This refers to therapeutic procedures as well as research and diagnostic uses. "Procedure" refers to human, veterinary, or other subjects (for example...) When applied to dogs, or research subjects, or cells, tissues, or organs, the present invention Antibodies and / or bispecific antibodies for, for example, dogs or other animals, cells, This includes contact with tissue, physiological compartments, or physiological fluids.

[0034] As used herein, the term "dog" means all households unless otherwise specified. This includes domestic dogs, including Canis lupus familialis or Canis familialis.

[0035] As used herein, the term “cat” refers to any member of the Felidae family. Members of this class include domestic cats, purebred and / or mixed breed pet cats, and show cats. This includes laboratory cats, cloned cats, and wild or feral cats. This includes members of the zoo and the household.

[0036] As used herein, the term "canine flame" refers to the CDR residue and This refers to the amino acid sequences of the heavy and light chains of canine antibodies, excluding the hypervariable region residues defined as such. Regarding canine antibodies, in most aspects, the amino acid sequence of natural canine CDRs is the same as that of canine CDRs. In the chain, the corresponding foreign CDR (e.g., derived from mouse or rat antibodies) is placed It can be replaced. The heavy and / or light chains of canine antibodies can be replaced, for example, as exemplified below. U.S. Article 10,106, and / or the entirety thereof, are incorporated herein by reference. As disclosed in No. 607, in order to preserve the three-dimensional structure of exogenous CDRs within canine antibodies, It may also contain several exogenous non-CDR residues to modify the Fc function.

[0037] Canine antibodies (also called immunoglobulin G or IgG) are large four-celled cells with a molecular weight of approximately 150 kD. It is a mass protein. Each IgG protein consists of two identical light chains, each approximately 25 kD long. Each of the four canine IgG molecules consists of two identical heavy chains, each approximately 50 kD in weight. There are known IgG heavy chain subclasses, which are IgGA, IgGB, IgGC, and I. It is called gGD. There are two types of light chains: κ chains and λ chains. Each consists of one variable domain (VL) and one constant domain (CL). Each of the two heavy chains consists of one variable domain (VH) and one heavy chain constant region 1 (CH1 or C). H-1), heavy chain steady region 2 (CH2 or CH-2) and heavy chain steady region 3 (CH3 or It consists of three constant domains called CH-3. The CH1 domain is a "hinge". Alternatively, it is connected to the CH2 domain via an amino acid sequence called the "hinge region". In this invention, the amino acid sequences for each of the four canine IgG Fc fragments are as follows: Tang et al.[Vet.Immunol.Immunopathol.80: The same CH1 domain and CH2 domain as determined by 259-270 (2001) It is based on defined boundaries. In humans, IgG is divided into IgG1, IgG2, IgG3, and It exists in one of the four subclasses called IgG4. The subclasses of IgG are This is primarily determined by the different hinge region sequences among the four subclasses of IgG. The heavy chains are linked to each other by disulfide bonds, and each heavy chain is connected by disulfide bonds It is also connected to one side of the light chain via this.

[0038] The digestion of IgG antibodies by the enzyme papain cleaves the antibody molecule at the hinge region, resulting in three segments. This results in the formation of fragments. Two of these fragments are identical, and each has the VH and It consists of a light chain combined with a CH1 domain. These fragments are called "Fab" fragments. It contains the antigen-binding site of the antibody. The Fab fragment is cross-linked with VH-CH1 by disulfide crosslinking. This is a VL-CL chain attached to the chain. The third fragment resulting from digestion with papain is "Fc It is called a "heavy chain" and contains the remainder of the two heavy chains that are joined together by a disulfide bond. Therefore, Fc is a dimer consisting of the CH2 and CH3 domains of the two heavy chains. It contains the body. Fab enables antibodies to bind to their homologous epitopes. Furthermore, Fc is involved in antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP). and can mediate immune effector functions such as complement-dependent cell-mediated cytotoxicity (CDC). To enable. The "Fab fragment" consists of one light chain (VL domain and CL domain) and It consists of one heavy chain CH1 and a variable region (VH). The heavy chain of the Fab molecule is another It cannot form disulfide bonds with heavy chain molecules.

[0039] The "fragment crystallizable region," abbreviated as "Fc," is a cell surface receptor called the Fc receptor. This corresponds to the CH3-CH2 portion of antibodies that interact with the body. The fragment crystallizable region (cFc) was prepared by Tang et al. [Vet. Immunol. It was first described in Immunopathol. 80:259-270 (2001). ,Bergeron et al.,Vet.Immunol.Immunopathy See also l.157:31-41(2014) and U.S. Patent No. 10,106,607. I want to be treated that way.

[0040] As used herein, canine Fc(cFc) "IgG-Bm" refers to the distribution of IgG-B The amino acid sequence of column number 14 contains two amino acid residue substitutions, D31A and N63A. This is canine IgG-B Fc (see below). The aspartic acid residue at position 31 of SEQ ID NO: 14. (D) and the asparagine residue at position 63 of Sequence ID No. 14 (N) are both IgG-Bm In this case, it is substituted by an alanine residue (A). These two amino acid residues Exchange is related to antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent reactions of naturally occurring canine IgG-B. Helps significantly reduce sex cell damage (CDC) [See this specification for the full details] See U.S. Patent No. 10,106,607 incorporated in this document. In IgG-B Similar to amino acid substitutions, it promotes heterodimer formation in bispecific antibodies. Further amino acid substitutions to IgG-Bm are also intended, which may include the amino acid substitutions shown.

[0041] When used herein, the amino acid sequence of an antibody may be altered by another amino acid residue. "Amino acid residue substitution" means, for example, "replacing an amino acid residue with another amino acid residue." They are equivalent, but a specific amino acid residue at a specific position in the amino acid sequence is a different amino acid. This indicates that a residue is replaced (or has been replaced) by another residue. This can be specifically designed, that is, for example, by recombinant DNA technology, amino Alanine at a specific position in the acid sequence can be intentionally replaced with serine. , specific amino acid residues or a series of amino acid residues of an antibody are selected through a more natural selection process. For example, an antibody produced by a cell can target a given region on its antigen, such as an epitope. Based on its ability to bind to a region containing or part thereof, and / or the antibody Includes a specific CDR that has the same canonical structure as the CDR that a particular CDR is replacing. It can be replaced by one or more amino acid residues. The substitution may result in a "mutant" CDR and / or mutant antibody.

[0042] As used herein, the term "antibody" refers to an antibody exhibiting a desired biological activity. This refers to any form. Antibodies can be monomers, dimers, or larger polymers. Therefore, the term "antibody" is used in the broadest sense, and monoclonal antibodies (full-length monoclonal antibodies) are used. (including noclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) Specifically, antibodies, canine antibodies, fully canine antibodies, chimeric antibodies, and camel-derived single-domain antibodies. This generally includes, but is not limited to, these. "Parental antibody" is used as an antibody for treating dogs. Before exposing the immune system to the antigen, modify the antibody for intended use, such as canine modification of the antibody. These are antibodies obtained by doing [this].

[0043] When used herein, for example, the binding of a canine receptor to its binding partner (ligand) The antibody of the present invention that "blocks" or "is blocking" binding or "is blocking binding". and / or bispecific antibodies can be used in standard binding assays (e.g., BIACore( Canine receptors are determined by (registered trademark), ELISA, or flow cytometry. Blocking (partially or completely) the binding to that canine ligand, and vice versa, It is a somatic and / or bispecific antibody.

[0044] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites are the same It possesses antigenic specificity.

[0045] As used herein, “bispecific antibody” is an antibody that simultaneously targets two different antigens. It is an artificial protein that can do this. One preferred type of bispecific antibody is four It is an IgG-like antibody composed of different polypeptide chains. Therefore, a bispecific antibody is, Each can be a heterodimer containing two monomers, one heavy chain and one light chain. First monomer The first monomer may be from an antibody against one specific antigen, while the second monomer may be from a different antigen. It may be derived from an antibody against an antigen. For example, certain bispecific antibodies of the present invention are Such antibodies can simultaneously target IL-31 and IL-22. One heavy chain and one light chain having specificity for L-31, and specificity for IL-22. It is formed by the association of heterozygous heavy and light chains. Furthermore, the heavy chain of the IL-22 antibody It promotes the association of the chain and light chain with each other, and the association of the heavy and light chains of the IL-31 antibody with each other. However, at the same time, association of one IL-22 heavy chain with another IL-22 heavy chain or another IL-31 heavy chain Rather than association with the IL-31 heavy chain, the heavy chain derived from the IL-22 antibody is associated with the heavy chain of the IL-31 antibody. To facilitate the association of the heavy and light chains, specific mutations in their amino acid sequences It can be modified.

[0046] In the context of bispecific antibodies, the "monomer" of an antibody is one heavy chain and one mono It consists of light chains.

[0047] As used herein, "artificial protein" and "artificial protein molecule" are interchangeable. Used for artificial fusion proteins or heterodimers of monomers derived from two different antibodies. Proteins that do not exist in nature, such as dimers, heterodimers, tetramers and This refers to protein polymers such as heterotetramers.

[0048] Typically, the antibody or antigen-binding fragment of the present invention is expressed in a molar basis. In combination, it retains at least 10% of its canine antigen-binding activity (compared to the parent antibody). Preferably, the antibody or antigen-binding fragment of the present invention has a canine antigen-binding affinity as the parent antibody. At least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more It retains more. The antibody or antigen-binding fragment of the present invention substantially retains its biological activity. Non-conservative or non-conservative amino acid substitutions ("conservative variants" or "functional variants" of antibodies) It is also intended to include variants that are considered "conserved variants."

[0049] "Isolated antibody" refers to a purified state, and in this context, the molecule is a nucleic acid, Proteins, lipids, carbohydrates and other biological molecules, or cell debris and growth medium This means that it contains virtually no other materials. Generally, the term "isolated" is used. This is present in an amount that substantially prevents the experimental or therapeutic use of the conjugated compounds described herein. If not present, it means that such material is completely absent, or that water, buffer, or salt is not present. It is not intended to indicate non-existence.

[0050] As used herein, "chimeric antibody" refers to a variable domain derived from the first antibody and The antibody has a constant domain derived from the second antibody, and the first and second antibodies are from different species. Derived from: [U.S. Patent No. 4,816,567; and Morrison et al.] ,Proc.Natl.Acad.Sci.USA 81:6851-6855(198 4) Typically, the resulting chimeric antibody is more potent than the parent (e.g., rodent) antibody. This reduces the likelihood of inducing a harmful immune response in humans or dogs. The variable domain is obtained from antibodies derived from experimental animals such as rodents ("parent antibodies"), and The constant domain sequence is obtained from animal target antibodies, such as human or dog antibodies.

[0051] As used herein, the term "canine antibody" refers to canine and non-canine antibodies (for example). This refers to an antibody form that contains sequences from both mouse and rat antibodies. Generally, Nu-type antibodies are those that make up all or virtually all of the hypervariable loop (for example, as illustrated below). It addresses the hypervariable loop of non-canine immunoglobulins (including six CDRs such as the one shown in the image), and the frame All or substantially all of the frame (FR) area (and typically the remaining frame) All or virtually all of the canine immunoglobulin sequences are in the framework (FR) region. It includes virtually all of at least one, typically two, variable domains. As illustrated in the detailed document, canine antibodies are derived from three types of mouse or rat anti-canine antigen antibodies. Both the heavy chain CDR and the three light chain CDRs are in canine flame or modified canine flame. It includes along with the canine flame. For example, a modified canine flame contains canine antibodies against its canine antigen. Blocking the binding of the canine antigen to its natural binding partner and / or to its natural binding partner. To further optimize the effectiveness of canine antibodies in order to increase the capabilities of canine antibodies, this specification Includes one or more example amino acid changes.

[0052] The variable region of each light / heavy chain pair forms the antibody binding site. Therefore, generally, the complete state Antibodies in this state have two binding sites. Except for bifunctional or bispecific antibodies, two binding sites The joining parts are generally the same.

[0053] Typically, both the heavy and light chain variable domains are relatively conserved frameworks. Three hypervariable regions, also known as complementary determination regions (CDRs), are located within the FR region. This includes. CDRs typically have aligned framework regions and specific epitopes. Enables binding. Generally, from the N-terminus to the C-terminus, the light chain variable domain and the heavy chain variable domain The IN models are FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 It includes. The assignment of amino acids to each domain is generally, Sequences of P roteins of Immunological Interest, Kabat, et al.,National Institutes of Health,Bet Hesda, Md., 5 th ed., NIH Publ. No. 91-3242 (19 91), Kabat, Adv. Prot. Chem. 32:1-75 (1978), Ka bat,et al.,J.Biol.Chem.252:6609-6616(197 7), Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-8 Follow the definition in 83(1989).

[0054] As used herein, the term “hypervariable region” refers to the part of an antibody involved in antigen binding. It refers to amino acid residues. The hypervariable region is called the "complementarity-determining region" or "CDR" [i.e., CDRL1 (or LCDR1), CDRL2 (or LCDR2) in the light chain variable domain ) and CDRL3 (or LCDR3) and CDRH1 (in the heavy chain variable domain) (or HCDR1), CDRH2 (or HCDR2), and CDRH3 (or HCDR 3) Contains amino acid residues derived from [Kaba, which defines the CDR region of the antibody by sequence]. t et al.Sequences of Proteins of Immunol ogical Interest,5th Ed.Public Health Ser vice,National Institutes of Health,Bethe See sda, Md. (1991); Chot defines the CDR region of antibodies by structure. hia and Lesk, J. Mol. Biol. 196:901-917 (1987 (See also) . When used herein, the term “framework” or “FR” residue is used. The term refers to variable domain residues other than hypervariable region residues, which are defined herein as CDR residues. It refers to.

[0055] There are four known IgG heavy chain subtypes of canine IgG: IgG-A, IgG-B, These are called IgG-C and IgG-D. The two known light chain subtypes are λ and κ. It is called. In certain aspects of the present invention, apart from the binding and activation of canine immune cells, the present invention For canine or canine antibodies against antigens, the optimal two are: It has the following attributes. 1. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC), etc. Lack of the Ejector function, and 2. Using industry-standard technologies such as Protein A chromatography technology It can be easily refined.

[0056] None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using protein A, but high levels of ADCC activity are possible. It possesses this property. On the other hand, IgG-A weakly binds to protein A but also exhibits ADCC activity. Furthermore, neither IgG-C nor IgG-D can be purified using a protein A column, IgG-D does not exhibit ADCC activity. (IgG-C has considerable ADCC activity.) One way in which the present invention addresses these problems is by using an effector function such as ADCC. It is deficient and can be easily purified using industry-standard Protein A chromatography. a modified canine IgG-B antibody specific to the antigen of the present invention, and / or This is achieved by providing bispecific antibodies.

[0057] In an alternative embodiment of the present invention, the canine IgG-B of the present invention is specific to the antigen of the present invention. Alternatively, IgG-C antibodies and / or bispecific antibodies are effectors such as ADCC. - Not intentionally modified to remove / substantially weaken the function, therefore AD It retains effects functions similar to CC.

[0058] As used herein, "anti-pruritic agent" "Antipuritic agents" or "antipruritic agents" inhibit and relieve itching. Compounds, polymers, and / or formulations that tend to neutralize and / or prevent the anti Itchy creams are colloquially called antipruritics.

[0059] When used herein, "antipruritic antibody" "Antipruritic antibody" or "antipruritic antibody" is In particular with regard to atopic dermatitis, including mammals such as humans, dogs, and / or cats. This is an antibody that can act as an antipruritic agent in animals. In a particular embodiment, anti Pruritic antibodies are IL-31 or its receptor IL-31RA, which are IL-31 signaling receptors. It binds to a specific protein in the pathway. The antipruritic antibody binds to its corresponding antigen (e.g., IL). Binding to IL-31 (or IL-31RA) is, for example, binding of IL-31 to IL-31RA. It inhibits and interferes with and / or prevents good signaling in this pathway, and Thus, it inhibits, alleviates, and / or prevents itch caused by the IL-31 signaling pathway without interference and / or prevention.

[0060] As used herein, an "anti-inflammatory agent" is a compound, polymer, and / or formulation that reduces inflammation by blocking the interaction of certain substances in the body that cause inflammation.

[0061] As used herein, an "anti-inflammatory antibody" is an antibody that can act as an anti-inflammatory agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In certain embodiments, the anti-inflammatory antibody binds to a specific protein in the IL-4 / IL-13 signaling pathway, such as IL-4 or the receptor IL-4R IL-4R α α α

[0062] As used herein, an "anti-proliferative agent" is a compound, polymer, and / or formulation that counteracts the induction of epithelial cell proliferation, particularly keratinocyte cell proliferation, particularly with respect to atopic dermatitis. Interleukin-22 binding protein (IL-22BP) is an example of a naturally occurring anti-proliferative agent.

[0063] As used herein, an "anti-proliferative antibody" is an antibody that, particularly with respect to atopic dermatitis, It acts as an antiproliferative agent in animals, including mammals such as dogs and / or cats. It is an antibody that can do this. In a particular embodiment, the anti-proliferative antibody is IL-22 or receptor Certain receptors in the IL-22 signaling pathway, such as the somatic IL-22 receptor (IL-22R), It binds to protein. The anti-proliferative antibody binds to its corresponding antigen (e.g., IL-22 or IL- Binding to 22R inhibits, for example, the binding of IL-22 to IL-22R, thus disrupting this pathway. Interfering with and / or preventing signal transmission, thereby preventing keratitis associated with atopic dermatitis It inhibits or prevents tinocyte cell proliferation.

[0064] "Homologousity" refers to the optimal relationship between two polynucleotide sequences or two polypeptide sequences. This refers to the similarity of the sequences when they are aligned. The positions in both of the two compared sequences are If the same base or amino acid monomer subunit is occupied, for example, two If each position within a DNA molecule is occupied by adenine, then the molecule is They are homologous in terms of position. The percentage of homology is the two positions divided by the total number of positions being compared. This is the number of homologous positions shared by the column multiplied by 100. For example, if the array is optimally sorted Sometimes, when 6 out of 10 positions in two arrays coincide or are homologous, The two sequences are 60% homologous. In general, comparisons are made when two sequences have the highest percentage homology. This is done when the objects are aligned to give a certain result.

[0065] "Isolated nucleic acid molecules" refers to genomes, mRNA, cDNA, or molecules of synthetic origin or other origins. These are some combinations of DNA or RNA, isolated polynucleotides The do is not accompanied by all or some of the polynucleotides found in nature. Potatoes, or isolated nucleic acid molecules, are polynucleotides that are not naturally linked in nature. This means linked together. In this disclosure, a specific nucleotide sequence "containing nucleic acid" Please understand that the term "molecule" does not include chromosomes in their original state. The specified nucleic acid sequence... Isolated nucleic acid molecules "containing" up to 10 or up to 20 in addition to the specified sequence. Or coding for more other proteins or parts or fragments thereof It may include columns or functionally linked to control the expression of coding regions of the described nucleic acid sequences. It may include a regulatory sequence and / or a vector sequence.

[0066] The term "regulatory sequence" refers to a functionally linked coding sequence in a particular host organism. This refers to the DNA sequence necessary for expression. Examples of regulatory sequences suitable for prokaryotes include promo It may contain an operator sequence and a ribosome binding site. It is known that cells use promoters, polyadenylation signals, and enhancers. That is the case.

[0067] Nucleic acids are "functionally linked" when they are arranged in a functional relationship with another nucleic acid sequence. For example, DNA for pre-sequences or secretion leaders is involved in polypeptide secretion. When expressed as a donor preprotein, it functionally contributes to the DNA for the polypeptide. If linked, the promoter or enhancer will affect the transcription of the sequence. Functionally linked to the coding sequence, or ribosome binding sites, facilitate translation. When arranged in this way, it is functionally linked to the code array. Generally, "functionally linked" "Linked to" means that the linked DNA sequences are continuous, and secretory In the case of Dar, it means that it is continuous and in the reading phase. However, En The Hansers do not need to be consecutive. Connections are made by connecting at convenient limiting points. It is achieved. If such a site does not exist, a synthetic oligonucleotide adapter or The linker is used according to conventional practice.

[0068] As used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable. The term is used interchangeably, and all such terms include offspring. Therefore, "transformed" and The term "transformed cells" refers to primary target cells and cells regardless of the number of times they have been transformed. Includes cultures derived therefrom. Due to intentional or unintentional mutations, all offspring It is also understood that grandchildren do not necessarily have exactly the same DNA composition. The cells that were screened for had mutations that exhibited the same function or biological activity as those that were screened. This includes heterologous offspring. If a different designation is intended, it should be clear from the context.

[0069] The present invention relates to the isolated canine antibody and / or bispecific antibody, and the treatment of symptoms. For example, antibodies and / or bispecific antibodies in the treatment of atopic dermatitis in dogs. Provides information on how to use the body. In dogs, four IgG heavy chains called A, B, C, and D They exist. These heavy chains are IgG-A (or IgGA), IgG-B (or IgG B) IgG-C (or IgGC) and IgG-D (or IgGD) This represents four different subclasses of IgG. Each of the two heavy chains has one variable domain. It consists of VH and three constant domains called CH-1, CH-2, and CH-3. The CH-1 domain is located via an amino acid sequence called the "hinge" or "hinge region". It is connected to the CH-2 domain.

[0070] The DNA and amino acid sequences of these four heavy chains were found in Tang et al. [Vet] .Immunol.Immunopathol.80:259-270(2001)] Therefore, they were the first to be identified. The amino acid and DNA sequences for these heavy chains are GenB It is also available from the ANK database. For example, the amino acid sequence of the IgGA heavy chain is available from the ANK database. Session number AAL35301.1, IgGB has accession number AAL3 5302.1 is present, IgGC has accession number AAL35303.1, and Therefore, IgGD has accession number (AAL35304.1). The canine antibody is, It also contains two types of light chains, κ and λ. The DNA and amino acid sequences of these light chains are G It can be obtained from the enBank Database. For example, κ light chain amino acid combination The column has accession number ABY57289.1, and the λ light chain has accession number AB It has Y55569.1.

[0071] Canine IL-31, IL-31RA, IL-22, or IL-4R α Inu-type ma Anti-canine antibodies, including but not limited to those of rats or dogs, include canine IgG-A, IgG-B, and Ig GC and IgG-D heavy chains and / or canine κ or λ light chains are used in mice or rats. The present invention includes the antibody and / or bispecific antibody, which are contained together with the anti-canine antigen CDR. Therefore, the present invention binds to their corresponding canine antigens and their natural binding partners. The isolated canine mouse or rat anti-infective of the present invention blocks the binding of its canine antigen to the canine antigen. Provides Nu antibodies and / or bispecific antibodies.

[0072] Therefore, the present invention further relates to canine-like mouse or rat antibodies, as well as treatment of symptoms. For example, the antibody and / or dual feature of the present invention in the treatment of atopic dermatitis in dogs. This document provides a method for using heterozygous antibodies.

[0073] The present invention can be further matched with the corresponding light chain to produce canine antibodies. The present invention provides a full-length canine heavy chain. Therefore, the present invention further provides canine mouse or rat antibody Canine antigen antibodies (including isolated canine-like mouse or rat anti-canine antibodies) and / or The present invention provides a bispecific antibody containing a canine antibody, and treatment of symptoms, for example, in dogs. Method of using the antibody and / or bispecific antibody of the present invention in the treatment of totopic dermatitis provide.

[0074] The present invention also aims to enhance, reduce, or remove one or more effect functions. The present invention contains a canine fragment crystallizable region (cFc region) in which cFc has been genetically modified. Provides a whole and / or bispecific antibody. In one embodiment of the present invention, genetically modified cFc reduces or eliminates one or more effect functions. Another aspect of the present invention In this case, the genetically modified cFc enhances one or more effector functions. In this manner, the genetically modified cFc region is a genetically modified canine IgGB Fc It is a region. In another such embodiment, the genetically modified cFc region is genetically modified This is a modified canine IgGC Fc region. In certain embodiments, the effector function is enhanced. Antibody-dependent cell-mediated cytotoxicity (ADCC) is reduced, eliminated, or removed. In another embodiment, In this context, the effector function is enhanced, reduced, or removed complement-dependent cell damage. It is harmful (CDC). In yet another embodiment, the cFc region is ADCC and CDC They have been genetically modified to enhance, reduce, or eliminate both.

[0075] To create mutant canine IgG lacking effector function, a large number of mutant canine Ig GB heavy chains were constructed. These mutants have the following single amino acid in the Fc portion of the heavy chain amino acid sequence. Or may include one or more of the following combined substitutions: P4A, D31A, N63A, G64P , T65A, A93G and P95A. Mutant heavy chains (i.e., such amino acid substitutions). Cloned into an expression plasmid containing the gene encoding the light chain It was transfused into HEK293 cells along with the plasmid. It was involved in the mediation of immunoeffector function. To evaluate those possibilities, Fc γ Regarding the binding of RI and C1q, H We evaluated the complete state of antibodies expressed and purified from EK293 cells [the whole content]. See U.S. Patent No. 10,106,607 incorporated herein by reference. ]

[0076] The present invention also includes, instead of its natural IgGD hinge region, a hinge region from the following: We provide modified canine IgGD. IgG-A:FNECRCTDTPPCPVPEP Sequence ID 26 IgG-B:PKRENGRVPRPPDCPKCPAPEM SEQ ID NO: 27; or IgG-C: AKECECKCNCNNCPCPGCGL SEQ ID NO: 28.

[0077] Alternatively, the IgG-D hinge region can be genetically modified by substituting serine residues with proline residues, i.e., PKESTCKCI PCPVPES, SEQ ID NO P 29 (the proline residues (P) are underlined and shown in bold and replace the naturally occurring serine residues). Such modifications can result in canine IgGD lacking Fab arm exchange. The modified canine IgGD can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, the nucleic acid encoding the amino acid sequence of canine IgGD can be modified to encode the modified IgGD. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression. iatis et al., Molecular Cloning, A Laborat ory Manual (1982)] can be used. To construct these variants, the nucleic acid encoding the amino acid sequence of canine IgGD can be modified to encode the modified IgGD. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0078] Sequence identity refers to the degree to which two polypeptides have the same amino acids at equivalent positions when the two sequences are optimally aligned. As used herein, one amino acid sequence is 100% "identical" to a second amino acid sequence when the amino acid residues of both sequences are the same. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparison is performed over contiguous blocks of amino acid residues encompassed by a given protein, e.g., the protein being compared, or a portion of the polypeptide. In certain embodiments ​​​​​​​​​​​​or alternatively, selected deletions or insertions that can change the correspondence between the two amino acid sequences are considered.

[0079] Sequence similarity includes identical residues and non-identical but biochemically related amino acids. Biochemically related amino acids that share similar properties and may be exchangeable are discussed.

[0080] A "conservatively modified variant" or "conservative substitution" refers to the substitution of an amino acid in a protein by another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) that allows frequent changes without changing the biological activity of the protein. One of ordinary skill in the art will generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change its biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. Furthermore, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are shown in Table A below.

[0081]

Table 1

[0082] Functionally conservative variants of the antibodies of the present invention are also contemplated by the present invention. As used herein, a "functionally conservative variant" changes a desired property such as antigen affinity and / or specificity This refers to an antibody or fragment in which one or more amino acid residues have been altered without any change in the antibody itself. As for mutants, amino acids with similar characteristics, such as the conservative amino acid substitutions in Table A above, are used. This includes, but is not limited to, replacing amino acids.

[0083] nucleic acid The present invention further comprises nucleic acids encoding the antibody and / or bispecific antibody of the present invention. (See, for example, the following examples.)

[0084] To give the greatest possible match between each array across the entire length of each reference array, The comparison is performed by the BLAST algorithm, which selects the parameters of the algorithm. In this case, the amino acid sequence of the canine antibody provided herein is at least about 70% identical, and is preferable. Ideally, they should be at least approximately 80% identical, more preferably at least approximately 90% identical, and most preferably At least approximately 95% are identical (for example, 95%, 96%, 97%, 98%, 99%, 10%). Nucleic acids encoding immunoglobulin polypeptides containing amino acid sequences that are 0% are also included in this invention. It is clearly included. The present invention provides the maximum between each sequence over the entire length of each reference sequence. The BLAST algorithm is selected so that its parameters give a match. When comparisons are made using this method, at least approximately 70% similarity to any of the reference amino acid sequences is required. Preferably at least about 80% similar, more preferably at least about 90% similar, most preferably Or at least about 95% similar (for example, 95%, 96%, 97%, 98%, 99%, 1%). Further nucleic acids encoding immunoglobulin polypeptides containing amino acid sequences that result in 00%) Provided and included in the present invention.

[0085] As used herein, percent identity of nucleotide and amino acid sequences is determined using the initial alignment parameters and identity parameters of C, MacVector (MacVector, Inc., Cary, NC 27519), Vector NTI (Informax, Inc., MD), Oxford Molecular Gr oup PLC (1996) and the Clustal W algorithm. It can also be determined using these commercially available programs with the same or similar initial parameters for determining sequence similarity. Alternatively, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program with initial parameters can be used, or an Advanced Blast search under the initial setting filter conditions can be used. The following references relate to the BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al. , J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden , T.L., et al., Meth. Enzymol. 266:131-141 (19 96); Altschul, S.F., et al., Nucleic Acids R [[ID= 20]]es. 25:3389-3402 (1997); Zhang, J., et al., Ge

[0086] al., Nature Genet. 20:126-131 (1998). , J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden , T.L., et al., Meth. Enzymol. 266:131-141 (19 96); Altschul, S.F., et al., Nucleic Acids R , T.L., et al., Meth. Enzymol. nome Res.7:649-656(1997);Wootton,J.C.,et al.,Comput.Chem.17:149-163(1993);Hancoc k,J.M.et al.,Comput.Appl.Biosci.10:67-70 (1994);ALIGNMENT SCORING SYSTEMS:Dayhoff ,M.O.,et al.,“A model of evolutionary ch ange in proteins.” in Atlas of Protein S equence and Structure,vol.5,suppl.3.M.O. Dayhoff(ed.),pp.345-352,(1978);Natl.Biom ed.Res.Found.,Washington,DC;Schwartz,R.M .,et al.,“Matrices for detecting distant relationships.”in Atlas of Protein Sequ ence and Structure,vol.5,suppl.3.”(1978) ,M.O.Dayhoff(ed.),pp.353-358(1978),Natl. Biomed.Res.Found.,Washington,DC;Altschul ,S.F.,J.Mol.Biol.219:555-565(1991);State s,D.J.,et al.,Methods 3:66-70(1991);Heni koff,S.,et al.,Proc.Natl.Acad.Sci.USA 89 :10915-10919(1992);Altschul,S.F.,et al., J.Mol.Evol.36:290-300(1993);ALIGNMENT ST ATISTICS:Karlin,S.,et al.,Proc.Natl.Acad .Sci.USA 87:2264-2268(1990);Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 90:5873-5877 (1993);Dembo,A.,et al.,Ann.Prob.22:2022- 2039 (1994); and Altschul, SF “Evaluating t he statistical significance of multiple distinct local alignments.”in Theoretica l and Computational Methods in Genome Re search(S.Suhai,ed.),pp.1-14,Plenum,New Y ork (1997).

[0087] The antibodies and / or bispecific antibodies of the present invention are recombinant by methods known in the art. It can be produced in a host for expressing the antibody or fragment disclosed herein. Mammalian cell lines available for use as American Typ are well known in the art, Many immortalized creatures are available from the e Culture Collection (ATCC) This includes cell lines, particularly Chinese hamster ovary (CHO) cells. NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells This includes cells, HEK-293 cells, and numerous other cell lines. Mammalian host cells include It contains cells from mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamsters. This allows us to determine which cell lines have high expression levels, thereby identifying particularly favorable details. Cell lines are selected. Other cell lines that can be used include insect cell lines such as Sf9 cells, and amphibian cell lines. Cells, bacterial cells, plant cells, and fungal cells. Heavy chains or their antigen-binding portion or cleavage. Recombinant expression vectors encoding fragments, light chains, and / or their antigen-binding fragments are used to host mammals. When introduced into chief cells, the antibody will suppress antibody expression in the host cells, or more preferably, the antibody will suppress antibody expression in the host cells. This is sufficient to allow the host cells to secrete antibodies into the culture medium in which they are growing. It is produced by culturing host cells over a certain period of time.

[0088] Antibodies can be recovered from the culture medium using standard protein purification methods. Furthermore, the expression of the antibody (or other part thereof) from the producing cell line is as follows: It can be enhanced using known techniques. For example, the glutamine synthetase gene. Expression systems (GS systems) are a common approach to enhance expression under specific conditions. The GS series is based on European Patent No. 0216846, No. 0256055, and No. 0323. In relation to Patent No. 997 and European Patent Application No. 89303964.4, in whole or in part It is being discussed in detail.

[0089] Generally, glycoproteins are produced in specific cell lines or transgenic animals. This is characteristic of the glycoproteins produced in that cell line or transgenic animal. It has a specific glycosylation pattern. Therefore, the specific glycosylation pattern of the antibody is It depends on the specific cell line or transgenic animal used to produce the antibody. However, the nucleic acid molecules provided herein are encoded by or are specified herein All antibodies containing the provided amino acid sequence may have a glycosylation pattern. The present invention is independent of the above. Similarly, in certain embodiments, the non-fucosylated Antibodies having a glycosylation pattern consisting solely of N-glycans are typically used in vitro. It has shown stronger efficacy than their fucosylated counterparts both in vivo and in vivo. These antibodies may be advantageous, as shown [e.g., Shinkawa et al.] al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent See issues No. 6,946,292 and No. 7,214,775.

[0090] antibody manipulation Antibodies also include light chain constant regions, such as the λ or κ canine light chain constant region or its variants. It may include a canine light chain steady region, for example, not limited to a canine heavy chain steady region. The region is a modified cFc such as IgG-B or IgG-Bm as used herein. [U.S. Patent No. 10,106,607, which is incorporated in its entirety herein by reference] [Reference] may originate from, and the canine light chain constant region may originate from the κ light chain.

[0091] The antibodies and / or bispecific antibodies of the present invention are, for example, used to improve the properties of antibodies. Canine phryphal in the variable domain of parental (i.e., mouse or rat) monoclonal antibody It can be manipulated to include modifications to muwerk and / or canine flame residues. ru.

[0092] Pharmaceutical composition and administration Prepare a pharmaceutical or sterile composition containing the antibody and / or bispecific antibody of the present invention. Therefore, these antibodies and / or bispecific antibodies are pharmaceutically acceptable carriers or It can be mixed with excipients. [For example, Remington's Pharmac eutical Sciences and USPharmacopeia:Nati onal Formulary,Mack Publishing Company,E See Aston, PA (1984).

[0093] Formulations of therapeutic and diagnostic agents include, for example, lyophilized powders, slurries, aqueous solutions, or Prepared by mixing with an acceptable carrier, excipient, or stabilizer in the form of a suspension. Obtain [For example, Hardman, et al. (2001) Goodman and G ilman's The Pharmacological Basis of The rapeutics,McGraw-Hill,New York,NY;Gennar o(2000) Remington: The Science and Practical e of Pharmacy, Lippincott, Williams, and Wi. lkins, New York, NY; Avis, et al. (eds.) (1993 )Pharmaceutical Dosage Forms: Parental Medications,Marcel Dekker,NY;Lieberman,e t al.(eds.)(1990)Pharmaceutical Dosage F orms:Tablets,Marcel Dekker,NY;Lieberman, et al.(eds.)(1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxi city ​​and Safety,Marcel Dekker,Inc.,New Y [See ork, NY]. In one embodiment, the antibody and / or bispecific antibody of the present invention Sodium acetate solution is diluted to an appropriate concentration at pH 5-6, and for isotonic purposes, NaCl or Sucrose is added. To improve stability, polysorbate 20 or polysorbate Further active ingredients such as 80 may be added.

[0094] Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with other active agents Sex is, for example, LD 50 (A lethal dose for 50% of the population) and ED 50 (group To determine the therapeutically effective dose (in 50% of cases) in cell cultures or experimental animals This can be determined by standard pharmaceutical procedures. The dose ratio between toxic effects and therapeutic effects. However, the treatment index (LD 50 / ED 50 ) In certain situations, it shows a high treatment index. Antibodies are preferred. The data obtained from these cell culture assays and animal studies are This can be used when formulating the dosage range for use in Nu. The dosage of the compound is preferably such that it has little to no toxicity during ED. 50 Circulation including The concentration is within the specified range. The dosage will be within this range, depending on the dosage form and route of administration used. It can fluctuate.

[0095] The mode of administration may vary. Appropriate routes of administration include oral, rectal, transmucosal, intestinal, and parenteral administration. Intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct ventricle, intravenous, abdominal, nasal, intraocular, inhalation , including by airflow, local, skin, transdermal or intra-arterial. In certain embodiments, the antibody of the present invention and The / or bispecific antibody can be administered via an invasive route such as injection. In a further embodiment of the present invention, the antibody and / or bispecific antibody of the present invention, or These pharmaceutical compositions are administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation or aerosol delivery. It is administered by non-invasive routes (e.g., orally; e.g., pills, capsules, or tablets). Administration by means of a drug is also within the scope of this invention.

[0096] The composition can be administered using medical devices known in the art. For example, this invention The pharmaceutical composition is administered by injection, for example, by subcutaneous needle, including a pre-filled syringe or an auto-injector. It can be administered by injection. The pharmaceutical compositions disclosed herein are also U.S. Patent No. 6,620,135; No. 6,096,002; No. 5,399,163; No. No. 5,383,851; No. 5,312,335; No. 5,064,413; No. 4 Disclosed in Nos. 941,880; Nos. 4,790,824 or Nos. 4,596,556. It can be administered using needleless subcutaneous injection devices such as those provided.

[0097] The pharmaceutical compositions disclosed herein may also be administered by infusion. Pharmaceutical compositions Well-known examples of implant and modular forms for administering drugs include drugs delivered at a controlled rate. U.S. Patent No. 4,487,603 discloses an embedded micro-injection pump for distributing materials. No. 4,4 discloses a drug infusion pump for delivering drugs at a precise infusion rate. Patent No. 47,233; U.S. Patent disclosing a variable flow rate implantable infusion device for continuous drug delivery. Patent No. 4,447,224; Disclosure of an osmotic drug delivery system having multiple chambers. This includes U.S. Patent No. 4,439,196. Many other such implants, delivery The system and modules are well known to those skilled in the art.

[0098] Alternatively, the antibodies and / or bispecific antibodies of the present invention may be local rather than systemically It can often be administered in depot formulations or sustained-release formulations.

[0099] The administration regimen involves serum or tissue turnover of therapeutic antibodies and / or bispecific antibodies. Speed, level of symptoms, immunogenicity of therapeutic antibodies and / or bispecific antibodies, and life It depends on several factors, including the reachability of target cells within the physical matrix. Furthermore, the administration regimen should simultaneously minimize undesirable side effects while targeting the disease / symptom. Deliver sufficient therapeutic antibodies and / or bispecific antibodies to bring about improvement in the condition. Therefore, the amount of biological agents delivered is, in part, specific therapeutic antibodies and / or bispecific antibodies, and depending on the severity of the symptoms being treated. Therapeutic antibodies Guidelines are available for selecting the appropriate dosage [for example, Wawrzynczak Antibody Therapy,Bios Scientific Pub.Ltd ,Oxfordshire,UK(1996);Kresina(ed.)Monocl onal Antibodies,Cytokines and Arthritis, Marcel Dekker,New York,NY(1991);Bach(ed. )Monoclonal Antibodies and Peptide Thera py in Autoimmune Diseases,Marcel Dekker, New York,NY(1993);Baert,et al.New Engl.J .Med.348:601-608(2003);Milgrom et al.New Engl.J.Med.341:1966-1973(1999);Slamon e t al.New Engl.J.Med.344:783-792(2001);Be niaminovitz et al.New Engl.J.Med.342:613 -619(2000);Ghosh et al.New Engl.J.Med.34 8:24-32(2003);Lipsky et al.New Engl.J.Me d.343:1594-1602 (2000)].

[0100] Determining the appropriate dosage is, for example, known in the art to affect the treatment. Or it is done by a veterinarian using the suspected parameter or factor. Generally, dosage It is recommended to start with a dose somewhat lower than the optimal dose, and then continue until the desired dose is achieved compared to any negative side effects. The dosage is increased in small increments until the optimal effect is achieved. Important diagnostic measures include symptoms. This includes the diagnostic scale.

[0101] The antibodies and / or bispecific antibodies provided herein are administered by serial injection. , or for example, daily, 1-7 times a week, weekly, bi-weekly, monthly, every other month, every three months, every six months, It may be provided in doses administered annually, etc. Dosages may be, for example, intravenous, subcutaneous, or topical. It may be administered orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. Total 1 week supply. The amount is generally at least 0.05 μg / kg body weight, and more generally at least 0.2 μg / kg, 0.5μg / kg, 1μg / kg, 10μg / kg, 100μg / kg, 0.2 5mg / kg, 1.0mg / kg, 2.0mg / kg, 5.0mg / ml, 10mg / k g, 25 mg / kg, 50 mg / kg or more [e.g., Yang, e t al.New Engl.J.Med.349:427-434(2003);He rold,et al.New Engl.J.Med.346:1692-1698( 2002);Liu,et al.J.Neurol.Neurosurg.Psych .67:451-456(1999);Portielji,et al.Cancer Immunol.Immunother.52:133-144(2003)]. 0. 1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more Furthermore, a predetermined target concentration of the antibody and / or bispecific antibody of the present invention in canine serum can be achieved. Doses for this purpose may also be provided. In other embodiments, the antibody and / or dual feature of the present invention Heterosexual antibodies are 10, 20, 50, 80, 100, 200, 500, 1000, or 250. 0 mg / target, administered subcutaneously weekly, bi-weekly, every four weeks, monthly, every other month, or every three months. It is administered intravenously.

[0102] As used herein, “inhibit,” “treat,” or “treat” means “impede.” This includes delay in the onset of symptoms related to and / or a decrease in the severity of symptoms of such disorders. These terms further describe improving existing uncontrolled or undesirable symptoms. To prevent further symptoms and to improve or prevent the underlying causes of such symptoms. This includes having a disorder, symptoms and / or signs. Therefore, these terms include having a disorder, symptoms and / or signs. or targeting vertebrates that are likely to develop such disorders, diseases or symptoms (e.g.) For example, it indicates that a beneficial result was given to the dog.

[0103] As used herein, "therapeutic effective dose," "therapeutic effective amount," and "effective dose" are used in this specification. The term refers to the use of cells, tissues, or subjects, either alone or in combination with additional therapeutic agents, for example, When administered to Nu, one or more symptoms of a disease or symptoms of such a disease or The antibodies and / or two antibodies of the present invention are effective in causing a measurable improvement in the progression of symptoms. This refers to the amount of heavy-specific antibodies. The therapeutically effective dose is further defined as at least partial improvement of symptoms, for example. If so, treatment, cure, prevention or improvement of related medical symptoms, or treatment of such symptoms , sufficient antibodies and / or dual traits to bring about an increased rate of healing, prevention, or improvement This refers to the amount of heterozygous antibodies. When applied to combinations, the therapeutically effective dose is the combined dose. Whether administered sequentially or simultaneously, the combination of active ingredients that produce a therapeutic effect This refers to the amount administered. An effective dose of the drug is at least 10% of the diagnostic scale or parameter; Usually, at least 20%; preferably at least about 30%; more preferably at least 40% %, most preferably at least 50%, improvement will be achieved. The effective dose also depends on the symptoms. To bring about improvement in subjective scales when subjective scales are used to assess severity. It is possible.

[0104] Other combination therapies The composition comprising the antibody and / or bispecific antibody of the present invention provides one or more further therapeutic effects. It can contain [a certain amount]. One such family of therapeutic components is Janus kinase (J It is an AK inhibitor. In a particular embodiment of this type, a JAK inhibitor has the following chemical formula, and its pharmaceutically acceptable salts: [ka] In the formula, R 1 C may be substituted with hydroxyl. 1~4 Alkyl [US Patent] [Patent No. 8,133,899; U.S. Patent No. 8,987,283]. More specifically, JAK The inhibitor is oclacitinib, and more specifically, oclacitinib maleate. Yes, there are other JAK inhibitors that preferentially inhibit JAK1 compared to JAK3. [ka] 1-[(3R,4S)-4-cyanotetrahydropyran-3-yl] has the chemical formula -3-[(2-fluoro-6-methoxy-4-pyridyl)amino]pyrazole-4-cal Boxamide and its pharmaceutically acceptable salts [International Publication No. 2018 / 1089] See issue 69.

[0105] Another therapeutic component that can be added to the composition of the present invention is spleen tyrosine kinase (SYK) inhibitor. It could be a drug. One such SYK inhibitor is (1S,4R)-4-hydroxy-2 ,2-dimethyl-4-{5-[3-methyl-5-(4-methylpyrimidine-2-yl Mino)-phenyl]-1,3-thiazol-2-yl}cyclohexanecarboxylic acid or It is a pharmaceutically acceptable salt [see, for example, U.S. Patent No. 8,759,366]. .

[0106] Furthermore, yet another therapeutic component that can be added to the composition of the present invention is, [ka] An antagonist to a chemoattractant receptor homologous molecule expressed on TH2 cells, having the chemical formula [chemical formula]. This can be a tagagonist or a pharmaceutically acceptable salt thereof [U.S. 7,699] No. 6,222, U.S. No. 8,546,422, U.S. No. 8,637,541, Country International Publication No. 2010 / 099039; International Publication No. 2010 / 031183; and United States See also Patent No. 8,546,422.

[0107] These further therapeutic components include compositions comprising the antibody and / or bispecific antibody of the present invention. It can be administered to dogs before, together with, or after the administration of other substances. [Examples]

[0108] General materials and methods: In all of the following examples, recombinant proteins are selected. The amino acid sequence of the modified protein was provided to commercial manufacturers (ATUM, Newark, Cali Obtained by providing (fornia), commercial manufacturers may use this amino acid sequence. I selected the appropriate nucleotide sequence to encode. The nucleotide sequence is from GenBank( It can also be obtained from publicly available DNA databases such as (registered trademark). Commercial manufacturers chemically synthesize nucleic acids, and then the synthesized nucleic acids are processed by ATUM. Then, to produce the corresponding recombinant protein, an expression plasmid (pD2610-v10 Cloned (available from AUTM). The plasmid was used in HEK-293 cells. Alternatively, place it in either a CHO cell and express the recombinant protein, and then express The recombinant protein was isolated using conventional methods.

[0109] [Example 1] Canine IL-22 and anti-canine IL-22 antibodies A nucleic acid encoding canine IL-22 with a C-terminal HIS tag was chemically synthesized, and HEK In eukaryotic cells, either -293 or CHO cells, the canine IL-22-HIS protein The protein was cloned into an expression plasmid suitable for quality production. The resulting canine protein was sequenced. It contains amino acid sequence number 1.

[0110] cIL-22-HIS:[Sequence ID 1] LPISSHCLDKSNFQQPYITNRTFMLAKEASLADNNTDV RLIGEKLFHGVNMGERCYLMKEVLNFTLEEVLLPQSDRFQ PYMQEVVPFLARLSNKLSQCHIENDDQHIQRNVQKLKDTV QKLGENGEIKAIGELDLLFMALRNACVHHHHHH

[0111] Furthermore, a human monoclonal antibody that binds to canine IL-22 [fezakinumab; WHO] Disclosed in Drug Information, Vol.24, No.1, 2010 The nucleic acids encoding the heavy and light chains of (HE) are chemically synthesized and used in eukaryotic cells. Expression that produces anti-canine IL-22 antibody (in either K-293 or CHO cells) The plasmids were cloned separately. The amino acid sequence of fezakinumab SEQ ID NO: 2 is included. The heavy chain and the light chain containing the amino acid sequence of SEQ ID NO: 3 are reproduced below.

[0112] Human fezakinumab heavy chain: [SEQ ID NO: 2] Prior art QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQ APGQGLEWVGWINPYTGSAFY AQKFRGRVTMTRDTSIST AYMELSRLRSDDTAVYYCAREPEKFDSDDSDVWGRGTLVT VS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYT LPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG

[0113] Human fezakinumab light chain: [SEQ ID NO: 3] Prior art QAVLTQPPSVVSGAPGQRVTISCTGSSSNIGAGYGVHWYQ QLPGTAPKLLIYGDSNRPSGV PDRFGSGSKSGTSASLAIT GLQAEDEADYYCQSYDNSLSGYVFGGGTQLTVLGQPKAAP SV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS SPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTECS

[0114] The complementarity-determining region (CDR), which is useful for constructing canine antibodies that bind to canine IL-22, is used as an anti- Tables 1A and 1B illustrate the LC CDR and HC CDR of fezakinumab. ru.

[0115] [Table 2]

[0116] [Table 3]

[0117] Anti-canine IL-22 antibodies useful in this invention also include the following heavy-chain and light-chain pairs. This can also be illustrated by the canine antibodies produced from the combination. The following are examples of canine antibodies. The amino acid sequences of the light and heavy chains that make up the antibody pair, i.e., SEQ ID NO: 20 and Items 21, 22 and 23 and 24 and 25 contain the respective CDRs of the antibody fezakinumab. The heavy chain contains the modified Fc, IgG-Bm as defined above.

[0118] cFezaVL1-cLC:[Sequence ID 20] QAVLTQPPSVVSAVLGQRVTISCTGSSSNIGAGYGVHWYQ QLPGKSPKTIIYGDSNRPSGVPDRFSGSKSGSTASLTITG LQAEDEADYYCQSYDNSLSGYVFGSGTQLTVLGQPKASPS VTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSP VTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCL VTHEGSTVEKKVAPAECS

[0119] cFezaVH1-cIgG-Bm:[SEQ ID NO: 21] EVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYYMHWVRQ APGAGLDWMGWINPYTGSAFYAQKFRGRVTLTADTSTSTA YMELSSLRAGDIAVYYCAREPEKFDSDDSDVWGQGTLVTV SSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVT VSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPS ETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAP EMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPE VQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQD WLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLP PSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESK YRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHE ALHNHYTQESLSHSPG

[0120] cFezaVL2-cLC:[Sequence ID 22] QAVLTQPPSVVSAVLGQRVTISCTGSSSNIGAGYGVHWYQ QLPGKSPKLLIYGDSNRPSGVPDRFSGSKSGSTASLTITG LQAEDEADYYCQSYDNSLSGYVFGGGTHLTVLGQPKASPS VTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSP VTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCL VTHEGSTVEKKVAPAECS

[0121] cFezaVH2-cIgG-Bm: [SEQ ID NO: 23] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQ APGAGLDWVGWINPYTGSAFYAQKFRGRVTLTRDTSTSTA YMELSSLRAGDIAVYYCAREPEKFDSDDSDVWGQGTLVTV SSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVT VSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPS ETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAP EMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPE VQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQD WLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLP PSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESK YRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHE ALHNHYTQESLSHSPG

[0122] cFezaVL3-cLC: [SEQ ID NO: 24] QSVLTQPPSVSGFLGQRVTISCTGSSSNIGAGYGVHWYQ QLPGTGPRLLIYGDSNRPSGVPDRFSGSRSGSTATLTISG LQAEDEADYYCQSYDNSLSGYVFGGGTHLTVLGQPKASPS VTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSP VTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCL VTHEGSTVEKKVAPAECS

[0123] cFezaVH3-cIgG-Bm:[SEQ ID NO: 25] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQ APGAGLDWVGWINPYTGSAFYAQKFRGRVTMTRDTSTSTA YMELSSLRAGDIAVYYCAREPEKFDSDDSDVWGQGTLVTV SSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVT VSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPS ETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAP EMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPE VQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQD WLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLP PSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESK YRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHE ALHNHYTQESLSHSPG

[0124] The binding of canine IL-22 HIS to the anti-IL-22 antibody fezakinumab is as follows: Decided by LISA: material: 1. Coating antigen: cIL-22-His, 2. Fezakinumab-mAb, 3. Mouse anti-human IgG, Fc peroxidase conjugate, Calbioche m 411550, lot D00167423, 4.1-Step(TM) Ultra TMB-ELISA, Thermo Scie NTific 34028, lot UF2782982, 5. NVSL PBST Lot: 5011318-0206, 6.1.5M Phosphate, MAH Lot 5011318-0219,

[0125] method: 1. Dilute cIL-22-His to 1 μg / mL in PBS and prepare 100 μL / well. Add to the ELISA plate. Incubate overnight at 2-7°C. 2. Wash with 200 μL / well / NVSL-PBST using an automated plate washer. Clean the coated plates (or similar) three times. Block the ELISA plate with 5% NFDM in 3,200 μL of NVSL-PBST. Wrap, seal, and incubate at 37°C for at least 60 minutes. 4. Wash with 200 μL / well / NVSL-PBST using an automated plate washer. Wash the clean, blocked ELISA plate three times. 5. Pre-dilute Feza-mAb to 5 μg / mL and dilute it three times across the dilution plate. Dilute the solution. Transfer 50 μL of diluted Feza-mAb to an ELISA plate and seal it. Then, incubate at 37°C for 30-60 minutes while rotating. 6. Wash with 200 μL / well / NVSL-PBST using an automated plate washer. Clean the plate three times. 7,100 μl / well of anti-human IgG, Fc peroxida diluted 1:2500. Add the -se conjugate, seal, and incubate at 37°C for 60 minutes. 8,100 μl / well of 1-Step® Ultra TMB-ELISA is added. Add. Incubate at 25°C for 10 minutes. Stop with 9,100 μl / well of 1.5 M phosphate. 10. Measure A450~A540 using a spectrophotometer.

[0126] The binding study plot is shown in Figure 1. The results show that the anti-IL-22 antibody dose-dependent on canine IL-22. This indicates that they are coupled in a dependent manner.

[0127] [Example 2] Antibodies against IL-4 receptor α A particularly preferred anti-IL-4 receptor α antibody useful in the composition of the present invention is antibody c152. H11VL3-cCLk-s / c152H11VH3-cIgG-Bm and antibody c14 Exemplify by 6E2VL3-cCLk-s / c146E2VH3-cIgG-Bm The individual light chain (LC) and heavy chain (HC) sequences of these antibodies are provided below.

[0128] c152H11VL3-cCLk-s(light chain):[Sequence ID 4] EIVMTQSPASLSLSQEEKVTITCKASQNVGTNVAWYQQK PGQAPKLLIYSASYRYSGLPDRFSGSGSGTDFSFTISSLE PEDVAEFFCQQYNSYPYTFGQGTKLEIKRNDAQPAVYLFQ PSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGI QESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKS LPSTLIKSFQRSECQRVD

[0129] c152H11VH3-cIgG-Bm(heavy chain):[SEQ ID NO: 5] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQ APDKRLQWVATISRGGDYTYYPDSVKGRFTISRDNAKNTL YLQMNSLRAEDTAMYYCARGTLNNRGFASWGQGTLVTVSS ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS WNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSET FTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPS REELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYR TTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG

[0130] c146E2VL3-cCLk-s(light chain):[Sequence ID 6] DIVLTQTPLSLSVSPGETASIYCRASESVDSYGNSFLNW YQQKPGQPPKLLIYRASNLASEIPDRFSGSGSRTEFTLKI SRVEADDAGVYYCQQNYENPRTFGQGTKLEIKRNDAQPAV YLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQ DTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEI THKSLPSTLIKSFQRSECQRVD

[0131] c146E2VH3-cIgG-Bm(heavy chain):[SEQ ID NO: 7] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQ APGAGLLDWIGMIHPDSGNINYNERFKTKATLTVDKSTSTA YMELSSLRAGDIAVYYCARQLRNAMDYWGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSRE ELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG

[0132] The ability to inhibit STAT-6 phosphorylation in DH82 cells is as follows: Antibodies against canine IL-4 receptor α were tested: material 1. Actively proliferating DH82 cells 2. DH82 Cell Growth Media (ATCC (Registered Trademark) 3020) 03 (trademark), thermo-inactivated fetal bovine serum was supplied to achieve a final concentration of 15% w / v. Eagle's Minimum Essential Medium) 3.AlphaLISA p-STAT6(Tyr641) Assay Kit:Pe rkin Elmer catalog: ALSU-PST6-A-HV 4. Recombinant Canine IL-4: R&D Systems, Catalog: 752-CL / CF 5. Recombinant Canine IL-13: R&D Systems, Catalog: 5894-CL / C F 6. Perkin Elmer Envision a. Canine-type anti-canine IL-4R α Monoclonal antibodies b.c146E2-H3L3 c.c152H11-H3L3 d.c4H3

[0133] method 1. Place 8 x 10⁶ cells per well in two tissue culture plates. 4 DH82 cells (4 × 10) 5 200 μL of cells were seeded at a density of cells / mL and incubated overnight at 37°C. 2. Pre-dilute the test antibody to 500 μg / mL, then use DH82 Cell Grow The culture medium was serially diluted 3-fold with th Media. The medium was removed from the cell culture plate, and 50 μL / The serially diluted test samples from the wells were transferred to each plate. 3. Increase canine IL-4 to 5 ng / mL using DH82 Cell Growth Media. Dilution was performed, and 50 μL was added to each well of one plate. DH82 Cell Gro Dilute canine IL-13 to 10 ng / mL with wth Media, and 50 μL is added to the second plate. The solution was added to each well of the plate. The plate was incubated at 37°C for 15 minutes. 4. Remove the culture medium from the plate and add 100 μL / well of AlphaLISA pS The freshly prepared 1x lysis buffer from the TAT-6 Assay Kit is attached to the plate. The plate was then stirred on a 350 rpm plate shaker at room temperature for 10 minutes. 5. Acceptor from AlphaLISA p-STAT6 Assay Kit Prepare the mix and add 30 μL of cell lysate to 96-well 1 / 2 Area Plates. 15 μL / well was added to the solution. The plate was sealed and stirred at 350 rpm for 2 minutes. Next, it was incubated at room temperature for 2 hours. 6. Under reduced laboratory lighting, AlphaLISA p-STAT6 Assay Donor Mix was prepared from the kit and 15 μL / well was added to each plate. Seal the sheet, cover with foil, stir at 350 rpm for 2 minutes, then let the ink steep at room temperature for 2 hours. I did it. 7. Use AlphaScreen settings in Perkin Elmer EnVision I used it to read the plate.

[0134] Regarding the ability to inhibit αSTAT-6 phosphorylation, c4H3 [International Publication No. 2016 / 1 [No. 56588], called c146E2-H3L3 and c152H11-H3L3 Different canine monoclonal anti-canine IL-4R α Antibodies, canine IL-4R α Dogs Evaluation was performed by blocking the binding of either IL-4 or canine IL-13. Figure 2 The data shown indicates that all three antibodies phosphorylated STAT-6 in the presence of IL-4. It results in dose-dependent inhibition of both c146E2-H3L3 and c152H11-H3L. The prior art anti-canine IL-4 receptor α antibody c4H3 [International Publication No. 2016 / 1565]

[88] indicates stronger bonding. IL-4Rα (IL-4R α ) In the absence of antibodies The IL-4 control in Figure 3 is shown in the upper left portion of the graph. All three data shown are from the same source. These antibodies induce dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13. c146E2-H3L3 and c152H11-H3L are both prior art anti-canine ILs. It shows that it binds more strongly to the IL-4 receptor α antibody c4H3. α The IL-13 control in the absence of the antibody is shown in the upper left portion of the graph.

[0135] [Example 3] Canine IL-31 receptor α and antibodies against IL-31 receptor α Nucleotide sequence The nucleotide sequence of Sequence ID No. 8 is the canine IL-31 receptor α fused to the HIS tag. Encodes the extracellular domain of IL-31RA. Canine IL-31RA ECD HIS The tagged protein contains the amino acid sequence of SEQ ID NO: 9. It is synthesized chemically to produce nucleotides. Prepare the sequence and then, in either eukaryotic cells, HEK-293 cells, or CHO cells... The corresponding protein was then cloned into an expression plasmid suitable for its production.

[0136] Canine IL-31RA ECD-10His:[Sequence ID 8] gtgctgcccgccaagcccgagaacatcagctgcatcttc tactacgaggagaacttcacctgcacctggagccccgaga aggaggccagctacacctggtacaaggtgaagagaaccta cagctacggctacaagagcgacatctgcagcaccgacaac agcaccagaggcaaccacgccagctgcagcttcctgcccc ccaccatcaccaaccccgacaactacaccatccaggtgga ggcccagaacgccgacggcatcatgaagagcgacatcacc tactggaacctggacgccatcatgaagatcgagccccccg agatcttcagcgtgaagagcgtgctgggcatcaagagaat gctgcagatcaagtggatcagacccgtgctggccccccac agcagcaccctgaagtacaccctgagattcagaaccatca acagcgcctactggatggaggtgaacttcaccaaggagga catcgacagagacgagacctacaacctgaccgagctgcag gccttcaccgagtacgtgatgaccctgagatgcgcccccg ccgagagcatgttctggagcggctggagccaggagaaggt gggcaccaccgaggaggaggccccctacggcctggacctg tggagagtgctgaagcccgccatggtggacggcagaagac ccgtgcagctgatgtggaagaaggccaccggcgcccccgt gctggagaaggccctgggctacaacatctggtacttcccc gagaacaacaccaacctgaccgagaccgtgaacaccacca accagacccacgagctgtacctgggcggcaagacctactg ggtgtacgtggtgagctacaacagcctgggcgagagcccc gtggccaccctgagaatccccgccctgaacgagaagacct tccagtgcatcgaggccatgcaggcctgcctgacccagga ccagctggtggtggagtggcagagcagcgcccccgaggtg gacacctggatggtggagtggttccccgacgtggacagcg agcccagcagcttcagctgggagagcgtgagccaggccag aaactggaccatccagaaggacgagctgaagcccctgtgg tgctacaacatcagcgtgtaccccgtgctgagagacagag tgggccagccctacagcacccaggcctacgtgcaggaggg catccccagcgccggccccgtgacccaggccgacagcatc ggcgtgaagaccgtgaccatcacctggaaggagatcccca agagcaagagaaacggcttcatcaagaactacaccatctt ctaccaggccgaggacggcaaggagttcagcaagaccgtg aacagcaacatcctgcagtacagactggagagcctgacca gaagaaccagctacagcctgcaggtgatggccagcaccaa cgccggcggcaccaacggcaccaagatcaacttcaagacc ctgagcatcagccaccaccaccaccaccaccaccacc ac

[0137] Expression and purification of IL-31 receptor αECD Using electroporation via MaxCyte equipment as recommended by the manufacturer, Sequence ID No. 8 Transplants containing the nucleotide sequence into HEK-293 or CHO cells Several days after translocation, the supernatant of translocated cells and non-translocated controls was collected and centrifuged. The solution was allowed to settle and cell debris was removed. Clarification from plasma-transferred cells was performed according to the manufacturer's recommendations. The collected liquid is then passed through a nickel column to obtain IL with a histidine tag. -31RA was purified from the cell culture medium. By measuring the absorbance of ultraviolet light at 280 nm... Then, the purified protein was quantified.

[0138] Canine IL-31RA ECD-10His:[Sequence ID 9] VLPAKPENISCIFYYEENFTCTWSPEKEASYTWYKVKRT YSYGYKSDICSTDNSTRGNSASCSFLPPTITNPDNYTIQV EAQNADGIMKSDITYWNLDAIMKIEPPEIFSVKSVLGIKR MLQIKWIRPVLAPHSSTLKYTLRFRTINSAYWMEVNFTKE DIDRDETYNLTELQAFTEYVMTLRCAPAESMFWSGWSQEK VGTTEEEAPYGLDLWRVLKPAMVDGRRPVQLMWKKATGAP VLEKALGYNIWYFPENNTNLTETVNTTNQTHELYLGGKTY WVYVVSYNSLGESPVATLRIPALNEKTFQCIEAMQACLTQ DQLVVEWQSSAPEVDTWMVEWFPDVDSEPSSFSWESVSQA RNWTIQKDELKPLWCYNISVYPVLRDRVGQPYSTQAYVQE GIPSAGPVTQADSIGVKTVTITWKEIPKSKRNGFIKNYTI FYQAEDGKEFSKTVNSNILQYRLESLTRRTSYSLQVMAST NAGGTNGTKINFKTLSISHHHHHHHHHH

[0139] Binding of canine IL-31RA to biotinylated canine IL-31: 1. By diluting the immunotherapy plate (or similar) to 10 μg / mL in PBS, the IL Coat with -31RA protein. Add 100 μL / well. Plate ( Incubate the samples (or similar) overnight at 2-7°C. Wash the plate three times with 2.275 μL / well PBST. 3. Gently shake (120±20 RPM) at 36±2℃ for 30-45 minutes. Plate with 00 μL / well of blocking buffer (1% Dry Milk in PBST). Block it. Wash the plate three times with 4.275 μL / well of PBST. 5. Dilute biotinylated IL-31 to 10 ug / mL in 1% NFDM in PBST. . 6. Biotinylated IL-31 (10 μg / mL) is diluted three times in 1% NFDM in PBST. Dissolve and transfer 100 μL / well to an immunoassay plate (or similar). Gently shake (120° Incubate at 36±2℃ for 30-45 minutes at ±20 RPM. Wash the plate three times with 7.275 μL / well of PBST. 8. In 1% NFDM in PBST, HRP-streptavidin was added in a 1:1000 final Dilute according to the dilution ratio. Add 9,100 μL / well of HRP-streptavidin to an immunoassay plate(s). Incubate at 36±2℃ for 30-45 minutes while gently shaking (120±20 RPM). To do. Wash the plate three times with 10.275 μL / well of PBST. 11. Assemble an equal volume of preheated TMB 2-Component substrate immediately before use. match. Add 12,100 μL / well of the prepared TMB substrate to the immunoassay plate(s), and gently immerse. Incubate in the dark for 10-15 minutes at 36±2℃ with gentle shaking (120±20 RPM). Bet. The reaction was stopped by adding 13,100 μL / well of 1 M H3PO4. ru. 14. Use a microplate reader with a wavelength of 450 nm and a reference wave of 540 nm. Read the plate using the long key.

[0140] Monoclonal antibody against canine IL-31 receptor α For 3-4 weeks, administer canine IL-31RA ECD (10 μg or 25 μg each time). By immunizing two Lewis rats multiple times using the antigen / rat, Monoclonal antibodies against IL-31RA were produced. After immunization, serum was obtained from each rat. The samples were collected and tested for canine IL-31RA by ELISA. The highest IL-31 Lymph node cells from rats that exhibit RA ECD responsiveness were fused with myeloma SP2 / 0 cell lines. Then, a hybridoma was created. About 10 days after fusion, from the proliferating hybridoma... The supernatant is then placed on a plate coated with IL-31RA ECD protein, as follows: Screening was performed using ELISA with the protocol described. In A, approximately 263 clones showing potential binding to IL-31RA were selected. Most of the clones had an OD > 1.

[0141] ELISA procedure: A 1.96-well half-region plate was treated with IL-31RA (1 μg / mL in PBS buffer). Coat with 25 μL / well. Incubate the plate overnight at 4°C. 2. Wash the plate three times with PBST (PBS + 0.05% Tween20). 3. Blocking buffer (PBS containing 5% FBS), 25 ul / well, plate Block it at room temperature for 30 minutes. Transfer 4.25 ul / well of hybridoma supernatant to a 96-well plate and allow to stand at room temperature for 60 minutes. Incubate for 1 minute. 5. Wash the plate three times with PBST. 6. Add 25 ul / well of anti-rat HR solution diluted 1:4000 to the blocking buffer. Add P to the plate and incubate at room temperature for 60 minutes. 7. Wash the plate five times with PBST. 8. Add the TMB reagent to the plate for a colorimetric reaction lasting 2-3 minutes. 9.0. The reaction is stopped with 16M sulfuric acid. 10. Read the plate using a plate reader.

[0142] Blocking activity of anti-IL-31 receptor α antibody Anti-canine IL-31RA hybridoma supernatant blocks the binding of IL-31 to IL-31RA. The ability to block was evaluated using the following blocking ELISA. Of the 263 clones that showed compatibility, approximately 24 clones were found to be I of IL-31RA. This demonstrated potential blockade of L-31 binding. A 1.96-well half-region plate was treated with IL-31RA (1 μg / mL in PBS buffer). Coat with 25 μL / well. Incubate the plate overnight at 4°C. 2. Wash the plate three times with PBST (PBS + 0.05% Tween20). . 3. Blocking buffer (PBS containing 5% FBS), 25 ul / well, plate Block it at room temperature for 30 minutes. Transfer 4.25 ul / well of hybridoma supernatant to a 96-well plate and allow to stand at room temperature for 60 minutes. Incubate for 1 minute. 5. Wash the plate three times with PBST. 6.25 μL / well of biotinylated IL-31 (0.5 μg / mL in blocking buffer) Transfer the contents to the appropriate container and incubate at room temperature for 60 minutes. 7. Wash the plate three times with PBST. 8. Add 25 μl / well of strepto solution diluted 1:5000 to the blocking buffer. Add Vigin-HRP to the plate and incubate at room temperature for 60 minutes. Wash the plate five times with PBST. Add the TMB reagent to the plate for a colorimetric reaction of 2-3 minutes. Stop the reaction with 0.16M sulfuric acid. • Read the plate using a plate reader.

[0143] Biological activity of anti-IL-31RA antibodies: Full-length canine IL-31RA chain and full-length canine oncostatin M receptor (OSMR) chain Using a Baf3 cell line transfected with the nucleotide sequence, STAT-3 activation was performed. The ability of the anti-IL-31RA antibody to inhibit it will be evaluated. This cell line also contains luciferase Translocation occurs via porter genes. The nucleotide sequences of these receptors are synthesized chemically. Prepare the solution, and then use an expression plasmid suitable for the expression of the corresponding protein in Baf3 cells. I cloned it into the mud.

[0144] The ability of anti-IL-31 receptor α antibody to inhibit STAT-3 activation in Baf3 cells The strength is evaluated as follows: 1.5 × 10 5 Prepare a cell suspension with a viable cell density of cells / mL, and use a 96-well set. Add 200 μL to each well of the culture plate (1 × 10 5 Cells / wells). Humidify Incubate the plates in a 37°C incubator for 22-24 hours. 2. Remove the culture medium from the plate and add 200 μL of Eagle's Minimum Essential Medium (EMEM). Add to each well of the cell plate. Return the plate to a 37°C incubator for 2 hours. 3. Remove the culture medium from the cell plate and grow the anti-IL-31RA hybrid cells in PBS. Add 50 μL / well supernatant from the doma. Incubate the plate at 37°C for 1 hour. Add 80 ng / mL of cIL-31 diluted in PBS to 50 μL / well. Next, incubate the plate at 37°C for 5 minutes. 4. Remove the culture medium from the plate and add 100 μL / well of freshly prepared 1× lysis buffer. Add the liquid to all wells. Shake the plate on a plate shaker at 350 rpm at room temperature. Stir for 10 minutes. At this point, the cell lysates can be frozen and stored at -20°C. 5. pSTAT-3 AlphaLISA: -Follow the instructions in the kit, AlphaLISA (registered trademark) SureFire (registered trademark) Prepare the reagents from the (Trademark) Ultra(Trademark) p-STAT3 Assay Kit. - Transfer 30 μL of cell lysate to a 96-well half-region plate and add 15 μL / well Ac Add ceptor mix to the cell lysate. - Seal the plate and leave at room temperature for 1 hour. Cube it. Add 15 μL / well of Donor Mix to the cell plate. Seal the plate. Then, cover with foil and incubate at room temperature for 1 hour or overnight. Note - Donor mix is It is photosensitive. - Perkin Elmer using Alpha Screen Assay settings Read the plate using EnVison.

[0145] Canine antibody against canine IL-31 receptor α (IL-31RA) IL-31 receptor αc10A12VH1-cIgGBm [SEQ ID NO: 42] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMAWVRQ APGKGLQWVASISTGGGNTYYRDSVKGRFTISRDNAKNTL YLQMNSLRAEDTAMYYCAKHGTLYFDYWGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSRE ELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPGK

[0146] IL-31 receptor αc10A12VL5-cCL [SEQ ID NO: 43] QPVLTQPPSLSASLGTTARLTCERSSGDIGDSYVSWYQQ KPGSPPRDLLYVDDQRPSGVSKSFSGSKDTSANAGLLLIS GLQPEDEADYYCQSYDSNIDGPVFGGGTHLTVLGQPKASP SVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGS PVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSC LVTHEGSTVEKKVAPAECS

[0147] Figure 4 shows the binding of IL-31 to the extracellular domain of IL-31R (IL-31RA). A graph is provided showing its ability to bind to canine IL-31. The extracellular domain (ECD) of IL-31RA was tested. The result was that IL-31RA ECD was 0.36 At an EC50 of 79 μg / ml, it was found to bind to biotinylated canine IL-31 in a dose-dependent manner. show.

[0148] [Example 4] Antibodies against canine IL-31 Antibodies that may be useful in the present invention are U.S. Patent No. 9,206,253 and U.S. Patent No. These are antibodies described in Patent No. 10,150,810. Preferably, these antibodies are , having the following light chain and heavy chain sequences: Canine heavy chain sequences from mouse antibody clone M14 and canine IgG-B: [Sequence ID 10] EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWIRK FPGNKLEYMGYISYSGITDYNPSLKSRITISRDTSKNQYY LQLNSVTTEDTATYYCARYGNYGYAMDYWGQGTLVTVSSA STTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSW NSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETF TCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPPEML GGPSVFIFPPKPKDTLLIARTPEVTCVWDLDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRWSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREE LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTP PQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPGK

[0149] Mouse antibody clone M14 and canine light chain sequence from the constant region of the canine light chain: [Sequence ID] 11] DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFMHW YQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTDFTLTI DPVQADDVATYYCQQSYEDPWTFGGGTKLEIKRNDAQPAV YLFQPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQD TGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEIT HKSLPSTLIKSFQRSECQRVD

[0150] Zoe-LC: Canine light chain sequence: [SEQ ID NO: 12] EIVMTQSPASLSLSQEEKVTITCKASQSVSFAGTGLMHW YQQKPGQAPKLLIYRASNLEAGVPSRFSGSGSGTDFSFTI SSLEPEDVAVYYCQQSREYPWTFGQGTKLEIKRNDAQPAV YLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQ DTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEI THKSLPSTLIKSFQRSEC

[0151] Zoe-HC: Canine heavy chain sequence: [SEQ ID NO: 13] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQ APGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTL YLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTLVTVSSA STTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSW NSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETF TCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPPEML GGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQI SWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLK GKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSR EELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRT TPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALH NHYTQESLSHSPG

[0152] [Example 5] bispecific antibody Bispecific antibodies are artificial molecules that can target two different antigens simultaneously. One preferred type of bispecific antibody is Ig, which consists of four different polypeptide chains. These are G-like antibodies. Examples of the bispecific antibodies of the present invention include IL-31RA and IL-4R α This includes antibody molecules that target both simultaneously. Such antibodies are IL-4R α In contrast to It has one HC and one LC chain with specificity, and has specificity for IL-31RA It is formed by the association of one HC and one LC. Each of the heavy and light chains IL-4R α Mutual association of HC and LC of antibodies, and HC of IL-31RA antibodies It facilitates meetings between LCs, and at the same time, it facilitates meetings between each HC and itself, rather than IL-4R. α anti To promote the association of HC derived from the body with HC of the IL-31RA antibody, their amino acids Modified by specific substitutions / mutations of the acid sequence.

[0153] Canine IgG-B Fc contains the amino acid sequence of SEQ ID NO: 14 provided below. Tang et al., [Vet Immunology & Immunopat] It was first defined in hology, 80:259-270 (2001).

[0154] [Table 4]

[0155] Canine IgG-Bm has two amino acid residue substitutions, namely the same as in IgG-B's SEQ ID NO: 14. Canine I, which is naturally present due to the inclusion of D31A and N63A in its amino acid sequence. Unlike γ-B, that is, the aspartic acid residue (D) at position 31 of sequence number 14 and The asparagine residue (N) at position 63 is replaced by an alanine residue (A). The positions of these residues in the noacid sequence are shown in bold and underlined. These two Amino acid residue substitutions are found in naturally occurring canine IgG-B antibody-dependent cell-mediated cytotoxicity (ADCC). and helps significantly reduce complement-dependent cell injury (CDC) [see the full details] See U.S. Patent No. 10,106,607 incorporated herein by reference.

[0156] Table 2 lists amino acid substitutions in antibodies that promote HC heterodimer formation. Listed in A. These substitutions are knuckles into hoes for heavy chain heterodimerization. Different weights are favorable to the Luz approach, or to enable heterodimerization. These amino acid substitutions are favorable for interchain electrostatic attraction. [For a comprehensive consideration of these amino acid substitutions, see Mo ore et al., Methods, 154:38-50 (2019) and Bri Refer to nkmann & Kontermann, MABS, 9:182-212 (2017). Please be informed. ] In the context of the amino acid substitutions listed in Table 2A below, in several aspects In this context, chain 1 refers to the anti-IL-31RA heavy chain (HC), and chain 2 refers to the anti-IL-4R α Refers to HC In another embodiment, chain 1 is anti-IL-4R α It refers to HC, and chain 2 is anti-IL-31RAHC In another embodiment, chain 1 points to IL-22HC and chain 2 points to anti-IL-31HC. This refers to... In another embodiment, chain 1 refers to anti-IL-31HC and chain 2 refers to IL-22H This refers to C.

[0157] [Table 5]

[0158] [Table 6]

[0159] Candidate light chains to be included in bispecific antibodies (such as those listed above) are, for example, If, without altering the light chain in the other antibody pair of the bispecific antibody pair, CL dominates in one of the antibody pairs. By swapping the position of the in-domain with the position of the CH1 domain, the light chain is incorporated into the antibody domain configuration. This can be made suitable for this purpose. CL-VL domain CH1- Other methods include exchange with the VH domain or exchange of the VL and VH domains in one of the antibody pairs. Options are available.

[0160] Therefore, variable light (VL) domains are used in domain exchange as variable heavy (VH) domains. It can be replaced with another domain, that is, one of the various domains within the antibody fab fragment can be replaced with another domain. It can be exchanged with the CH1 domain. In another example, the CH1 domain is exchanged with the (constant light) CL domain. VL-CH1 and VH-CL1 can be obtained. Table 2B shows such domain exchanges. (i) Amino acid sequence at the junction of the VH-CL domain after exchange and (ii) VL- An example of the amino acid sequence at the CH1 domain junction is provided. Another example is VH-CH The entire fab fragment is exchanged so that one domain is exchanged for a VL-CL domain. can.

[0161] Therefore, in order to optimally provide IgG-like bispecific antibodies, a) heavy chain homodimer Modify the antibody's Fc to promote heterodimerization rather than ionization, and b) two light chains There are two types of modifications: one involves modifying the light chain to facilitate association with its corresponding heavy chain. Modifications can be made. The amino acid substitutions listed in Table 2A modify the Fc chain 1 It can be used with respect to chain 2. Suitable for incorporating light chains into bispecific antibodies. To achieve this, you can use a method called cross-mab [Kle in et al.,Methods:154:21-23(2019);Klein et al., MABS, 8:1010-1020 (2016); and Schaefe r et al.,Proc.Nat'l Acad.Sci.108:11187-1 1192 (2011). Using cross-mab technology, it was incorporated into bispecific antibodies. Candidate light chains for this purpose (such as the light chain of the present invention) are, for example, bispecific to the other antibody. Without altering the light chain, the position of the CL domain in one of the antibody pairs is changed to the position of the CH1 domain. By incorporating a light chain into the antibody domain configuration that is replaced by the other configuration, a suitable solution for this purpose can be achieved. This can be done by exchanging the CL-VL domain with the CH1-VH domain or by antibody pairing. Other options are also possible, such as exchanging VL and VH domains in one of them.

[0162] Examples of modified HCs of the antibody of the present invention are as follows:

[0163] Modified HC amino acids of canine M14: [SEQ ID NO: 15] EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWIRK FPGNKLEYMGYISYSGITDYNPSLKSRITISRDTSKNQYY LQLNSVTTEDTATYYCARYGNYGYAMDYWGQGTLVTVSSA STTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSW NSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETF TCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPPEML GGPSVFIFPPKPKDTLLIARTPEVTCVWDLDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRWSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP C REE LSKNTVSL W CLIKDFFPPDIDVEWQSNGQQEPESKYRTTP PQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPGK

[0164] Modified HC of Zoe-HC canine antibody: [SEQ ID NO: 16] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQ APGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTL YLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTLVTVSSA STTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSW NSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETF TCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPPEML GGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQI SWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLK GKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP C R EELSKNTVSL W CLIKDFFPPDIDVEWQSNGQQEPESKYRT TPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALH NHYTQESLSHSPG

[0165] Modified cFezaVH3-cIgG-Bm[SEQ ID NO: 17] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQ APGAGLDWVGWINPYTGSAFYAQKFRGRVTMTRDTSTSTA YMELSSLRAGDIAVYYCAREPEKFDSDDSDVWGQGTLVTV SSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVT VSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPS ETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAP EMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPE VQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQD WLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV C VLP PSREELSKNTVSL S C A IKDFFPPDIDVEWQSNGQQEPESK YRTTPPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHE ALHNHYTQESLSHSPG

[0166] Modified IL-4 receptor αc152H11VH3-cIgG-Bm (heavy chain): [sequence] Number 18] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQ APDKRLQWVATISRGGDYTYYPDSVKGRFTISRDNAKNTL YLQMNSLRAEDTAMYYCARGTLNNRGFASWGQGTLVTVSS ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS WNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSET FTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVC VLPPS REELSKNTVSL S C A IKDFFPPDIDVEWQSNGQQEPESKYR TTPPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG

[0167] Modified IL-4 receptor αc146E2VH3-cIgG-Bm:(heavy chain):[sequence] Number 19] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQ APGAGLLDWIGMIHPDSGNINYNERFKTKATLTVDKSTSTA YMELSSLRAGDIAVYYCARQLRNAMDYWGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSV C VLPPSRE ELSKNTVSL S C A IKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFL V SKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG

[0168] Modified IL-31 receptor α-canine heavy chain 10A12VH1-cIgGBm [SEQ ID NO:] 44] EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMAWVRQ APGKGLQWVASISTGGGNTYYRDSVKGRFTISRDNAKNTL YLQMNSLRAEDTAMYYCAKHGTLYFDYWGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP C RE ELSKNTVSL W CLIKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPGK

[0169] [Table 7]

[0170] The present invention is not limited in scope by the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention are derived from the foregoing description. It will be obvious to those skilled in the art. Such modifications are included within the scope of the attached claims. That is the intention.

Claims

1. A composition for treating atopic dermatitis in dogs, comprising canine antipruritic antibodies and canine antiproliferative antibodies, Here, the canine antipruritic antibody and the canine antiproliferative antibody are part of the bispecific antibodies, Here, the bispecific antibody comprises the monomer of the canine antipruritic antibody and the monomer of the canine antiproliferative antibody, The aforementioned canine antipruritic antibody is a canine interleukin-31 receptor α (IL-31RA) antibody, The canine IL-31RA antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 43 and a heavy chain containing the amino acid sequence of SEQ ID NO: 44, and The aforementioned canine antiproliferative antibody is a canine interleukin-22 (IL-22) antibody, The canine IL-22 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain containing the amino acid sequence of SEQ ID NO:

17. composition.

2. The light chain of the canine IL-31RA antibody comprises a heavy chain constant domain 1 (CH1) instead of a light chain constant domain (CL), and The composition according to claim 1, wherein the heavy chain of the canine IL-31RA antibody comprises CL instead of CH1.

3. A composition for treating atopic dermatitis in dogs, comprising canine anti-inflammatory antibodies and canine anti-proliferative antibodies, Here, the canine anti-inflammatory antibody and the canine anti-proliferative antibody are part of the bispecific antibodies, Here, the bispecific antibody comprises a monomer of the canine anti-inflammatory antibody and a monomer of the canine anti-proliferative antibody, The aforementioned canine anti-inflammatory antibody is a canine interleukin-4 receptor α (IL-4Rα) antibody, The aforementioned canine IL-4Rα antibody, It comprises a light chain containing the amino acid sequence of SEQ ID NO: 4 and a heavy chain containing the amino acid sequence of SEQ ID NO: 5, or It comprises a light chain containing the amino acid sequence of SEQ ID NO: 6 and a heavy chain containing the amino acid sequence of SEQ ID NO: 7, and here, The aforementioned canine antiproliferative antibody is a canine interleukin-22 (IL-22) antibody, The canine IL-22 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain containing the amino acid sequence of SEQ ID NO:

17. composition.

4. The composition according to any one of claims 1 to 3, further comprising one or more further components selected from the group consisting of Janus kinase (JAK) inhibitors, spleen tyrosine kinase (SYK) inhibitors, or antagonists for chemoattractant receptor homologous molecules expressed on TH2 cells.

5. The aforementioned JAK inhibitor 【Chemistry 1】 In the formula, R １ C may be substituted with hydroxyl. １～４ Alkyl compounds, and their pharmaceutically acceptable salts, as 【Chemistry 2】 and its pharmaceutically acceptable salts, A composition according to claim 4, selected from the group consisting of the following.

6. A method for treating atopic dermatitis in a dog having atopic dermatitis, comprising administering to the dog a composition according to any one of claims 1 to 5.

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