Antibodies that specifically bind to interleukin-13 and their applications

Abstract

To provide an anti-canine interleukin-13 antibody that blocks or effectively neutralizes the signaling pathway of canine interleukin-13. [Solution] An antibody or an antigen-binding fragment thereof is provided based on one embodiment of the present disclosure. In one embodiment, it is possible to provide an anti-interleukin-13 antibody that specifically recognizes interleukin-13 (IL-13), or an antigen-binding fragment thereof, a pharmaceutical composition for the treatment of an immune disease or cancer containing the antibody, a host cell that produces the antibody, and a vector containing nucleic acid encoding the antibody.

Description

Technical Field

[0001] The present disclosure relates to an antibody that specifically binds to Canis Interleukin-13 (cIL-13) and its uses. Specifically, it relates to an anti-canis Interleukin-13 antibody or an antigen-binding fragment thereof, and a pharmaceutical composition containing the above antibody or antigen-binding fragment as an active ingredient.

Background Art

[0002] Interleukin-13 is a protein encoded by the IL13 gene in humans. It was first cloned in 1993, has a length of 1.4 kb, and is located on chromosome 5q31.1. The above interleukin-13 has a mass of 13 kDa and is folded to form four α-helix bundles. Its secondary structural characteristics are similar to those of interleukin-4 (IL-4), but it has 25% sequence identity with IL-4 and can transmit signals independently of IL-4. The above interleukin-13 is known to be a cytokine secreted by T helper type 2 (Th2) cells, CD4 cells, natural killer T cells, mast cells, basophils, eosinophils, and macrophages. Interleukin-13 is also known to cause IgE synthesis, hyperplasia of goblet cells, excessive secretion of mucus, airway hypersensitivity reaction, fibrosis, and upregulation of chitinase. Therefore, interleukin-13 is suspected to be a factor in the physiological changes induced by allergic inflammation in many tissues.

[0003] Therefore, despite the need to develop anti-interleukin-13 drugs, the development and manufacture of such drugs can be a complex and challenging process due to several factors. For example, because these antibody drugs are highly specific, they require extensive knowledge of the target and the development of highly selective antibodies. Furthermore, antibody drugs are subject to stringent regulatory requirements, including the need to demonstrate safety, efficacy, and consistent quality, which necessitates extensive testing and documentation throughout the entire development and manufacturing process. Despite these difficulties, the development and manufacture of antibody drugs specific to the interleukin-13 target can lead to successful treatment of a variety of diseases in humans and animals, including cancer and autoimmune diseases.

[0004] In connection with this, the Republic of Korea Published Patent No. 10-2019-0053184 has been devised. [Overview of the project] [Problems that the invention aims to solve]

[0005] The purpose of this disclosure is to provide an anti-canine interleukin-13 antibody that blocks or effectively neutralizes the signaling pathway of canine interleukin-13.

[0006] Furthermore, this disclosure aims to provide pharmaceutical compositions using anti-canine interleukin-13 antibodies, as well as compositions for the prevention and / or treatment of inflammatory diseases.

[0007] The technical challenges described herein are not limited to those mentioned above. A person skilled in the art can clearly understand other challenges based on the following description. [Means for solving the problem]

[0008] Based on one embodiment of the present disclosure, an anti-canis interleukin-13 antibody or its antigen-binding fragment that specifically recognizes canine interleukin-13 (cIL-13) is provided.

[0009] According to one embodiment of the present disclosure, the above anti-canine interleukin-13 antibody can be made chimeric.

[0010] According to one embodiment of the present disclosure, the above-mentioned anti-canine interleukin-13 antibody can be a caninized antibody.

[0011] According to one embodiment of the foregoing disclosure, the above-mentioned anti-canine interleukin-13 antibody may include a monoclonal antibody.

[0012] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may have a heavy chain constant region derived from canine immunoglobulin GA, B, C, or D type.

[0013] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may have a light chain constant region derived from a canine kappa or lambda chain.

[0014] According to one embodiment of the present disclosure, the amino acid sequence of the light chain variable region of the anti-canine interleukin-13 antibody or its antigen-binding fragment can be selected from SEQ ID NOs: 12, 23, 24, 25, 26, 27, and 28.

[0015] According to one embodiment of the present disclosure, the amino acid sequence of the light chain of the anti-canine interleukin-13 antibody or its antigen-binding fragment can be selected from SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.

[0016] According to one embodiment of the present disclosure, the amino acid sequence of the heavy chain variable region of the anti-canine interleukin-13 antibody or its antigen-binding fragment can be selected from SEQ ID NOs: 10, 17, 18, 19, 20, 21, and 22.

[0017] According to one embodiment of the present disclosure, the amino acid sequence of the heavy chain of the anti-canine interleukin-13 antibody or its antigen-binding fragment can be selected from SEQ ID NOs: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77.

[0018] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may have at least one of the following characteristics: the characteristic of suppressing the proliferation of TF-1 cell lines, or the characteristic of suppressing STAT-6 phosphorylation.

[0019] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may comprise at least one of a heavy chain having the amino acid sequence of SEQ ID NO: 69 or 71, or a variant thereof, and a light chain having the amino acid sequence of SEQ ID NO: 70 or 72, or a variant thereof.

[0020] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may include an antibody consisting of two light chains and two heavy chains.

[0021] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or an antigen-binding fragment thereof may include a Fab fragment, an Fv fragment, a diabody, a triabody, a tetrabody, or at least one fragment thereof.

[0022] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or an antigen-binding fragment thereof can be used for treating or preventing diseases in animals.

[0023] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or an antigen-binding fragment thereof may have the use of treating or preventing at least one of inflammatory diseases, immune diseases, autoimmune diseases, atopic dermatitis, and cancers in animals including dogs.

[0024] Based on other embodiments of the present disclosure, a pharmaceutical composition for treating immune diseases or cancers including the antibody according to the present disclosure and a pharmaceutically acceptable carrier can be provided.

[0025] Based on other embodiments of the present disclosure, the pharmaceutical composition for treating immune diseases or cancers including the antibody according to the present disclosure and a pharmaceutically acceptable carrier can be processed into an injection form.

[0026] Based on other embodiments of the present disclosure, host cells for producing the antibody according to the present disclosure can be provided.

[0027] Based on other embodiments of the present disclosure, the host cells for producing the antibody according to the present disclosure can be selected from the group consisting of bacteria including Escherichia coli, yeast, CHO cells, and HEK cells.

[0028] Based on other embodiments of the present disclosure, the host cells for producing the antibody according to the present disclosure may include a gene encoding a chaperone protein or a glycosylation enzyme as a result of genetic engineering operations for improving antibody expression and secretion.

[0029] Based on other examples of the present disclosure, a vector containing nucleic acid encoding the antibody of the present disclosure can be provided. [Effects of the Invention]

[0030] This disclosure can provide pharmaceutical compositions for blocking or neutralizing interleukin-13 signaling, as well as compositions for the prevention and / or treatment of inflammatory diseases.

[0031] On the other hand, the effects derived from this disclosure are not limited to those described above, and a person with ordinary skill in the art to which this disclosure belongs can clearly understand effects other than those described above based on the following description. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows the gene sequence of Canis IL-13 optimized for CHO cells, based on one embodiment of the present invention. [Figure 2] Figure 2 shows an expression vector prepared by treating the CHO-optimized canine IL-13 cloning vector (pUC-GW-Amp) with XhoI / EcoRI double restriction enzymes and then inserting it into a pcDNA3.4 plasmid, based on one embodiment of the present invention. [Figure 3] Figures 3a to 3c are IMAC chromatograms showing the results of purifying recombinant cIL-13 protein cultured in ExpiCHO-S based on one embodiment of the present invention. [Figure 4] Figure 4 shows the electrophoresis results of cIL-13 when N-linked glycans are cleaved by PNGase, based on one embodiment of the present invention. [Figure 5] Figure 5 is a graph evaluating the TF-1 proliferation inhibitory ability of purified cIL-13 based on one embodiment of the present invention. [Figure 6] Figure 6 shows the results of analyzing the binding sites of anti-canine interleukin-13 monoclonal antibodies by Western blotting after removing N-linked or O-linked glycans by treating with PNGase-F or O-glycosidase. [Figure 7] Figure 7 shows a sequence obtained by codon-optimizing the light chain and heavy chain variable region gene sequences identified from a 32A10 hybridoma, based on one embodiment of the present invention, to suit CHO cells. [Figure 8] Figure 8 is a graph showing the purity of two mouse / dog chimeric purified antibodies, injected into an SE-HPLC column and evaluated, based on one embodiment of the present invention. [Figure 9] Figure 9 is a graph evaluating the STAT-6 phosphorylation inhibitory ability of a chimeric purified antibody based on one embodiment of the present invention. [Figure 10] Figure 10 shows the result of modeling the tertiary structure of a chimeric antibody based on one embodiment of the present invention. [Figure 11] Figures 11a to 11d are graphs evaluating the antigen-binding affinity of canine antibodies based on one embodiment of the present invention. [Figure 12] Figure 12 is a graph showing the thermal stability of a canine antibody based on one embodiment of the present invention. [Figure 13] Figures 13a to 13c are graphs evaluating the TF-1 cell inhibitory ability of a canine antibody based on one embodiment of the present invention. [Modes for carrying out the invention]

[0033] Various embodiments will be described below with reference to the drawings. In this specification, various explanations are provided to facilitate understanding of the disclosure. In the specific descriptions for implementing the disclosure, configurations not directly related to the technical essence of the disclosure have been omitted to the extent that the technical essence of the invention is not obscured. Furthermore, terms and words used in this specification and in the claims should be interpreted as meanings and concepts consistent with the technical idea of ​​the invention, based on the principle that inventors may define the concepts of terms as appropriate to best describe their invention.

[0034] Furthermore, the terms “or,” “or else,” and “or else” shall mean “or,” “or else,” and “or else” in an implicational sense, rather than in an exclusive sense. In other words, unless otherwise specified or made clear from the context, “X uses A or B” shall mean one of the natural implicational substitutions. That is, if X uses A; X uses B; or X uses both A and B, then “X uses A or (or else) B” can apply to any of these cases. Also, the terms “and / or” and “at least one” in this specification shall refer to and include all possible combinations of one or more items from the multiple related items discussed. For example, the terms “at least one of A or B” and “at least one of A and B” should be interpreted as “including only A,” “including only B,” and “including a combination of A and B.”

[0035] Furthermore, the term “includes” as a predicate and / or as a modifier should be understood to mean that the feature and / or component in question exists. However, the term “includes” as a predicate and / or as a modifier should be understood not to exclude the existence or addition of one or more other further features, components and / or groups thereof. Also, where the number is not specifically identified or where it is not clear from the context to indicate a singular form, the singular in this specification and the claims should generally be interpreted to mean “one or more.”

[0036] The descriptions relating to the embodiments presented herein are provided so that a person with ordinary skill in the art of this disclosure may utilize or practice the invention. Various modifications to such embodiments will be readily apparent to a person with ordinary skill in the art of this disclosure. The general principles defined herein can be applied to other embodiments without departing from the scope of this disclosure. Accordingly, the invention is not limited to the embodiments presented herein. The invention should be interpreted in the broadest sense, consistent with the principles and novel features presented herein.

[0037] Interleukins are a group of cytokines primarily expressed and secreted by immune cells, involved in facilitating communication between different types of cells and regulating immune responses. Interleukins mediate communication between leukocytes involved in immune responses and transmit signals that regulate the proliferation, differentiation, and activation of immune cells. While interleukins influence both innate and adaptive immune responses, they can stimulate or suppress immune cell activity, modulate inflammation, and contribute to the resolution of immune responses. Interleukins are often released in response to immune problems such as infection and inflammation. Interleukin production is tightly controlled to prevent excessive immune responses that can lead to autoimmune reactions or chronic inflammation.

[0038] Interleukin dysregulation is associated with a variety of diseases. Recombinant antibodies designed to recognize and neutralize interleukins are used as therapeutic agents to modulate the activity of specific interleukins or their receptors. For example, tocilizumab and sarilumab are two drugs that block interleukin-6. Secukinumab and ixekizumab are recombinant antibodies that target IL-17A and are used to treat diseases such as psoriasis and psoriatic arthritis. Ustekinumab is a recombinant antibody that targets the p40 subunit shared by IL-12 and IL-23 and is used to treat diseases such as psoriasis and Crohn's disease. Canakinumab is an example of a recombinant antibody that targets IL-1β and is used to treat certain autoinflammatory diseases. Dupilumab is an example of a recombinant antibody that targets both interleukin-4 and interleukin-13 receptors. Dupilumab is used to treat conditions such as atopic dermatitis with nasal polyps, asthma, and chronic sinusitis.

[0039] Among the diverse subgroups of interleukins, interleukin-13, primarily produced by T helper 2 (Th2) cells, is involved in the development and maintenance of allergic inflammation by promoting IgE antibody production and enhancing eosinophil recruitment. IL-13 can modulate immune responses by influencing fibroblast activity to participate in tissue repair, suppressing the production of inflammatory cytokines, and promoting anti-inflammatory signals. IL-13 exerts its effects by binding to the IL-13 receptor complex, which consists of IL-13 receptor α1 (IL-13Rα1) and IL-4 receptor α (IL-4Rα). When IL-13 binds to the receptor, an intracellular signaling cascade is initiated, inducing a variety of cellular responses.

[0040] Appropriate regulation of interleukin-13 can also be used in the treatment of atopic dermatitis, which is both an inflammatory and an autoimmune disease. Atopic dermatitis is a chronic, recurrent skin eczema characterized by intense itching, and is believed to develop due to a combination of factors, including genetic predisposition, environmental factors, immunological abnormalities in the patient, and abnormalities in the skin barrier. Mild cases of atopic dermatitis can be improved with moisturizing, but treatment with corticosteroids, calcineurin inhibitors, or systemic immunosuppressants may be necessary depending on the severity. In recent years, the antibody therapies dupilumab and tralokinumab, which suppress interleukin-4 and / or interleukin-13 signaling, have been approved in the United States and Europe as treatments for moderate to severe atopic dermatitis, providing a safe and effective new treatment opportunity for patients requiring systemic therapy.

[0041] Canine atopic dermatitis (CAD) is a common allergic skin disease in dogs. CAD is characterized by a hypersensitive immune system reaction to environmental allergens, resulting in chronic inflammation of the skin, similar to atopic dermatitis or eczema in humans. Common allergens that cause canine atopic dermatitis include pollen, fungal spores, dust mites, and certain food components. There are various methods for managing canine atopic dermatitis. These may include avoiding or minimizing exposure to allergens, feeding hypoallergenic foods, alleviating symptoms with medications such as antihistamines and steroids, and addressing secondary infections with antibiotics and antifungals. Immunotherapy (allergy injections) to desensitize the dog to specific allergens over time may also be recommended.

[0042] In severe cases of atopic dermatitis in dogs, systemic treatment is necessary. Traditionally, steroids and immunosuppressants (cyclosporine) have been widely used to treat atopic dermatitis in pet dogs. In recent years, drugs such as Cytopoint (Lokivetmab) and Apoquel (Oclacitinib) have been released and are in use. However, because Apoquel is a JAK inhibitor that suppresses various cytokines, it cannot be used in individuals with immunosuppression, such as those with hyperadrenocorticism or progressive malignant tumors, and can induce various side effects, including diarrhea and vomiting. Cytopoint has attracted attention for its ability to specifically neutralize canine interleukin-31 and effectively suppress itching, but it has the limitation of not being a fundamental cure. The anti-cIL-13 antibody provided in this invention can be used as a therapeutic agent for atopic dermatitis in dogs, but its purpose is not limited to this.

[0043] TF-1 cells are a cell line derived from human erythroleukemia cells, which are isolated and immortalized to obtain a stable cell line for experimental research. TF-1 cells exhibit proliferation dependence on granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3. Because TF-1 cells respond to a wide range of cytokines and carry a broad spectrum of endogenous receptors, they are a useful tool for studying cytokine signaling mechanisms.

[0044] One of the major signaling pathways induced by IL-13 involves the activation of STAT-6. STAT-series proteins carry signals from their receptor complexes to the nucleus, activating gene expression. Activation of STAT-6 proteins by cytokine exposure is mediated by JAK (Janusfamily) tyrosine kinases. Activated STAT-6 regulates the transcription of various genes involved in immune responses, inflammation, and tissue remodeling. Many genes regulated by the IL-13 / STAT-6 pathway are associated with the characteristics of allergic inflammation. Dysregulation of this pathway is linked to the pathogenesis of allergic diseases, and therapeutic agents targeting IL-13 or STAT-6 are associated with diseases such as asthma and atopic dermatitis.

[0045] term antibody Antibodies, also known as immunoglobulins, are large Y-shaped proteins produced by the immune system in response to foreign substances called antigens. Antibodies that recognize and bind to specific antigens play a crucial role in the immune response by labeling the antigen so that it can be destroyed or neutralized by other components of the immune system. An antibody molecule consists of two heavy chains, which are larger polypeptide chains, and two light chains, which are smaller polypeptide chains. The heavy chains determine the type of antibody (e.g., IgG, IgA, IgM) and its effector function, and are composed of an invariant region (HC) and a variable region (HV). There are two types of light chains, kappa (κ) and lambda (λ), and similarly consist of an invariant region (LC) and a variable region (LV). The antigen-binding site of an antibody is determined by the variable regions of the heavy and light chains. Antibodies can be separated into enzymatically functional fragments, and these antibody fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv.

[0046] Hybridoma Developed in 1975 by Georges J.F. Köhler and César Milstein, hybridoma technology revolutionized immunology and antibody production. Hybridoma technology involves fusing B cells and myeloma cells to create stable, immortalized cell lines capable of producing large quantities of monoclonal antibodies. B cells can produce antibodies but have low sustained proliferative capacity. Myeloma cells have a low antibody-producing capacity but can divide continuously. After fusing the two types of cells, the hybridoma is cultured in a medium that supports the growth of only the fused cells. It is also screened to identify cells that produce antibodies with the desired specificity against a particular antigen. Once the hybridoma has been created and screened, it can uniformly produce highly specific monoclonal antibodies that recognize a single antigenic determinant (epitope) in the target antigen.

[0047] Subclone A subclone is a subpopulation of genetically identical cells derived from a parent clone or original clone. Subcloning involves the process of isolating and culturing specific cells from an original clone to create a new population with identical genetic material. A parent clone is a genetically identical population of cells containing the target DNA or genetic material. This genetic material may include the target gene, a specific DNA fragment, or a plasmid containing a specific insertion. Specific cells are isolated from this parent clone according to desired characteristics. This process may involve cell sorting, limiting dilution, or other methods for selecting cells with the desired genetic features. Subsequently, the isolated cells are cultured to grow subclonal colonies, and the genetic identity and characteristics of the subclones can be verified through polymerase chain reaction (PCR), DNA sequencing, etc.

[0048] Chimeric antibodies A chimeric antibody is an artificial antibody molecule that creates a hybrid structure by combining genetic material from one species with that of another. While chimeric antibodies are typically designed to minimize potential immune responses when administered to humans while retaining the specificity of non-human antibodies, this specification describes the development of a chimeric antibody specifically for use in dogs. The mouse-derived variable region retains the antibody's inherent specificity. To reduce the risk of immunogenicity (immune response to the antibody itself), the invariant region of the chimeric antibody is generally derived from a human antibody, and in the examples described herein, it is derived from a dog. The invariant region mediates effector functions and interacts with other components of the immune system.

[0049] Complementarity Determining Region (CDR) Complementarity-determining regions (CDRs) are structures located in the variable regions of the heavy and light chains of antibodies. They interact with the antigenic determinants (epitopes) on the antigen surface through a combination of morphological complementarity, electrostatic interactions, hydrogen bonding, and van der Waals forces. The immune system produces antibodies with different antigen-binding specificities based on the diverse sequences of CDRs, enabling the recognition and neutralization of various pathogens and foreign molecules. Generally, there are three CDRs in both the heavy and light chains.

[0050] Framework (framework region) In antibodies, the framework refers to the region that surrounds and supports the complementarity-determining region within the antibody's variable region. While the CDR directly interacts with the antigen and determines the specificity of antigen recognition, the framework provides the structural backbone of the antibody molecule and plays a crucial role in maintaining overall stability and integrity. This disclosure describes a canine substitution process in which framework residues are replaced with canine counterparts to reduce immunogenicity and improve the suitability of the therapeutic antibody.

[0051] canonical structures Standard structures are a series of repeating morphologies or structural motifs observed in antibody variable regions (CDRs), as defined by Chothia et al. CDR standard structures are broadly classified into six types, A through F, each characterized by specific backbone dihedral angles (phi and psi angles) and hydrogen bonding patterns that define the overall shape and orientation of the loop. These standard structures are considered a framework for understanding the structural diversity of CDR loops and are widely used in computer modeling and antibody engineering for predicting and designing antibody-antigen interactions. However, standard structures are not strictly conserved across all CDR loops, and deviations may occur depending on the specific sequence and context of the loop.

[0052] anchor residue Anchor residues are specific amino acid residues within the complementarity-determining region (CDR) of an antibody, which play a crucial role in antigen recognition and binding. Typically, anchor residues are in direct contact with the antigenic determinant (epitope) at key locations within the CDR loop.

[0053] Vernier Zone The vernier zone refers to specific amino acid residues located adjacent to the CDR or at specific positions within the CDR loop that contribute to antigen binding. Because the vernier zone structurally influences the shape of the CDR and the fine-tuning of antigen recognition, residues in this zone are replaced during the canine antibody design process to prevent loss of affinity or antibody stability due to structural incompatibility between the CDR sequence of the mouse antibody (CDR donor) and the canine antibody framework (receptor). In other words, they are replaced with amino acid residues derived from the mouse antibody.

[0054] Post-Translational Modification (PTM) Post-translational modification (PTM) refers to the chemical changes that occur in a protein molecule after it has been synthesized from an mRNA template. In antibody technology, PTM can have a significant impact on the structure, function, stability, and activity of an antibody. The types of PTM mainly mentioned in antibody technology include: glycosylation, disulfide bond formation, fatty acid acylation, pegylation, asparagine deamidation, or glutamine deamidation; methionine oxidation, aspartic acid (D) isomerization, N-terminal glutamine cyclization, cysteine ​​bonding, lysine side-chain glycosylation, and tryptophan residue oxidation.

[0055] host cell A host cell is a living cell capable of replicating and expressing foreign genetic material such as DNA or RNA, and contains enzymes and ribosomes necessary for the transcription and translation of foreign genes. Host cells are transfected (in the case of eukaryotic cells) or transformed (in the case of prokaryotic cells) to introduce foreign genetic material, and are designed to express and produce specific proteins or antigens. The produced proteins are harvested and purified and used in the manufacture of therapeutic proteins, vaccines, or other biotechnology products. Commonly used host cell examples include Escherichia coli (E. coli) for bacterial systems, Saccharomyces cerevisiae (yeast) for eukaryotic systems, and various mammalian cell lines such as HEK293 (human embryonic kidney) or CHO (Chinese hamster ovary) cells for more complex eukaryotic systems.

[0056] vector In molecular biology, a vector refers to a DNA molecule that serves as a medium for transporting foreign genetic material (e.g., genes, DNA fragments) into a host organism. Vectors are widely used in genetic engineering for gene manipulation and research, recombinant DNA technology, and various applied fields of molecular biology. The use of vectors facilitates the replication, proliferation, and expression of foreign genetic material within host cells. Common types of vectors include plasmids, bacteriophages, cosmids, artificial chromosomes (e.g., bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs)), and viral vectors.

[0057] The following inventions will be described in more detail later in the embodiments of this disclosure, which are shown in the accompanying drawings.

[0058] IL-13 signaling is believed to play a crucial role in various immune diseases, including atopic dermatitis, and in the symptoms of cancer. The inventors of this invention produced an antibody that recognizes and neutralizes canine IL-13 and verified its effectiveness. First, the inventors developed recombinant canine IL-13 (recombinant cIL-13). Next, the inventors administered the developed recombinant cIL-13 to mice to induce an immune response. The inventors removed the spleens of the mice that had induced the immune response, prepared a cell suspension, and fused it with myeloma cells using PEG to produce hybridomas. The inventors evaluated the hybridoma pool that formed colonies using ELISA, TF-1 proliferation inhibitory activity, and STAT-6 phosphorylation inhibitory activity, and finally obtained a monoclonal antibody named "32A10-#20".

[0059] The binding of cIL-13, from which N-linked or O-linked glycans were removed using PNGase F and O-glycosidase, to the 32A10-#20 monoclonal antibody was confirmed. The results showed that 32A10-#20 reacted with cIL-13 regardless of the binding site.

[0060] In one embodiment of the present invention, the gene sequence was cloned from the obtained 32A10-#20 hybridoma. The antigen-binding specificity and biological activity of the monoclonal antibody are determined by the variable region, which includes the complementarity-determining region (CDR). When the sequences were confirmed using three types of primers for both the heavy and light chains, the CDR regions of the heavy and light chains were identical, but two types of framework sequences were detected for each. The confirmed variable region gene sequences were optimized as codons that fit the host CHO (Chinese Hamster Ovary) cells. Transformation was induced in ExpiCHO-S cells via an expression vector, and the chimeric antibody was purified by injecting the culture medium into a column. After analyzing the purity and activity of the purified antibody and finally determining the effective sequence, a method was applied to re-verify the activity of the selected antibody.

[0061] The examples are described in further detail below.

[0062] Acquisition of Canis Interleukin-13 The interleukin-13 of the genus Canis described herein consists of 131 amino acids, with a signal peptide located at the N-terminus and N-linked glycans attached to asparagine amino acids 38, 48, 56, and 71. Cysteine ​​molecules 47-75 and 63-89 are linked by disulfide bonds, forming a tertiary structure. The interleukin-13 precursor gene sequence of animals of the genus Canis was obtained using the database of the National Center for Biotechnology Information (NCBI) (GeneBank:AF244915.1). Subsequently, codon optimization was performed by excluding restriction enzyme sites necessary for cloning to make it suitable for CHO (Chinese hamster ovary, hereafter CHO) cells used as the host. For example, such codon optimization was performed using known optimization tools or by outsourcing to an external organization. Subsequently, the XhoI enzyme site (C TCCGA) was inserted at the 5' end for cloning, and the Kosack sequence (GCCACC), start codon (ATG), IL-13 coding sequence (CDS), polyhistidine (CATCATCACCATCACCAC), stop codon (TGA), and EcoRI enzyme site (G AATTC) necessary for translation were sequentially added, and synthesis proceeded (Figure 1). The final gene sequence of the canine IL-13 optimized for CHO is as follows.

[0063] [Table 1]

[0064] cIL-13 Recombinant Protein Production A cloning vector (pUC-GW-Amp) was obtained by synthesizing a canine IL-13 optimized for the above CHO. The insertion DNA was obtained and purified by XhoI / EcoRI double restriction enzyme digestion, and then inserted into a plasmid to complete the expression vector (Figure 2). In this case, for example, the pcDNA3.4 vector may be used as the plasmid, but this example is not intended to limit the scope of the rights of this disclosure.

[0065] Subsequently, the inventors of the present invention transformed CHO cell lines with the prepared vector and temporarily cultured them in a medium supplemented with L-glutamine. At this time, for example, the ExpiCHO® expression system kit (ThermoFisher, #A29133) may be used, but this example is not intended to limit the scope of the rights of the present disclosure.

[0066] [Table 2]

[0067] The culture medium, after cultivation was complete, was diluted 20-fold with distilled water and then sterilized and filtered to prepare the injection sample.

[0068] Recombinant cIL-13 Purification In one embodiment of the present disclosure, a sample was injected into a resin column, and the target histagged canine interleukin-13 (his-tagged canine IL-13) protein was purified by the imidazole gradient method. A Histrap HP 5mL column (Cytiva, #29051021) may be used, but this example is not intended to limit the scope of the rights described herein.

[0069] Based on the method according to one embodiment of this disclosure, the eluted protein was dialyzed to pH 7.0, 20 mM phosphate equilibrium buffer to exchange the buffer, and quantified using an extinction coefficient of 0.766, and used for immunoantigen testing, screening, etc. (Figures 3a to 3c).

[0070] The quantitative formula is as follows: cIL-13 concentration (mg / mL) = (A280 × dilution factor) / 0.766

[0071] *In the diagram, the blue solid line represents the absorbance value at 280 nm, the green solid line represents the gradient, and the brown solid line represents the electrical conductivity (ms / cm).

[0072] 1. Purity analysis cIL-13 is a heavily glycosylated protein with four N-linked glycosylation sites. Electrophoretic analysis of the IMAC-purified protein did not clearly show a main band, but cleaving the N-linked glycosylation caused the main band to shift to the 10-15 kDa range, which is consistent with the molecular weight of 13.3 kDa calculated based on amino acid information (Figure 4).

[0073] 2.Activity analysis Based on a method relating to one embodiment of this disclosure, recombinant cIL-13 was treated with different concentrations of human erythroleukemia cell line TF-1 at various concentrations to confirm the activity of cIL-13, and the growth rate was evaluated after culturing for 3 days. Ez-Cytox (DoGENBio, #EZ-500) can be used to evaluate the growth rate, but this example is not intended to limit the scope of the rights of this disclosure. The manufactured cIL-13 induced TF-1 cell proliferation in a concentration-dependent manner, and showed similar activity even under conditions of three freeze-thaw cycles (Figure 5).

[0074] Hybridoma fabrication Based on the method described in one embodiment of this disclosure, an immunoassay was prepared by mixing histagged cIL-13 with ODN1585-TLR9 ligand to produce an antibody targeting the recombinant protein cIL-13. Next, based on the method described in one embodiment of this disclosure, immunization was performed on 6-week-old female BALB / c mice by intraperitoneal injection three times at intervals of 4 weeks / 3 weeks / 3 weeks. Based on the method described in one embodiment of this disclosure, orbital blood was collected one week after the third immunization and plasma was extracted. The immune response was confirmed by Conventional ELISA, and high reactivity to the recombinant antigen cIL-13 was observed in all five mice. Based on the method described in one embodiment of this disclosure, a recombinant antigen was produced for hybridoma development and additional immunization was performed. Three days later, the spleen of the mice was removed and a cell suspension was prepared.

[0075] [Table 3]

[0076] Based on the method described in one embodiment of this disclosure, the Kohler-Milstein method was used to prepare DMEM (Dulbeco's modified Eagle's medium) for 10 minutes. 8 10 spleen cells and 10 7 Cell fusion was induced in individual myeloma cell lines using 50% polyethylene glycol 400. While the myeloma cell line used could be X63-Ag8.653 (ATCC, PTA-8431), a myeloma cell-8-azaguanine (Sigma-Aldrich, #SML2963) resistant mouse, this example is not intended to limit the scope of the rights of this disclosure. Following the method described in one embodiment of this disclosure, the fused cells were washed and resuspended in DMEM culture medium (HAT culture medium) supplemented with 20% fetal bovine serum, 100 μm hypoxanthine, 0.44 μm aminopterin, and 16 μm thymidine. The cells were then dispensed into 96-well plates and cultured for 2 weeks in an incubator supplied with 37°C and 5% CO2.

[0077] Subsequently, based on the method according to one embodiment of this disclosure, the hybridoma pool in which colony formation was observed was evaluated for antigen reactivity and TF-1 proliferation inhibitory activity, and primary limiting dilution was performed. Then, the antigen reactivity and TF-1 proliferation inhibitory activity were re-evaluated, and after evaluating the STAT-6 phosphorylation inhibitory activity, secondary limiting dilution was performed. Finally, based on the method according to one embodiment of this disclosure, the TF-1 proliferation inhibitory activity was evaluated again, and five types of monoclonal purified antibodies were obtained.

[0078] Monoclonal antibody evaluation 1. Purity evaluation Based on the method described in one embodiment of this disclosure, the physicochemical and biological activities of five established anti-canine IL-13 monoclonal antibody subclones were compared and evaluated. First, based on the method described in one embodiment of this disclosure, the five purified antibodies were injected into an SE-HPLC column and their purity was evaluated. One antibody molecule, consisting of two heavy chains and two light chains, eluted at a retention time of approximately 15.6 minutes, and all showed high purity of 96% or more.

[0079] [Table 4]

[0080] 2. Antigen reactivity evaluation Conventional ELISA was performed to evaluate the antigen-binding reactivity of the purified antibody, using an antigen (cIL-13)-coated antibody. Among the clones analyzed, clone 32A10-#20 showed the highest reactivity, with an EC50 of 463 pM.

[0081] [Table 5]

[0082] 3. Evaluation of TF-1 proliferation inhibitory ability Canine IL-13 and human IL-13 share 61.8% similarity based on their amino acid sequences, and both are reported to be able to interact with the human IL-13 receptor (JOURNALOF INTERFERON AND CYTOKINE RESEARCH 20:779-785, 2000). Therefore, to confirm the biological activity of the 32A10 monoclonal antibody, its ability to inhibit cell proliferation by canine IL-13 was evaluated using the TF-1 cell line.

[0083] The inventors of this invention cultured the TF-1 cell line in 10% fetal bovine serum RPMI medium containing GM-CSF, and washed it on day 1 to remove the GM-CSF. Furthermore, the inventors of this invention cultured 2 × 10⁶ cells in a flat-bottomed 96 microplate. 4 TF-1 cells were dispensed at 50 μL / well. Then, the antibody was sequentially diluted threefold from 50 μg / mL in a medium containing 50 ng / mL of recombinant cIL-13 protein, and incubated for 1 hour. A mixture of interleukin-13 and antibody was added to the TF-1 cell line at 100 μL / well, and the cells were cultured for an additional 72 hours. The degree of cell culture was then evaluated by testing. The 32A10 monoclonal antibody was found to inhibit TF-1 proliferation in a concentration-dependent manner, with an IC50 in the range of 2.2–3.3 nM.

[0084] [Table 6]

[0085] 4.Binding site To confirm the binding site of the 32A10 antibody, the inventors of the present invention treated recombinant cIL-13 protein with PNGase-F or O-glycosidase to remove N-linked or O-linked glycans, and then analyzed whether the 32A10 monoclonal antibody was bound to the protein via Western blotting. The main band of the sample from which the N-linked glycan was removed was in the 13 kDa range, the same as the theoretical molecular weight of cIL-13, while the main bands of the sample with unremoved glycans and the sample from which the O-linked glycan was removed were observed in the 20-35 kDa range. Since the 32A10 antibody reacted regardless of the presence or absence of antigenic glycans, it was determined that the 32A10 monoclonal antibody recognizes and binds to the canine interleukin-13 peptide site regardless of the glycan (Figure 6).

[0086] 32A10 gene sequence obtained The antigen-binding specificity and biological activity of monoclonal antibodies are determined by a variable region including the Complementarity Determining Region (CDR). To confirm the amino acid sequence of the rodent antibody 32A10 monoclonal antibody, the gene sequence was cloned and obtained from an established 32A10-20 hybridoma.

[0087] First, the inventors of this invention isolated total RNA from 32A10 hybridomas. For total RNA isolation, for example, the RneasyPlus Mini Kit (Qiagen) may be used, but this example is not intended to limit the scope of the rights of this disclosure. Next, RT-PCR was performed using IgG invariant region-specific primers to obtain a DNA template. PCR amplification was performed using the obtained DNA template and variable region-specific primers, and amplified variable region vectors were obtained through TA cloning. At this time, the ProgenMouse IgG library primer set and the TAKARAMighty TA Cloning Kit may be used, but this example is not intended to limit the scope of the rights of this disclosure. Finally, sequencing analysis was requested for three types of variable region heavy chains and three types of variable region light chains to confirm the variable region sequences.

[0088] Sequence analysis revealed that the CDR sequences were identical regardless of the primer type, but two different framework 1 sequences were identified for each type.

[0089] [Table 7]

[0090] *The CDR sequence is shown underlined. It is arranged in the order of CDR1, CDR2, and CDR3.

[0091] 32A10 Chimeric Antibody Cloning The variable region gene sequence identified from 32A10 hybridomas was optimized as a codon suitable for CHO cells. An XhoI restriction enzyme site was added to the 5' end for gene transfer to the expression vector, a Kozak sequence for smooth translation induction, and a human albumin-derived signal peptide was added to ensure extracellular secretion after protein expression. A stop codon and an EcoRI restriction enzyme site were added to the 3' end to terminate expression. In one embodiment of the present invention, a canine IgGB type is used as the heavy chain invariant region; however, this is not intended to limit the scope of the present invention. Canine IgG A, B, C, or D-type can be used to construct the chimeric antibody heavy chain invariant region. Similarly, in one embodiment of the present invention, a kappa chain from the genus Canis is used in the light chain invariant region. However, this is not intended to limit the scope of the present invention, and kappa or lambda chains from the genus Canis can be used in the composition of the light chain invariant region of a chimeric antibody (Figure 7).

[0092] [Table 8]

[0093] [Table 8 continued] TIFF2026509905000010.tif239170

[0094] [Table 8 continued] TIFF2026509905000011.tif153170

[0095] The pcDNA3.4 vector, with its multiple cloning site modified to XhoI / EcoRI, was then modified to insert the four chimeric genes listed in Table 8, thereby completing the expression vectors.

[0096] To produce vectors expressing the chimeric genes listed in Table 8, the four DNAs were first treated with double restriction enzymes using XhoI / EcoRI according to one embodiment of the present disclosure, followed by electrophoresis and gel extraction to prepare the inserted DNA. Next, the pcDNA3.4 vector was similarly treated with double restriction enzymes using XhoI / EcoRI according to one embodiment of the present disclosure, followed by electrophoresis and gel extraction. Next, two heavy chains and two light chains were inserted into the pcDNA3.4 vector using T4 DNA ligase, and the cells were transformed into DH5α Escherichia coli cells to produce the following four cell lines: pcDNA3.4-VHC-B, pcDNA3.4-VHF-B, pcDNA3.4-VLN-kappa, and pcDNA3.4-VLO-kappa. Based on the method according to one embodiment of this disclosure, electrophoresis was performed again after double restriction digestion of XhoI / EcoRI to confirm whether the target gene was inserted into the vector, and the sequences of the light chain variable region and heavy chain variable region were confirmed by requesting sequencing analysis.

[0097] Production of 32A10 mouse / canine chimeric antibodies Based on the method described in one embodiment of this disclosure, transformation of CHO cells was induced by combining two heavy chains (C and F types) and two light chains (N and O types), and the cells were cultured until their viability was 70% or less. Based on the method described in one embodiment of this disclosure, the culture medium was collected and injected into a chromatography column to purify the chimeric antibody. At this time, a MabselectSure (Cytiva, #29049104) column may be used, but this example is not intended to limit the scope of the rights of this disclosure. Based on the method described in one embodiment of this disclosure, antibodies were produced only in two of the four combinations, including the O-type light chain, and it was determined that the N-type light chain gene was an incorrect sequence that did not produce antibody expression. Based on the method described in one embodiment of this disclosure, the OD280nm of the purified antibody was measured and quantified to evaluate its purity and activity.

[0098] [Table 9]

[0099] 1. Purity evaluation The inventors of the present invention evaluated the purity of two types of chimeric purified antibodies by injecting them into an SE-HPLC column. While TOSOH and TSKgelG3000SWXL could be used as SE-HPLC columns, this example is not intended to limit the scope of the rights of this disclosure. Each antibody molecule, composed of two heavy chains and two light chains, eluted at a retention time of approximately 15.6 minutes, and all showed high purity of over 99% (Figure 8).

[0100] 2. Antigen reactivity evaluation (quantification of EC50 value) Based on the method of one embodiment of the present disclosure, histidine-tagged cIL-13 recombinant protein was added to a nickel-coated plate (ThermoFisher, #15442) at a rate of 100 μL per well (100 ng / 100 μL / well) and reacted at room temperature for 1 hour. Subsequently, based on the method of one embodiment of the present disclosure, the plates were washed three times with 1× PBST, and then the chimeric antibody was added after serial dilution with 1× PBS, and the antigen / antibody reaction was induced at room temperature for 1 hour. Based on the method of one embodiment of the present disclosure, after washing three times with 1× PBST, an HRP-conjugated secondary antibody that cross-reacts with canis antibodies was diluted 100-fold in casein buffer, added at a rate of 100 μL / well, and reacted at 37°C for 30 minutes. In this case, JacksonImmunoResearch, #115-036-071 can be used as the HRP-conjugated secondary antibody, and ThermoFisher Scientific, #37528 can be used as the casein buffer; however, this example is not intended to limit the scope of the rights of the present disclosure. Based on the method of one embodiment of the present disclosure, the mixture was washed three times with 1×PBST, TMB was added to induce color development, and the reaction was terminated by adding an equal amount of 1.0N sulfuric acid (sulfuricacid). Based on the method of one embodiment of the present disclosure, it was confirmed that two chimeric antibodies showed EC50 values ​​similar to the parent antibody (mouse 32A10).

[0101] [Table 10]

[0102] 3. TF-1 proliferation inhibitory activity The inhibitory activity of the chimeric antibody on the proliferation of TF-1 cell lines was evaluated.

[0103] Based on the method according to one embodiment of this disclosure, 2 × 10⁻¹⁰ 4TF-1 cells were cultured in GM-CSF-free medium at a rate of 50 μL / cell / well. Based on the method described in one embodiment of this disclosure, the parent antibody or chimeric antibody FO was sequentially diluted threefold the following day to prepare 10 samples ranging from 50,000 to 2.54 ng / mL. Based on the method described in one embodiment of this disclosure, an equal volume of the prepared antibody test solution containing canine IL-13 recombinant antigen (300 ng / mL) was mixed with the sample to induce an antigen / antibody neutralization reaction (37°C, 1 hour). 100 μL of the mixture was then transferred to the TF-1 cell line and cultured for 72 hours. Subsequently, the growth rate of the TF-1 cell line was measured.

[0104] The chimeric antibody FO suppressed the proliferation of the TF-1 cell line, similar to the parent antibody.

[0105] [Table 11]

[0106] 4. STAT-6 phosphorylation inhibitory activity To confirm the cIL-13 sub-signaling blockade effect of the chimeric antibody, its ability to inhibit STAT-6 phosphorylation was evaluated.

[0107] Based on the method according to one embodiment of this disclosure, on the day before the test (Day-1), the TF-1 cell line was suspended in GM-CSF-free medium and washed, and 4 × 10 5Cells were dispensed at 50 μL / well and cultured in a 37°C cell incubator for 24 hours. Based on the method of one embodiment of this disclosure, chimeric antibodies were sequentially diluted in interleukin-13 supplemented medium at a concentration of 100 ng / mL to prepare nine samples ranging from 50,000 to 7.62 ng / mL. Based on the method of one embodiment of this disclosure, 200 μL of the mixture was left in a 37°C CO2 cell incubator for 1 hour to induce an antigen / antibody reaction, and the mixture was added to TF-1 cells (50 μL / well). The mixture was allowed to react for 5 minutes (37°C, CO2 5%) to induce signal transduction into TF-1 cells, and the TF-1 cell line was harvested and washed with 1× PBS. Based on the method of one embodiment of this disclosure, three wells were prepared under the same conditions, and phosphorylated STAT-6 was quantified by combining the three wells into one sample to ensure sensitivity. Based on a method according to one embodiment of this disclosure, the quantification of intracellular phosphorylated STAT-6 can be tested and measured using the PathScan® Phospho-Stat6 (Tyr641) Sandwich ELISA Kit (Cell Signaling Technology, #7275C) according to the manual; however, this example is not intended to limit the scope of rights of this disclosure. The chimeric antibody FO effectively inhibited interleukin-13-mediated STAT-6 phosphorylation in a concentration-dependent manner, and the IC50 was evaluated to be 2,418 pM (Figure 9).

[0108] [Table 12]

[0109] Design of canine antibodies In order to develop recombinant antibodies as a treatment for atopic dermatitis in pet dogs, the need arose to further caninize established chimeric antibodies. In this disclosure, caninization refers to the process of substituting a certain portion of the mouse-derived variable region of a chimeric antibody with the canine variable region in order to reduce immunogenic side effects when the antibody is administered to dogs. In order to maintain antigen binding ability while reducing immunogenicity, the mouse-derived complementarity-determining region (CDR) of the chimeric antibody was transplanted into a canine germline-derived framework using CDR grafting technology, based on a method according to one embodiment of this disclosure. At this time, the antibody that provides the CDR site was called the donor, and the antibody that receives the transplanted CDR site as its own skeleton was called the recipient.

[0110] Based on the method according to one embodiment of this disclosure, homology model building was first performed in order to carry out CDR grafting. Homology model building is a method for constructing a three-dimensional model of an antibody using the established sequence of the chimeric antibody, and is considered to be particularly useful for predicting the structure of the CDR and predicting the structural compatibility between the framework region and the CDR. Furthermore, homology model building can provide a basis for the entire canine transformation process by contributing to the decision of whether to preserve donor residues or substitute them with receptor residues.

[0111] Based on the method according to one embodiment of this disclosure, in order to construct a homology model, a protein structure with high homology to the chimeric antibody variable region light chain sequence and heavy chain sequence was first searched using the Protein Data Bank (PDB). The structure of PDB code 5IKC was selected as the light chain variable region framework template, and the structure of 5YFI was selected as the heavy chain variable region framework template. A deep learning-based analysis system was used for the analysis of the CDR loop structure. Ablooper can be used as the deep learning-based CDR loop structure analysis system, but is not limited to it. Based on the method according to one embodiment of this disclosure, the corresponding Chothia canonical structures were identified for the three CDR structures of the light chain region and heavy chain region, and a protein structure to be adopted as a template for each CDR was separately selected. Next, a tertiary structure model that fits the variable region heavy chain and light chain sequence was identified using a protein binding prediction program. According to one embodiment of this disclosure, the tertiary structure that fits the variable region of the chimeric antibody is a protein structure coded as 1HIN in the Protein Databank and has a relative packing angle of -46.9°. PAPS and ABangle can be used as the prediction programs, but are not limited to these. Based on the method according to one embodiment of this disclosure, the backbone coordinates of the chimeric antibody anchor residue were aligned with the structure of 1HIN to assemble the final model, and an energy minimization process was performed by molecular mechanics simulation. A schematic diagram of the final model is shown in Figure 10.

[0112] When designing canine antibody sequences, care must be taken in retaining mouse-derived sequences or substituting them with canine-derived sequences to prevent loss of affinity and antibody stability due to structural incompatibility between mouse-derived CDR sequences and canine-derived frameworks. This is particularly important in the vernier zone, which is structurally located in or adjacent to the CDR and is considered to affect the morphology of the CDR and the fine-tuning of antigen recognition. Substitution with canine-derived sequences aims to enhance sequence identity with canine genes without impairing antibody affinity. Furthermore, comprehensive sequence optimization strategies aimed at improving the physicochemical properties, manufacturability, and pharmacological properties of the antibody should also be considered during antibody candidate design. Given the fragility of the physical structure of proteins, antibodies should ideally be designed to maintain chemical and structural integrity during manufacturing, distribution, and storage. Therefore, the possibility of various post-translational modifications (PTMs) should also be considered. The potential for methionine oxidation, N- and O-glycosylation, asparagine deamidation and aspartic acid (D) isomerization, N-terminal glutamine cyclization, cysteine ​​crosslinking, lysine side-chain glycosylation, and tryptophan residue oxidation were each examined. Some residues were canineized or retained in mouse-derived sequences to avoid the possibility of post-translational modifications.

[0113] In one embodiment of the method described herein, similar sequences of the variable region heavy and light chains of chimeric antibodies were searched using the Canine Gene Database and the Canine Genome Project to select a receptor framework for transplanting the CDR of a chimeric antibody. Selection was based on the sequence identity of the entire framework, the agreement and compatibility of the variable region heavy and light chain contacts, and the standard structure of the CDR. Based on the analysis results, the canine germline-derived genes IGHV1-30*01 and IGHV3-23*01 were selected as suitable templates for the variable region heavy chain. The canine germline-derived genes IGK3-18*01 and IGKV2-10*01 were selected as suitable templates for the variable region light chain. Three canine sequences were designed for each of the selected templates.

[0114] [Table 13]

[0115] [Continued from Table 13] TIFF2026509905000017.tif112170

[0116] Therefore, according to one embodiment of the present disclosure, based on the results of the canine antibody design described above, the antibodies to be produced were selected as shown in Table 14.

[0117] [Table 14]

[0118] Canine antibody production The inventors of this disclosure attempted to manufacture canine antibodies (listed in Table 14) selected by an external laboratory. Canine antibodies Ab1-Ab14 and Ab21-Ab24 were successfully manufactured, but unfortunately, Ab15-Ab20 could not be manufactured. All antibodies that were successfully manufactured showed a purity of 98% or higher, and the endotoxin level was less than 1 EU. The purity and endotoxin levels of Ab1-Ab24 are shown in Table 15.

[0119] [Table 15]

[0120] Canine antibody evaluation Based on one embodiment of the information disclosed herein, the function of the manufactured canine antibody was verified, and the binding ability of the antibody to cIL-13, the thermal stability of the antibody, and the ability to inhibit TF-1 cell proliferation were evaluated in order to identify clinical candidate substances.

[0121] 1. Antigen reactivity (EC50) A standard ELISA test was used to evaluate the antigenic reactivity of the canine antibody. The inventors of the present invention coated a 96-well nickel plate with histag-conjugated cIL-13 at a concentration of 1 μg / mL and incubated it at room temperature for 1 hour. The wells were washed three times with 200 μL of washing buffer each time. 1×PBS containing 0.1% tween 20 was used as the washing buffer. Based on the method according to one embodiment of the present disclosure, the diluted antibody was dispensed into 100 μL portions per well and incubated again at room temperature for 1 hour. A solution of 25 mL of 1× casein solution mixed with 25 mL of 1× PBS was used as the dilution buffer. Based on the method according to one embodiment of the present disclosure, 50 μL of HRP-conjugated Goat-Anti-human IgG (HRP conjugated) was diluted in 10 mL of dilution buffer, dispensed into 100 μL portions per well, and incubated again at room temperature for 1 hour. The wells were washed three times with 200 μL of washing buffer per wash. 50 μL of TMB substrate was added to each well, and the mixture was reacted at room temperature for 10 minutes. 50 μL of 1N sulfuric acid was dispensed into each well as a stop solution, and the absorbance was measured at 450 nm using a spectrophotometer. Based on the results, the EC50 was calculated as shown in Table 16. Ab1 to Ab5 showed superior reactivity compared to the chimeric antibodies, and Ab21 to Ab24 were confirmed to have similar antigenic reactivity to the chimeric antibodies.

[0122] [Table 16]

[0123] 2.Thermal stability Antibody instability under thermal conditions can lead to reduced production yield, loss or damage of efficacy, adverse immune responses, and patient-related complications. Furthermore, function may be limited or lost when working under extreme conditions or during long-term storage. Therefore, the thermal stability of antibodies is evaluated as an important indicator that may act as a variable in future applications. Based on one embodiment of this disclosure, 200 μL of each test sample was dispensed and stored for 1 hour at 60°C or 4°C. The inventors then dispensed the samples into an opaque 96-well plate, 180 μL per well. 20 μL of 0.2 mg / mL ANS solution was dispensed into each well and incubated in the dark for 30 minutes. The plates were read using a fluorescence analyzer. (Excitation: 375 nm, Emission: 470 nm)

[0124] The following formula was used to quantify the thermal stability of the antibody. Relative instability = (thermal stress at 60°C - untreated) / untreated × 100

[0125] The thermal stability comparison results for each candidate antibody are shown in Figure 12 and Table 17 as follows. The thermal stability of Ab21 to Ab24 was equivalent to or better than that of the chimeric antibody.

[0126] [Table 17]

[0127] 1. TF-1 proliferation inhibitory activity Based on one embodiment of the present disclosure, the cell proliferation inhibitory effect of a canine antibody was evaluated. The inventors of the present invention prepared 2 × 10⁶ solutions at 50 μL per well, excluding B2, B3, and B4, the day before the experiment. 4Cell-level TF-1 was dispensed under GM-CSF-free conditions. The cells were then incubated overnight in a 5% CO2 incubator at 37°C. Next, 90 μL of 300 μg / ml antibody diluent was placed in wells B11, C11, D11, E11, F11, and G11 of a clear 96-well V-bottom plate. Using a multichannel pipette, 30 μL of antibody diluent was transferred from wells B11, C11, D11, E11, F11, and G11 to wells B10, C10, D10, E10, F10, and G11, respectively, and pipetted several more times to ensure thorough mixing. The above procedure was repeated, and 30 μL was removed from the final wells B2, C2, D2, E2, F2, and G2 to adjust the final volume to 60 μL. 60 μL of culture medium was added to wells A2-A7 and H2-H7 according to one embodiment of the present disclosure. 60 μL of cIL-13 solution, prepared at a concentration of 100 ng / mL, was added to each well containing 60 μL of diluted antibody solution and IL-13 only. 120 μL of culture medium was added to the wells containing only TF-1 cells and the wells containing only GM-CSF, and the cells were incubated in a 5% CO2 incubator at 37°C for 1 hour. The TF-1 cell plates that had been cultured overnight were removed, and 100 μL of cIL-13 and antibody mixture was added to each well to a final volume of 150 μL. The cells were incubated in a CO2 incubator at 37°C for 72 hours. Before the end of the culture, a substrate solution for growth inhibition analysis was prepared. 20 μL of substrate per well was mixed with 30 μL of culture medium. 50 μL of the substrate solution per well was added to the completed culture plate, and the cells were incubated in a CO2 incubator at 37°C for an additional 3 hours. After maintaining equilibrium at room temperature for 15 minutes, the absorbance was measured using a spectrophotometer. Based on the measurement results, the IC50 was calculated as follows. The cell proliferation inhibitory ability of Ab3-Ab5 and Ab21-Ab24, including the chimeric antibody, is shown in Figures 13a-13c. The comparison revealed that Ab21 and Ab22 had a similar level of TF-1 cell proliferation inhibitory ability to the chimeric antibody 32A10, but Ab3-Ab5 and Ab23 and Ab24 did not have sufficient proliferation inhibitory ability.

[0128] [Table 18]

[0129] [Table 19]

[0130] conclusion Based on a method according to one embodiment of this disclosure, we successfully manufactured and produced a canine interleukin-13 histidine-tagged recombinant protein. The cIL-13 recombinant protein polypeptide, excluding the glycans, had a molecular weight of approximately 13 kDa and was expressed as a glycoprotein with an excess of N-linked glycans. cIL-13 showed biological activity that induced TF-1 cell proliferation.

[0131] Based on a method described in one embodiment of this disclosure, we subsequently succeeded in developing a monoclonal antibody 32A10 that specifically recognizes canine interleukin-13. Among the three subclones, the "32A10-#20" clone showed the highest antigen affinity, and its binding EC50 to the antigen was evaluated to be 463 pM. The 32A10 monoclonal antibody suppressed interleukin-13-dependent TF-1 cell proliferation, and its IC50 value was confirmed to be in the range of 2.9 to 4.2 nM. The 32A10 monoclonal antibody was confirmed to detect the antigen regardless of N and O-linked glycans and to recognize recombinant protein polypeptides.

[0132] Based on a method according to one embodiment of this disclosure, two variable region sequences each for the heavy chain and light chain were obtained from a 32A10 monoclonal antibody, and mouse / dog chimeric antibodies were produced. Among the four combinations, the FO-type chimeric antibody showed similar antigen-binding affinity and TF-1 proliferation inhibitory activity to the parent clone. Furthermore, the FO chimeric antibody showed an effect of inhibiting STAT-6 phosphorylation by interleukin-13. Therefore, it was finally confirmed that the 32A10 heavy chain variable region sequence is of type F and the light chain variable region sequence is of type O.

[0133] The 32A10 monoclonal antibody neutralized cIL-13 activity and effectively inhibited TF-1 cell line proliferation in the IC50 range of 2.2–3.3 nM. The excellent cIL-13 neutralizing activity of the 32A10 monoclonal antibody, demonstrated at low concentrations, suggests that the 32A10 antibody can be developed as a treatment for pet atopic dermatitis. The mouse / dog chimeric antibody, exhibiting activity similar to the parent antibody, indicates that the variable region gene sequence derived from the 32A10 hybrid cell line is error-free, and the extremely high purity of the 32A10 chimeric antibody can be understood as a stable physical characteristic that meets the requirements for development.

[0134] Based on the method described in one embodiment of this disclosure, the 32A10 chimeric antibody was canine-modified. In the canine-modification design process, the mouse-derived complementarity-determining region (CDR) of the chimeric antibody was transplanted into a canine germline-derived framework using CDR grafting technology. Based on the method described in one embodiment of this disclosure, the tertiary structure of the chimeric antibody was first predicted by constructing a homology model. Considering the possibility of vernier zones and post-translational modifications, the chimeric antibody sequence was maintained or replaced with a canine-modified sequence. Based on the method described in one embodiment of this disclosure, the canine germline genes IGHV1-30*01 and IGHV3-23*01 were selected as variable region heavy chain templates and IGK3-18*01 and IGKV2-10*01 were selected as variable region light chain templates by searching a canine gene database. Three versions of the canine-modified sequence were designed for each template.

[0135] A total of 36 sequences were obtained by combining heavy chain and light chain sequences. Of the 24 named antibodies, Ab1-Ab14 and Ab21-Ab24 were successfully obtained. Based on the method described in one embodiment of this disclosure, the antigen reactivity, thermal stability, and TF-1 cell proliferation inhibitory ability of the antibodies were tested and evaluated. As a result, canine antibodies Ab3-Ab5 and Ab21-Ab24 were selected as candidate substances considering productivity and biological activity.

[0136] On the other hand, the above-mentioned antibodies may be, but are not limited to, chimeric antibodies, humanized antibodies, caninized antibodies, bivalent antibodies, bispecific molecules, minibodies, domain antibodies, bispecific antibodies, antibody mimetic antibodies, diabodies, triabodies, tetrabodies, or fragments thereof.

[0137] In this disclosure, "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody and the invariant region of a canine antibody are rearranged, resulting in an antibody that exhibits a significantly improved immune response compared to a mouse antibody.

[0138] In this disclosure, "humanized antibody" refers to an antibody whose protein sequence, derived from a non-human species, has been modified to resemble a naturally occurring antibody variant in humans. As an example, the above-mentioned humanized antibody can be produced by creating a humanized variable region by rearranging a mouse-derived CDR with a human antibody-derived FR, and then rearranging this variable region with the invariant region of a desired human antibody.

[0139] The antibodies described in this disclosure are also applicable to veterinary medicine. For example, the term "caninized antibody" refers to an antibody whose protein sequence, derived from a species other than dogs, has been modified to resemble an antibody variant naturally produced by dogs.

[0140] Furthermore, this disclosure can provide a pharmaceutical composition for the treatment of an immune disease or cancer, comprising the above-mentioned antibody and a pharmaceutically acceptable carrier.

[0141] The antibodies described herein may take the form of antibody-drug conjugates. The term "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without impairing the biological activity of the antibody and the drug.

[0142] The chemically linked form described above in this disclosure may be a covalent bond. The antibody-drug conjugate described above in this disclosure refers to a form in which the drug is bound to the N-terminal amino acid residue of the heavy chain and / or light chain of the antibody, specifically a form in which the drug is bound to the N-terminal α-amino group of the heavy chain and / or light chain of the antibody.

[0143] The pharmaceutical composition described herein is characterized in that it is in the form of an injectable preparation, and the pharmaceutical composition may be characterized in that it is intended for at least one of humans or animals.

[0144] The administration routes of the pharmaceutical compositions described herein are not limited to those described herein and include oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration, but administration other than oral administration is preferred.

[0145] In this disclosure, “other than oral” includes injection or infusion techniques for subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-bursal, intrasternal, intradural, intrafocal, and intracranial administration. The pharmaceutical compositions of this disclosure may also be administered in the form of suppositories for rectal administration.

[0146] The pharmaceutical compositions described herein may vary depending on several factors, including the activity of the specific compound used, age, weight, general health status, sex, diet, administration time, route of administration, elimination rate, drug combination, and the severity of the specific disease being prevented or treated. The dosage of the pharmaceutical compositions may vary depending on the patient's condition, weight, severity of the disease, form of administration, route of administration, and duration, but can be appropriately selected by those skilled in the art.

[0147] In this disclosure, "immune-related diseases" refers to diseases induced by the overactivation and expression of various immune and inflammatory cells, including, but not limited to, autoimmune diseases; graft-versus-host diseases; organ transplant rejection; asthma; atopic dermatitis; or acute or chronic inflammatory diseases.

[0148] Furthermore, the term "autoimmune disease" in this disclosure may include, but is not limited to, one or more diseases selected from the group consisting of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behçet's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

[0149] In this disclosure, "cancer" refers to a disease characterized by the rapid and uncontrolled proliferation of mutated cells, and may be at least one selected from the group consisting of malignant melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymph node cancer, gallbladder cancer, hematological cancer, thyroid cancer, endocrine cancer, oral cancer, liver cancer, bile duct cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, gastric cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, anaplastic thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma.

[0150] Furthermore, this disclosure can provide host cells that produce the above-mentioned antibodies.

[0151] The host cells used in the manufacture of antibody drugs may vary depending on the specific antibody and intended use. Typically, antibody drugs are produced using living cells such as bacteria, yeast, or mammalian cells to produce antibody proteins, and the choice of host cell can affect the yield, quality, and glycosylation pattern of the final antibody product. For example, the host cells may be bacteria such as E. coli, or mammalian cells such as yeast, CHO cells, or HEK cells.

[0152] On the other hand, in addition to selecting host cells, genetic engineering of host cells can also be performed in the production of antibody drugs to improve antibody expression and secretion. For example, inserting genes that encode chaperone proteins or glycosylation enzymes can improve the yield and quality of the final antibody product.

[0153] Furthermore, this disclosure can provide a vector containing nucleic acid encoding the above-mentioned antibody.

[0154] A vector is a DNA molecule used to transmit foreign genetic material, such as a gene encoding an antibody protein, to a host cell for the purpose of producing a desired protein. The choice of vector may vary depending on the specific host cell and the intended use of the recombinant protein.

[0155] The descriptions relating to the embodiments presented herein are provided so that any person with ordinary skill in the art of this disclosure may utilize or implement the disclosure. Various variations of such embodiments are readily apparent to a person with ordinary skill in the art of this disclosure, and the general principles defined herein can be applied to other embodiments without departing from the scope of this disclosure. Accordingly, this disclosure is not limited by the embodiments presented herein and should be interpreted in the broadest sense consistent with the principles and novel features presented herein.

[0156] As described above, the relevant information was stated based on the best mode for carrying out the invention. [Industrial applicability]

[0157] It can be used to treat atopic dermatitis in individuals, including dogs. [Sequence Listing Free Text]

[0158] In the sequence listing of this application, sequence identification number 5 is a sequence of fewer than four amino acids, and is described separately as follows. Sequence identification number 5 Leu Alaser

Claims

1. An anti-canis interleukin-13 antibody or its antigen-binding fragment that specifically recognizes and neutralizes canis interleukin-13 (cIL-13).

2. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a monoclonal antibody.

3. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a mouse antibody, a chimeric antibody, or a caninized antibody.

4. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from canine immunoglobulin G of type A, B, C, or D.

5. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a canine kappa or lambda chain.

6. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a light chain variable region having at least one amino acid sequence selected from SEQ ID NOs: 12, 23, 24, 25, 26, 27, and 28.

7. The anti-canine interleukin-13 antibody or antigen-binding fragment according to claim 6, wherein the light chain of the anti-canine interleukin-13 antibody or antigen-binding fragment thereof has at least one amino acid sequence selected from SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.

8. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a heavy-chain variable region having at least one amino acid sequence selected from SEQ ID NOs: 10, 17, 18, 19, 20, 21, and 22.

9. The anti-canine interleukin-13 antibody or antigen-binding fragment according to claim 8, wherein the heavy chain of the anti-canine interleukin-13 antibody or antigen-binding fragment thereof has at least one amino acid sequence selected from SEQ ID NOs: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77.

10. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof has at least one of the characteristics of inhibiting the proliferation of TF-1 cell lines or inhibiting STAT-6 phosphorylation.

11. The aforementioned anti-canine interleukin-13 antibody or its antigen-binding fragment is Heavy chains or variants thereof having the amino acid sequence of SEQ ID NO: 69 or 71; and The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, comprising at least one of a light chain having the amino acid sequence of SEQ ID NO: 70 or 72, or a variant thereof.

12. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises an antibody consisting of two light chains and two heavy chains.

13. The anti-canine interleukin-13 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-canine interleukin-13 antibody or antigen-binding fragment thereof comprises a Fab fragment, an Fv fragment, a single-chain binding fragment (scFv), a diabody, a triabody, a tetrabody, or at least one fragment thereof.

14. A method for using the anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1 for the treatment or prevention of a disease in an animal.

15. The method according to claim 14, wherein the method is applied to the animal and has the use of treating or preventing at least one of the diseases, including inflammatory diseases, immune diseases, autoimmune diseases, and cancer.

16. The method according to claim 15, wherein the disease includes atopic dermatitis.

17. The method according to claim 14, wherein the animal includes a dog.

18. A pharmaceutical composition comprising the anti-canine interleukin-13 antibody or its antigen-binding fragment as described in claim 1, The aforementioned pharmaceutical composition is a pharmaceutical composition used to treat or prevent diseases in animals.

19. The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition is processed into the form of an injectable preparation.

20. A host cell used for producing an anti-canis interleukin-13 antibody or its antigen-binding fragment that specifically recognizes and neutralizes canis interleukin-13 (cIL-13).

21. The host cell according to claim 20, wherein the host cell is selected from the group consisting of bacteria including Escherichia coli, yeast, CHO cells and HEK cells.

22. The host cell according to claim 20, wherein the host cell comprises a gene encoding a chaperone protein or glycosylation enzyme as a result of genetic engineering manipulation to enhance antibody expression and secretion.

23. A vector applied to host cells for the purpose of producing an anti-canine interleukin-13 antibody or an antigen-binding fragment thereof as described in claim 20.

24. The vector according to claim 23, wherein the vector is a DNA molecule containing nucleic acid that encodes all or part of the anti-canine interleukin-13 antibody.

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