Antibody specifically binding to interleukin-13 and use thereof

Abstract

According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In one embodiment, a specific recognition of interleukin-13 (Interleukin-13; the present invention relates to an anti-interleukin-13 antibody or antigen-binding fragment thereof for the treatment of an immune disease or cancer comprising said antibody, a host cell generating said antibody, and a vector comprising a nucleic acid encoding said antibody.

Description

Technical Field

[0001] This disclosure relates to antibodies that specifically bind to canine interleukin-13 (cIL-13) and their uses, and more specifically, to anti-interleukin-13 antibodies or antigen-binding fragments thereof and pharmaceutical compositions comprising said antibody or antigen-binding fragment as an active ingredient. Background Technology

[0002] Interleukin-13 is a protein encoded by the IL13 gene in humans. First successfully replicated in 1993, it is 1.4 kb in length and located on chromosome 5q31.1. Interleukin-13 has a mass of 13 kDa, folds into four α-helical bundles, and its secondary structural features are similar to interleukin-4 (IL-4), sharing 25% sequence identity with IL-4. It can independently transduce signals with IL-4. Interleukin-13 is known to be a cytokine secreted by helper T cells 2 (Th2) cells, CD4 cells, natural killer T cells, mast cells, basophils, eosinophils, and macrophages. Furthermore, interleukin-13 is known to contribute to IgE synthesis, excessive goblet cell formation, excessive mucus secretion, respiratory allergic reactions, fibrosis, and chitinase upregulation. Therefore, it is suspected that interleukin-13 is a central mediator of physiological changes induced by allergic inflammation in many tissues.

[0003] Therefore, despite the necessity to develop anti-interleukin-13 drugs, the development and preparation of such drugs can be complex and challenging due to various factors. For example, antibody drugs are highly specific, thus requiring extensive knowledge of the target and the development of highly selective antibodies. Furthermore, due to the need for strict regulatory compliance, including the verification of safety, efficacy, and consistent quality, the entire development and preparation process requires extensive testing and documentation. Despite these difficulties, the development and preparation of antibody drugs specific to the interleukin-13 target are sure to lead to success in the treatment of a variety of diseases, including cancers and autoimmune diseases in humans and animals.

[0004] Relatedly, this has been disclosed in Korean Patent Publication No. 10-2019-0053184. Summary of the Invention

[0005] Technical issues

[0006] The purpose of this disclosure is to provide an anti-canine interleukin-13 antibody that effectively blocks or neutralizes the signal transduction of canine interleukin-13.

[0007] Furthermore, the purpose of this disclosure is to provide pharmaceutical compositions utilizing anti-canine interleukin-13 antibodies and compositions for the prevention and / or treatment of inflammatory diseases.

[0008] The technical problems addressed in this disclosure are not limited to those mentioned above. Those skilled in the art to which this invention pertains can clearly understand other technical problems not mentioned through the following description.

[0009] Technical solution

[0010] According to one embodiment of the present disclosure, an anti-canis interleukin-13 antibody or its antigen-binding fragment that specifically recognizes and neutralizes canine interleukin-13 (cIL-13) is provided.

[0011] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody may be chimeric.

[0012] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody may be a caninized antibody.

[0013] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody may comprise a monoclonal antibody.

[0014] According to one embodiment of the present disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may include a heavy chain constant region derived from canine immunoglobulin G type A, B, C or D.

[0015] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may have a light chain constant region derived from the Canis Kappa or lambda chain.

[0016] According to one embodiment of this disclosure, the amino acid sequence of the light chain variable region of the anti-canine interleukin-13 antibody or its antigen-binding fragment may be selected from sequence listings 12, 23, 24, 25, 26, 27 and 28.

[0017] According to one embodiment of this disclosure, the amino acid sequence of the light chain of the anti-canine interleukin-13 antibody or its antigen-binding fragment may be selected from sequence listings 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.

[0018] According to one embodiment of this disclosure, the amino acid sequence of the heavy chain variable region of the anti-canine interleukin-13 antibody or its antigen-binding fragment may be selected from sequence listings 10, 17, 18, 19, 20, 21 and 22.

[0019] According to one embodiment of this disclosure, the amino acid sequence of the heavy chain of the anti-canine interleukin-13 antibody or its antigen-binding fragment may be selected from sequence listings 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.

[0020] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may have at least one of the features of inhibiting the proliferation of TF-1 cell lines or inhibiting STAT-6 phosphorylation.

[0021] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may comprise at least one of a heavy chain or a variant thereof having an amino acid sequence having sequence number 69 or 71 and a light chain or a variant thereof having an amino acid sequence having sequence number 70 or 72.

[0022] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may comprise an antibody consisting of two light chains and two heavy chains.

[0023] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may include a Fab fragment, an Fv fragment, a diabody, a triabody, a tetrabody, or at least one of these fragments.

[0024] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment can be used to treat or prevent diseases in animals.

[0025] According to one embodiment of this disclosure, the anti-canine interleukin-13 antibody or its antigen-binding fragment may be used to treat or prevent at least one of diseases in animals, including dogs, including inflammatory diseases, immune diseases, autoimmune diseases, atopic dermatitis, and cancer.

[0026] According to another embodiment of the present disclosure, a pharmaceutical composition for treating immune diseases or cancer may be provided, comprising the antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0027] According to another embodiment of this disclosure, a pharmaceutical composition comprising the antibody of this disclosure and a pharmaceutically acceptable carrier for treating immune diseases or cancer can be formulated into an injectable dosage form.

[0028] According to another embodiment of this disclosure, a host cell can be provided to generate the antibody disclosed herein.

[0029] According to another embodiment of this disclosure, the host cell for generating the antibody disclosed herein can be selected from a group consisting of bacteria including Escherichia coli, yeast, CHO cells, and HEK cells.

[0030] According to another embodiment of this disclosure, the host cell from which the antibody of this disclosure is generated may be the result of genetic engineering operations performed to enhance antibody expression and secretion, and may contain genes encoding chaperone proteins or glycosylation enzymes.

[0031] According to another embodiment of this disclosure, a vector comprising a nucleic acid encoding an antibody of this disclosure is provided.

[0032] The effects of the invention

[0033] This disclosure may provide pharmaceutical compositions for blocking or neutralizing interleukin-13 signal transduction and compositions for the prevention and / or treatment of inflammatory diseases.

[0034] On the other hand, the effects that can be obtained from this disclosure are not limited to the effects described above, and those skilled in the art to which this disclosure pertains can clearly understand other effects not mentioned through the following description. Attached Figure Description

[0035] Figure 1 The gene sequence of canine IL-13 optimized in CHO cells according to an embodiment of the present invention is shown.

[0036] Figure 2 This invention illustrates an expression vector prepared by treating the CHO optimal canine IL-13 cloning vector (pUC-GW-Amp) with Xhol / EcoRI double restriction enzyme and then inserting it into the pcDNA 3.4 plasmid according to an embodiment of the present invention.

[0037] Figures 3a to 3c An IMAC chromatogram showing the results of purifying recombinant cIL-13 protein cultured in ExpiCHO-S according to an embodiment of the present invention.

[0038] Figure 4 The electrophoretic results of cIL-13 when N-glycan chains are cleaved by PNGase according to an embodiment of the present invention are shown.

[0039] Figure 5 A graph to evaluate the TF-1 proliferation inhibition ability of purified cIL-13 according to an embodiment of the present invention.

[0040] Figure 6 The results show the removal of N-glycans or O-glycans by treating PNGase-F or O-glycans and the analysis of the binding sites of anti-canine interleukin-13 monoclonal antibodies by Western blot assay.

[0041] Figure 7 This invention illustrates a sequence from which the light and heavy chain variable region gene sequences identified in 32A10 hybridomas are adapted for codon optimization in CHO cells, according to an embodiment of the invention.

[0042] Figure 8 A graph illustrating the purity assessment of two mouse / dog chimeric purified antibodies injected into an SE-HPLC column according to an embodiment of the present invention.

[0043] Figure 9 A graph illustrating the ability of a chimeric purified antibody to inhibit STAT-6 phosphorylation according to an embodiment of the present invention.

[0044] Figure 10 The results of modeling the tertiary structure of a chimeric antibody according to an embodiment of the present invention.

[0045] Figures 11a to 11d A graph illustrating the antigen-binding ability of canine-derived antibodies according to an embodiment of the present invention.

[0046] Figure 12 A graph illustrating the thermostability of a canine antibody according to an embodiment of the present invention.

[0047] Figures 13a to 13c A graph illustrating the ability of a canine-derived antibody to inhibit TF-1 cells according to an embodiment of the present invention. Detailed Implementation

[0048] The following describes various embodiments with reference to the accompanying drawings. Various descriptions will be provided in this specification to aid in understanding the present disclosure. Before describing the specific content used to implement the present disclosure, it should be noted that structures not directly related to the technical essence of the present disclosure will be omitted from description to the extent that they do not obscure the technical essence of the invention. Furthermore, in order to best illustrate the invention, the inventors may appropriately define the concepts of terms; based on this principle, the terms or vocabulary used in this specification and the scope of the invention claims should be interpreted as meanings and concepts consistent with the technical concept of the invention.

[0049] Furthermore, the term "or" is not an exclusive "or," but rather an implicit "or." That is, unless otherwise specified or explicitly stated in the context, "X uses A or B" indicates one of the natural implicit substitutions. Specifically, "X uses A or B" can apply to any of the following: X uses A; X uses B; or X uses both A and B. Moreover, the terms "and / or" and "at least one" as used in this specification should be understood to refer to and include all possible combinations of more than one of the listed related items. For example, the terms "at least one of A or B" or "at least one of A and B" should be interpreted as "the case including only A," "the case including only B," or "the case consisting of A and B."

[0050] Furthermore, the terms "comprising" and / or "comprising..." should be understood to indicate the presence of the relevant feature and / or structural element. However, the terms "comprising" and / or "comprising..." should be understood not to exclude the presence and addition of more than one other feature, structural element, and / or combination thereof. And, unless the context otherwise specifically indicates or indicates a singular form, the singular form in this specification and the claims of the invention generally means "one or more".

[0051] The description of the proposed embodiments is provided to enable those skilled in the art to utilize or practice the invention. Those skilled in the art will understand variations relating to these embodiments. The general principles defined herein can be applied to other embodiments without departing from the scope of this disclosure. Therefore, the invention is not limited to the embodiments presented herein. The invention should be interpreted within the broadest possible scope consistent with the principles and novel features set forth herein.

[0052] Interleukins are a group of cytokines primarily expressed and secreted by immune cells, participating in the regulation of immune responses by promoting communication between different cell types. Interleukins mediate communication between leukocytes involved in the immune response, transducing signals that regulate the proliferation, differentiation, and activation of immune cells. Interleukins influence both innate and adaptive immune responses, playing a role in stimulating or inhibiting immune cell activity, regulating inflammation, and resolving immune responses. Interleukins are released in response to various infection- or inflammation-related immune problems. The production of interleukins is strictly controlled to prevent excessive immune responses that could lead to autoimmune or chronic inflammation.

[0053] Disorders of interleukin regulation are associated with a variety of diseases. Recombinant antibodies designed to recognize and neutralize interleukins are used as therapeutic agents aimed at modulating the activity of specific interleukins or their receptors. For example, tocilizumab and thalidomab are two drugs that block interleukin-6. Secukinumab and ixekizumab, as recombinant antibodies targeting IL-17A, are used in the treatment of diseases such as psoriasis and psoriatic arthritis. Ustekinumab, as a recombinant vector targeting the p40 subunit shared by IL-12 and IL-23, is used in the treatment of diseases such as psoriasis and Crohn's disease. Canatumab, as an example of a recombinant vector targeting IL-1β, is used in the treatment of certain autoinflammatory diseases. Dupilumab is an example of a recombinant antibody that simultaneously targets the receptors for both interleukin-4 and interleukin-13. Dupilumab is used in the treatment of diseases such as atopic dermatitis with nasal polyps, asthma, and chronic sinusitis.

[0054] Among the various downstream groups of interleukins, interleukin-13, mainly produced by helper T cells 2, participates in the occurrence and maintenance of allergic inflammation by promoting the production of IgE antibodies and enhancing the aggregation of eosinophils. IL-13 plays a role in repairing tissue structure by affecting the activity of fibroblasts, and it can regulate the immune response by inhibiting the production of pro-inflammatory cytokines and promoting anti-inflammatory signaling. 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 its receptor, it initiates an intracellular signal transduction cascade to induce a variety of cellular responses.

[0055] Appropriate regulation of interleukin-13 can be utilized in the treatment of atopic dermatitis, which is both an inflammatory skin disease and an immune disorder. Atopic dermatitis is a chronic, relapsing eczematous skin disease characterized by severe pruritus, and its pathogenesis is known to be influenced by a combination of factors, including genetic factors, environmental factors, immune abnormalities in the patient, and abnormalities in the skin's protective barrier. In mild cases of atopic dermatitis, adequate hydration can provide relief, but depending on the severity, treatment with corticosteroids, calcineurin inhibitors, or systemic immunosuppressants is required. Recently, the antibody therapies dupilumab and troroculumab, which inhibit interleukin-4 and / or interleukin-13 signaling, have gained recognition in the United States and Europe as treatments for moderate to severe atopic dermatitis, providing safe and effective new treatment opportunities for patients requiring systemic therapy.

[0056] Canine atopic dermatitis (CAD) is a common allergic skin disease in dogs. CAD is characterized by chronic inflammation of the skin caused by an allergic reaction of the dog's immune system to environmental allergens, a condition similar to post-atopic dermatitis eczema in humans. Common allergens that trigger canine atopic dermatitis include pollen, mold spores, dust mites, and certain food ingredients. Management of canine atopic dermatitis involves various methods. These may include avoiding or minimizing exposure to allergens, using a hypoallergenic diet, using antihistamines or steroids to provide symptom relief, and using antibiotics or antifungals to address secondary infections. Immunotherapy (allergy injections) that desensitizes the dog to specific allergens over time can also be recommended.

[0057] Severe cases of canine atopic dermatitis require systemic treatment. Treatment for canine atopic dermatitis widely utilizes steroids and immunosuppressants (cyclosporine), and recently, new treatments have been introduced... (Lokivetmab) and Novel drugs such as Oclacitinib have been introduced and put into use. However, Ibac, being a JAK inhibitor that inhibits multiple cytokines, cannot be used in individuals exhibiting hyperadrenocorticism or progressive malignant tumors, and may induce various side effects including diarrhea and vomiting. Cetirizine has attracted attention for its effective suppression of itching by specifically neutralizing canine interleukin-31, but it has limitations as it is not a fundamental treatment. The anti-cIL-13 antibody provided in this invention can be used as a treatment for canine atopic dermatitis, but its purpose is not limited thereto.

[0058] TF-1 cells are a cell line derived from human erythroleukemia cells. These cells were isolated and immortalized to prepare stable cell lines for laboratory research. TF-1 cells exhibit growth dependence on granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3. TF-1 cells respond to a variety of cytokines and transport a wide range of intrinsic receptors, making them an effective tool for studying cytokine signaling mechanisms.

[0059] One of the major signal transduction pathways induced by IL-13 includes STAT-6 activation. STAT family proteins transduce signals from receptor complexes to the nucleus and activate gene expression. STAT-6 activation induced by cytokine exposure is mediated by JAK (Janus family) tyrosine kinases. Activated STAT-6 regulates the transcriptional regulation of multiple genes involved in immune responses, inflammation, and tissue regeneration. Many genes regulated through the IL-13 / STAT6 pathway are associated with characteristics of allergic inflammation. Disruptions in this pathway are associated with the pathogenesis of allergic diseases, and therapeutics targeting IL-13 or STAT-6 are associated with diseases such as asthma and atopic dermatitis.

[0060] the term

[0061] Antibody

[0062] Antibodies, called immunoglobulins, are large "Y"-shaped proteins formed by the immune system in response to foreign substances called antigens. Antibodies that recognize and bind to specific antigens play a decisive role in the immune response by labeling antigens so that they can be destroyed or synthesized by other components of the immune system. An antibody molecule consists of two heavy chains (larger polypeptide chains) and two light chains (smaller polypeptide chains). The heavy chains determine the type of antibody (e.g., IgG, IgA, IgM) and its effector function, and consist of a constant region (HC) and a variable region (HV). Light chains, of two types (κ and λ), also consist of a constant region (LC) and a variable region (LV). The antigen-binding site of the antibody is determined by the variable regions of the heavy and light chains. Antibodies can be isolated into fragments with enzymatic functions; antibody fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv.

[0063] Hybridoma

[0064] The hybridoma technology, developed in 1975 by George Köhler and César Milstein, revolutionized immunology and antibody production. Hybridoma technology refers to the technique of creating stable and immortalized cell lines for the mass production of monoclonal antibodies by fusing B cells with myeloma cells. While B cells can produce antibodies, their ability to continuously divide is low. Myeloma cells, while also having limited antibody production capacity, can continuously divide. After the two cell lines are fused, the hybridoma is cultured in a medium that only supports the growth of the fused cells. Furthermore, screening is performed to identify cells capable of producing antibodies with desired specificity against a particular antigen. The prepared and screened hybridomas can uniformly produce highly specific monoclonal antibodies that recognize a single antigenic determinant (epitope) targeting a specific antigen.

[0065] Subcloning

[0066] A subclone is a subset of genetically homologous cells derived from a maternal or primary clone. Subcloning involves the process of isolating specific cells from a primary clone and culturing them to form new groups with identical genetic material. The maternal clone is a genetically identical group of cells containing the DNA or genetic material of interest. This genetic material may include the gene of interest, a specific DNA fragment, or a plasmid with a specific insert. Specific cells can be isolated from the maternal clone according to desired characteristics. This process may include cell sorting, limiting dilution, or other methods used to select cells with the desired genetic traits as listed in the table below. The isolated cells are then cultured to proliferate subclonal colonies, and the genetic identity and characteristics of the subclones can be examined using polymerase chain reaction (PCR), DNA base sequence analysis, etc.

[0067] chimeric antibodies

[0068] Chimeric antibodies are artificial antibody molecules created by combining genetic material from one species with that of another to form a hybrid structure. Originally, chimeric antibodies were designed to minimize potential immune responses when administered to humans while retaining the specificity of non-human antibodies; however, the chimeric antibodies described in this specification are developed for canine administration. The variable region, derived from mice, retains the original specificity of the antibody. To reduce the risk of immunogenicity (an immune response to the antibody itself), the constant region of chimeric antibodies is typically derived from human antibodies; however, the examples in this specification are derived from canine antibodies. The constant region mediates effector functions and interacts with other structural elements of the immune system.

[0069] Complementarity Determining Region (CDR)

[0070] Complementarity-determining regions (CDRs) are variable regions located in the heavy and light chains of antibodies. They interact with antigenic determinants (epitopes) on the antigen surface through a combination of morphological complementarity, electrostatic interactions, hydrogen bonds, and van der Waals forces. The immune system produces antibodies with different antigen-binding specificities through the diverse sequences of CDRs, enabling the recognition and neutralization of various pathogens and foreign molecules. Typically, CDRs exist in three parts in both the heavy and light chains.

[0071] Framework region

[0072] In antibodies, the framework region refers to the complementarity-determining region (CDR) within the variable region that surrounds and supports the antibody. While the CDR directly interacts with the antigen and determines the specificity of antigen recognition, the framework region provides the structural backbone of the antibody molecule, playing a crucial role in maintaining overall stability and integrity. In this disclosure, to reduce immunogenicity and improve the compatibility of therapeutic antibodies, a canine derivatization process is described, involving the replacement of framework region residues with canine counterparts.

[0073] Standard structures (canonical structures)

[0074] The standard structure is a series of repeating morphological or structural motifs observed in the variable region (CDR) of an antibody, as defined by Chothia et al. Six standard CDR structures, from A to F, have been identified. Each structure is characterized by an overall ring shape and orientation defined by specific scaffold dihedral angles (phi and psi angles) and hydrogen bonding patterns. These standard structures serve as a framework for understanding the structural diversity of CDR rings and are widely used in computer modeling and antibody engineering for predicting and designing antibody-antigen interactions. However, the standard structure is not strictly preserved in all CDR rings and can vary depending on the specific sequence and veining of the ring.

[0075] Anchored residues

[0076] Anchor residues are specific amino acid residues within the complementarity-determining region (CDR) of an antibody that play a crucial role in antigen recognition and binding. Anchor residues typically occupy a major position within the CDR ring that is in direct contact with the epitope.

[0077] Vernier District

[0078] The Vernier region consists of specific amino acid residues at specific positions adjacent to or within the CDR ring that are responsible for antigen binding. Structurally, the Vernier region influences subtle adjustments to the morphology of the CDR and antigen recognition. In canine antibody design, to prevent loss of affinity or antibody stability due to structural incompatibility between the CDR sequence of the mouse antibody (as the CDR donor) and the framework region of the canine antibody (as the recipient), residues at these positions are replaced. That is, amino acid residues derived from mouse antibodies are used as substitutes.

[0079] Post-translational modification (PTM)

[0080] Post-translational modifications (PTMs) refer to the chemical modifications that occur in protein molecules after synthesis from an mRNA template. In the field of antibody technology, PTMs can significantly impact the structure, function, stability, and activity of antibodies. The main types of PTMs mentioned in antibody technology include: glycosylation, disulfide bond formation, fatty acid acylation, polyethylene glycolation, asparagine deamidation, or glutamine deamidation; methionine oxidation; aspartic acid (D) isomerization; N-terminal glutamine cyclization; cysteine ​​binding; lysine side-chain glycosylation; and tryptophan residue oxidation.

[0081] host cells

[0082] A host cell is a living cell capable of replicating and expressing exogenous genetic material such as DNA or RNA, containing the enzymes and ribosomes required for the transcription of exogenous genes. Host cells are designed to introduce exogenous genetic material through transfection (in the case of eukaryotic cells) or transformation (in the case of prokaryotic cells) to express and produce specific proteins or antigens. The produced proteins are harvested and purified for use in the production of therapeutic proteins, vaccines, or other bioengineering products. Commonly used host cells include various mammalian cell lines such as *E. coli* for bacterial systems, *Saccharomyces cerevisiae* (yeast) for eukaryotic systems, and HEK293 (human embryonic kidney) or CHO (Chinese hamster ovary) cells for more complex eukaryotic systems.

[0083] carrier

[0084] In molecular biology, a vector is a DNA molecule used as a medium to transport and carry exogenous genetic material (e.g., genes, DNA fragments) into a host organism. Vectors are widely used in genetic engineering, recombinant DNA technology, and various other molecular biology applications for manipulating and studying genes. The use of vectors facilitates the replication, proliferation, and expression of exogenous genetic material within host cells. Common types of vectors include plasmids, bacteriophages, phages, artificial chromosomes (e.g., bacterial artificial chromosomes or BACs, yeast artificial chromosomes or YACs), and viral vectors.

[0085] The present invention will now be described in detail in the embodiments described in this disclosure, which will be illustrated in the accompanying drawings.

[0086] IL-13 signal transduction is considered to play a central role in the symptoms of various immune diseases, including atopic diseases, and cancers. To verify its efficacy, the inventors prepared an antibody that recognizes and neutralizes canine IL-13. First, they developed a canine recombinant cIL-13. Then, they injected mice with the developed recombinant cIL-13 to induce an immune response. The spleens of the induced mice were harvested to prepare cell suspensions, and hybridomas were prepared by fusing polyethylene glycol (PEG) with myeloma cells. After assessing the formation of hybridoma pools using enzyme-linked immunosorbent assay (ELISA), TF-1 proliferation inhibition, and STAT-6 phosphorylation inhibition, a monoclonal antibody named 32A10-#20 was finally produced.

[0087] The binding of cIL-13 with N- or O-glycan chains 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.

[0088] One embodiment of the present invention describes a method for cloning gene sequences from obtained 32A10-#20 hybridomas. The antigen-binding specificity and biological activity of monoclonal antibodies are determined by a variable region containing a complementarity-determining region (CDR). When confirming the sequence using three heavy chain and three light chain primers, although the CDRs for the heavy and light chains were identical, two different framework region sequences were found. The confirmed variable region gene sequence was optimized using codons suitable for CHO cells as the host. After inducing transformation of ExpiCHO-S cells using an expression vector, the chimeric antibody was purified by injecting culture medium into a column. The activity of the selected antibody was retested after the effective sequence was finally determined by analyzing the purity and activity of the purified antibody.

[0089] The experimental example will be explained in more detail through the following illustrations.

[0090] Acquisition of Canis Interleukin-13

[0091] The canine interleukin-13 disclosed herein consists of 131 amino acids, with a signal peptide at the N-terminus and N-glycans linked to asparagine amino acids at positions 38, 48, 56, and 71. Cysteines at positions 47-75 and 63-89 are linked by disulfide bonds to form a tertiary structure. The complete genome sequence of canine interleukin-13 was obtained using the National Center for Biotechnology Information (NCBI) database (GeneBank: AF244915.1). Codon optimization was then performed, adapted to the host CHO (Chinese hamster ovary) cells, except for the restriction enzyme sites required for subsequent cloning. For example, this codon optimization was performed using well-known optimization tools or by relying on external institutions. Then, for cloning, an XhoI enzyme site (C TCCGA) is inserted at the 5' direction, followed by the sequential addition of the Kozak sequence (GCCACC), start codon (ATG), IL-13 coding sequence (CDS), polyhistidine (CATCATCACCATCACCAC), stop codon (TGA), and EcoRI enzyme site (GAATTC) required for translation. Figure 1 The optimal gene sequence for canine IL-13 in CHO is as follows.

[0092] Table 1. CHO-optimized canine IL-13 sequence

[0093]

[0094]

[0095] Production of cIL-13 recombinant protein

[0096] The cloning vector (pUC-GW-Amp) was obtained by synthesizing the most optimized canine IL-13 from the stated CHO. After ensuring the insertion of DNA by treating with the Xhol / EcoRI double restriction enzyme, the DNA was purified and inserted into the plasmid, thus completing the expression vector. Figure 2 In this case, for example, the plasmid can be a pcDNA3.4 vector, and the scope of protection of this disclosure is not limited to such examples.

[0097] The inventors then transformed the prepared vector into a CHO cell line and temporarily cultured it in a medium supplemented with L-glutamate. In this case, for example, ExpiCHO could be used. TM Expression system kit (ThermoFisher, #A29133), the scope of the invention claims of this disclosure is not limited to such examples.

[0098] Table 2

[0099] Temporary cultivation

[0100]

[0101] The culture medium after the culture was completed was diluted 20 times with distilled water and then filtered through a sterile filter to prepare the injection sample.

[0102] Purification of recombinant cIL-13

[0103] After injecting a sample into a resin column using the method of one embodiment of this disclosure, the target histidine-tagged canine interleukin-13 protein is purified by an imidazole gradient method. In this case, a His-tag HP 5 mL column (Cytiva, #29051021) can be used, and the scope of protection of this disclosure is not limited to such examples.

[0104] One embodiment of this disclosure involves dialyzing the eluted protein in a 20mM phosphate-balanced buffer at pH 7.0, then replacing the buffer, quantifying the protein using an absorbance coefficient of 0.766, and then using it in immunoassays, clones, etc. Figures 3a-3c ).

[0105] The quantitative formula is as follows.

[0106] The concentration of cIL-13 (mg / mL) = (A280 × dilution factor) / 0.766

[0107] *In the attached figure, the blue solid line represents the absorbance at 280 nm; the green solid line represents the gradient; and the brown solid line represents the conductivity (ms / Cm).

[0108] 1. Purity Analysis

[0109] cIL-13 is known to have four N-glycan binding sites, classifying it as a highly glycosylated protein. IMAC purification of the protein did not show a clearly defined main band during electrophoresis; however, upon cleavage of the N-glycans, the main band shifted to the 10–15 kDa range and was observed, consistent with the calculated molecular weight of 13.3 kDa based on amino acid information. Figure 4 ).

[0110] 2. Activity Analysis

[0111] To confirm the activity of cIL-13, one embodiment of this disclosure describes a method that treats the human erythroleukemia cell line TF-1 with recombinant cIL-13 at different concentrations and cultures them for 3 days to assess proliferation. In this case, Ez-Cytox (DoGENBio, #EZ-500) can be used to assess proliferation, but the scope of this disclosure is not limited to such examples. The prepared cIL-13 concentration-dependently induced TF-1 cell proliferation and showed similar activity under three freeze-thaw cycles. Figure 5 ).

[0112] Preparation of hybridoma

[0113] To prepare antibodies targeting the recombinant protein cIL-13, an embodiment of this disclosure describes a method for preparing an immunogenic substance by mixing His-tag cIL-13 with ODN 1585-TLR9 ligand. Then, the method of this disclosure immunizes 6-week-old female BALB / c mice via intraperitoneal injection three times at 4-week, 3-week, and 3-week intervals. One week after the third immunization, plasma was collected via orbital blood sampling, and the immune response was confirmed by conventional ELISA. High responsiveness to the recombinant antigen cIL-13 was observed in all five mice. For hybridoma development, the method of this disclosure prepares the recombinant antigen and performs a booster immunization, with the spleen of the mice harvested three days later to prepare a cell suspension.

[0114] Table 3

[0115] Preparation of additional immune substances

[0116] Substance Name Usage Recombinant protein His-tag cIL-13 50 μg / mouse ODN 1585-TLR9 ligand 25 μg / mouse Rat anti-mouse CD40 mAb (1C10) 30 μg / mouse

[0117] One embodiment of this disclosure describes a method for processing 10 liters of material in Dulbecco's modified Eagle's medium (DMEM) using the Kohler-Milstein method. 8 10 spleen cells and 10 7 A myeloma cell line was induced to fuse using 50% polyethylene glycol 400. In this case, the myeloma cell line could be X63-Ag8.653 (ATCC, PTA-8431) from myeloma cells resistant to 8-azoguanine (Sigma-Aldrich, #SML2963), but the scope of this disclosure is not limited to such examples. One embodiment of this disclosure involves washing fused cells, resuspending them in DMEM medium (HAT medium) supplemented with 20% fetal bovine serum, 100 μm hypoxanthine, 0.44 μm aminopterin, and 16 μm thymidine, aliquoting the cells into 96-well plates, and culturing them at 37°C with a 5% CO2 supply for 2 weeks.

[0118] Then, the method of one embodiment of this disclosure performs a first limiting dilution after evaluating the antigen reactivity and TF-1 proliferation inhibitory capacity of the observed hybridoma pools forming colonies. Then, a second limiting dilution is performed after re-evaluating the antigen reactivity and TF-1 proliferation inhibitory capacity, and evaluating the STAT-6 phosphorylation inhibitory capacity. Finally, the method of one embodiment of this disclosure yields five monoclonal purified antibodies after further evaluation of the TF-1 proliferation inhibitory capacity.

[0119] Evaluation of monoclonal antibodies

[0120] 1. Purity assessment

[0121] One embodiment of this disclosure uses a method to compare and evaluate the physicochemical and biological activities of five established anti-canine IL-13 monoclonal antibody subclones. First, the method of one embodiment of this disclosure assesses purity by injecting the five purified antibodies into an SE-HPLC column. An antibody molecule consisting of two heavy chains and two light chains eluted at a retention time of approximately 15.6 minutes, all showing high purity of over 96%.

[0122] Table 4

[0123] Monoclonal antibody purity analysis

[0124]

[0125]

[0126] 2. Assess antigen reactivity

[0127] To assess the antigen-binding reactivity of the purified antibodies, a routine ELISA assay with the coated antigen (cIL-13) was performed. Among the clones analyzed, clone 32A10-#20 was evaluated with an EC50 of 463 pM, showing the highest reactivity.

[0128] Table 5

[0129] Antigen reactivity of mouse monoclonal antibodies

[0130]

[0131] 3. Assess the proliferation-inhibiting ability of TF-1

[0132] According to reports, canine IL-13 shares 61.8% similarity with human IL-13 based on amino acid sequence, and both can interact with the human IL-13 receptor (JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 20:779-785, 2000). Therefore, to confirm the biological activity of the 32A10 monoclonal antibody, its cell proliferation inhibition ability via canine IL-13 was evaluated using the TF-1 cell line.

[0133] The inventors cultured TF-1 cell lines in 10% fetal bovine serum RPMI medium containing GM-CSF, and removed the GM-CSF by washing on the first day. Furthermore, the inventors used 2 × 10⁻⁶ cells in flat-bottomed 96-well microplates. 4 TF-1 cells were dispensed at 50 μL / well. The antibody was then serially diluted 3-fold with medium containing 50 ng / mL cIL-13 recombinant protein, starting at a concentration of 50 μg / mL, and reacted for 1 hour. A mixture of interleukin-13 and antibody was added to 100 μL / well of TF-1 cell lines and cultured for an additional 72 hours. Cell culture success was then assessed. The 32A10 monoclonal antibody was confirmed to inhibit TF-1 proliferation in a concentration-dependent manner, with an IC50 range of 2.2–3.3 nM.

[0134] Table 6

[0135] Inhibitory ability of mouse monoclonal antibodies against TF-1 cell proliferation

[0136]

[0137] 4. Joint area

[0138] To confirm the binding site of the 32A10 antibody, the inventors treated the recombinant cIL-13 protein with PNGase-F or O-glycosidase to remove the N-glycans or O-glycans, and analyzed the binding of the 32A10 monoclonal antibody using Western blotting. The main band of the sample with the N-glycans removed was observed in the 13 kDa range, which is theoretically the same as the molecular weight of cIL-13. The main bands of both the glycan-removed and O-glycan-removed samples were observed in the 20–35 kDa range. The 32A10 antibody reacted independently of the presence or absence of the antigen glycans; therefore, it was determined that the 32A10 monoclonal antibody recognizes and binds to the canine interleukin-13 peptide independently of the glycans. Figure 6 ).

[0139] Obtain the 32A10 gene sequence

[0140] The antigen-binding specificity and biological activity of monoclonal antibodies are determined by the variable region containing the CDR (Complementarity Determining Region). To confirm the amino acid sequence of the rodent antibody 32A10 monoclonal antibody, the gene sequence was obtained by cloning from the established 32A10-20 hybridoma.

[0141] First, the inventors isolated total RNA from 32A10 hybridomas. For example, total RNA isolation can be performed using the RneasyPlus Mini Kit (Qiagen), but the scope of this disclosure is not limited to such examples. Then, a DNA template was obtained by RT-PCR using IgG constant region-specific primers. Following PCR amplification using the obtained DNA template and variable region-specific primers, the amplified variable region vector was obtained by TA cloning. In this case, the Progen Mouse IgG library primer set and the TAKARA Mighty TA Cloning Kit can be used, but the scope of this disclosure is not limited to such examples. Finally, the variable region sequence was confirmed by sequencing the three variable region heavy chains and three variable region light chains.

[0142] The sequencing results showed that the CDR sequences were identical regardless of the primer type, but two different sequences were identified for sequence 1 in the frame region.

[0143] Table 732A10 Variable Region Sequence

[0144]

[0145] *The underline indicates the CDR sequence. They are arranged in the order of CDR1, CDR2, CDR3.

[0146] Cloned 32A10 chimeric antibody

[0147] The variable region gene sequence identified from 32A10 hybridomas was optimized using codons suitable for CHO cells. It was designed as follows: an XhoI restriction enzyme site was appended to the 5' end for gene transfer to the expression vector; a Kozak sequence was appended to induce smooth translation; and a signal peptide derived from human albumin was appended to induce protein expression followed by extracellular secretion. A stop codon and an EcoRI restriction enzyme site were appended to the 3' end to terminate expression. According to one embodiment of the invention, canine IgG type B is used as the heavy chain constant region, but the scope of the invention is not limited thereto. Canine IgG types A, B, C, or D can be used in the construction of the chimeric antibody heavy chain constant region. Similarly, according to one embodiment of the invention, the light chain constant region uses the canine kappa chain, but the scope of the invention is not limited thereto; canine kappa or λ chains can be used in the construction of the chimeric antibody light chain constant region. Figure 7 ).

[0148] Table 832A10 antibody optimized gene sequence

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] Expression vectors were created by inserting the pcDNA3.4 vectors with XhoI / EcoRI modified multiple cloning sites into the four chimeric antibodies described in Table 8.

[0155] To prepare vectors expressing the chimeric genes described in Table 8, one embodiment of this disclosure first treats four DNAs with XhoI / EcoRI using double restriction enzymes, followed by electrophoresis and gel extraction to prepare the inserted DNA. Then, another embodiment of this disclosure treats the pcDNA3.4 vector with XhoI / EcoRI using double restriction enzymes, followed by electrophoresis and gel extraction. Next, using T4 DNA ligase, the pcDNA3.4 vector is inserted with two heavy chains and two light chains, and then transfected into DH5α *E. coli* cells to prepare four cell lines: pcDNA3.4-VHC-B, pcDNA3.4-VHF-B, pcDNA3.4-VLN-kappa, and pcDNA3.4-VLO-kappa. After another XhoI / EcoRI double digestion, electrophoresis is used to confirm the insertion of the target gene into the vector, and then commissioned sequencing is used to confirm the sequences of the variable regions of the light and heavy chains.

[0156] Production of 32A10 mouse / canine chimeric antibodies

[0157] One embodiment of this disclosure involves combining two heavy chains (C-type and F-type) with two light chains (N-type and O-type) to induce CHO cell transformation, followed by culture until the viability falls below 70%. The method of this disclosure also involves recovering the culture medium and injecting it into a chromatographic column to purify the chimeric antibody. In this case, the column can be a Mabselect Sure (Cytiva, #29049104), and the scope of protection of this disclosure is not limited to such examples. The method of this disclosure produces antibodies only in two of the four combinations containing the O-type light chain, thus identifying the N-type light chain gene as an erroneous sequence that does not express antibodies. The method of this disclosure quantifies and assesses the purity and activity of the purified antibody by measuring its OD280 nm.

[0158] Table 9

[0159] Temporary culture of mouse / dog 32A10 chimeric monoclonal antibody

[0160]

[0161] 1. Purity assessment

[0162] The inventors assessed purity by injecting two chimeric purified antibodies into an SE-HPLC column. In this case, the SE-HPLC column could be TOSOH or TSKgel G3000SWXL, and the scope of this invention is not limited to such examples. Elution of one molecule of antibody consisting of two heavy chains and two light chains at a retention time of approximately 15.6 minutes showed a high purity of over 99%. Figure 8 ).

[0163] 2. Assess antigen reactivity (quantitative EC50 value)

[0164] One embodiment of this disclosure involves adding 100 μL (100 ng / 100 μL / well) of histidine-tagged cIL-13 recombinant protein to each well of a nickel-plated culture plate (Thermo Fisher Scientific, #15442) and reacting at room temperature for 1 hour. Next, the method involves washing three times with 1×PBST, serially diluting the chimeric antibody with 1×PBS, adding it, and inducing an antigen / antibody reaction at room temperature for 1 hour. Another embodiment involves washing three times with 1×PBST, then diluting a horseradish peroxidase (HRP)-conjugated secondary antibody cross-reactive with canine antibodies 100-fold in casein buffer and adding 100 μL / well, reacting at 37°C for 30 minutes. In this case, the HRP-conjugated secondary antibody can be Jackson Immuno Research #115-036-071, and the casein buffer can be Thermo Fisher Scientific #37528. The scope of this disclosure is not limited to such examples. One embodiment of the method disclosed herein uses 1×PBST to wash three times, followed by the addition of TMB to induce color development, and then the addition of an equal volume of 1.0N sulfuric acid to terminate the reaction. This embodiment of the method confirmed that two chimeric antibodies have EC50 values ​​similar to the parent antibody (mouse 32A10).

[0165] Table 10

[0166] Canine interleukin-13 binding reaction

[0167] Antibody EC50 (ng / mL) EC50 (nM) Mother antibody (mouse 32A10) 217.9 1.45nM Chimeric antibody (CO type) 162.4 1.08 Chimeric antibody (FO type) 147.3 0.98nM

[0168] 3. TF-1 proliferation inhibitory activity

[0169] To evaluate the proliferation-inhibiting activity of the chimeric antibody in TF-1 cell lines.

[0170] One embodiment of the method disclosed herein uses a 2×10⁻⁶ rpm method on Day 1, the day before the test. 4TF-1 cells were aliquoted into a medium without GM-CSF and cultured at a rate of 50 μL / well. The following day, ten samples were prepared by serially diluting the parent antibody or chimeric antibody FO three-fold in a concentration range of 50,000 to 2.54 ng / mL, according to an embodiment of this disclosure. In another embodiment, an equal volume of canine IL-13 recombinant antigen (300 ng / mL) solution was mixed with the prepared antibody test solution 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. The proliferation of the TF-1 cell line was then measured.

[0171] The chimeric antibody FO inhibited the proliferation of TF-1 cell line in a similar manner to the parent antibody.

[0172] Table 11 Inhibition effect on TF-1 cell line proliferation

[0173] Antibody IC50(pM) Mother antibody (mouse 32A10) 3969pM Chimeric antibody (FO type) 3278pM

[0174] 4. STAT-6 phosphorylation inhibitory activity

[0175] To confirm the blocking effect of the chimeric antibody on downstream signal transduction of cIL-13, the ability to inhibit STAT-6 phosphorylation was evaluated.

[0176] The day before the experiment (Day-1), a method according to one embodiment of this disclosure was used to suspend and wash the TF-1 cell line in a medium without GM-CSF, at 4 × 10⁻⁶. 5 Cells were aliquoted at 50 μL / well and cultured for 24 hours at 37°C. In one embodiment of this disclosure, the chimeric antibody was serially diluted in a medium supplemented with interleukin-13 at a concentration of 100 ng / mL to prepare nine samples in the range of 50,000–7.62 ng / mL. In another embodiment, 200 μL of the mixture was incubated in CO2 cell culture medium at 37°C for 1 hour to induce an antigen / antibody reaction, and then the mixture was added to TF-1 cells (50 μL / well). The reaction was allowed to proceed for 5 minutes (37°C, 5% CO2) to induce signal transduction in the TF-1 cells. The TF-1 cell line was then recovered and washed with 1×PBS. In another embodiment, three wells were prepared under identical conditions. To ensure sensitivity, phosphorylated STAT-6 was quantified by combining three wells into one sample for analysis. The method described in this embodiment allows for the quantification of phosphorylated STAT-6 in cells using [the following method is missing from the original text]. The Phospho-Stat6 (Tyr641) Sandwich ELISA Kit (Cell Signaling Technology, #7275C) was used for assays according to the instructions. The scope of protection of this disclosure is not limited to such examples. The chimeric antibody FO effectively inhibited STAT-6 phosphorylation induced by interleukin-13 in a concentration-dependent manner, with an IC50 assessment of 2418 pM. Figure 9 ).

[0177] Table 1232A10 Variable Region Amino Acid Sequence

[0178]

[0179] Canine antibody design

[0180] To prepare recombinant antibodies as therapeutic agents for atopic dermatitis in dogs, the necessity of further caninizing established chimeric antibodies has been proposed. In this disclosure, caninization refers to the process of replacing a defined portion of the mouse-derived variable region of a chimeric antibody with a canine variable region to reduce immunogenic side effects when administering the antibody to dogs. To maintain antigen binding capacity while reducing immunogenicity, one embodiment of this disclosure utilizes CDR grafting technology to transplant the mouse-derived complementarity-determining region (CDR) of a chimeric antibody into a canine germline-derived framework region. In this case, the antibody providing the CDR site is called the donor, and the antibody receiving the CDR site with its own backbone is called the recipient.

[0181] To perform CDR grafting, one embodiment of this disclosure first constructs a homology model. Homology model building is a method for constructing a three-dimensional model of the antibody using the established sequence of the chimeric antibody, which is particularly helpful for predicting the structure of the CDR or the structural compatibility between the framework region and the CDR. Furthermore, homology model building helps determine whether to preserve donor residues or replace them with recipient residues, providing a basis for the entire canine derivatization process.

[0182] To construct a homology model, one embodiment of this disclosure first utilizes a protein data bank (PDB) to retrieve structures with high homology to the light and heavy chain sequences of the variable region of a chimeric antibody. The structure encoding 5IKC in the PDB is selected as the template for the light chain variable region frame region, and the structure of 5YFI is selected as the template for the heavy chain variable region frame region. Analysis of the CDR loop structure uses a deep learning-based analysis system. The deep learning-based CDR loop structure analysis system can use Ablooper, but is not limited to it. For three CDR structures each for the light and heavy chain regions, the method of one embodiment of this disclosure identifies relevant canonical structures (Chothia structures) and individually screens protein structures suitable for templates of each CDR. Then, tertiary structure models suitable for the heavy and light chain sequences of the variable region are identified using protein binding prediction software. According to one embodiment of this disclosure, the tertiary structure suitable for the variable region of the chimeric antibody is the structure of the protein numbered 1HIN in the protein data bank, with a relative stacking angle of -46.9°. The prediction software can use PAPS and ABangle, but is not limited to them. One embodiment of this disclosure assembles a final model by corresponding the backbone coordinates of the chimeric antibody anchoring residues to the structure of 1HIN, and then performs an energy minimization process through molecular mechanical simulations. A diagram of the final model is shown below. Figure 10 As shown.

[0183] In the design of canine-derived antibody sequences, to prevent loss of affinity or antibody stability caused by structural incompatibility between mouse-derived CDR sequences and canine-derived framework regions, careful attention must be paid to the preservation of mouse-derived sequences or their replacement with canine-derived sequences. This operation is particularly considered to be performed in the Vernier zone. The Vernier zone, structurally located within or adjacent to the CDR, is considered to influence subtle adjustments to CDR morphology and antigen recognition. Replacement with canine-derived sequences aims to increase sequence identity with the canine gene without sacrificing antibody affinity. Additionally, when dealing with antibody candidates, sequence optimization strategies to improve the physicochemical properties, preparability, pharmaceutical properties, etc., should also be considered. Given the fragility of antibodies as proteins, they are preferably designed to maintain chemical and structural integrity during preparation, distribution, and storage. Therefore, the possibility of various post-translational modifications (PTMs) should also be considered. The possibilities of methionine oxidation, N- and O-glycosylation, asparagine deamidation and aspartic acid (D) isomerization, N-terminal glutamine cyclization, cysteine ​​binding, lysine side-chain glycosylation, and tryptophan residue oxidation were examined. To avoid the possibility of post-translational modifications, some residues were canine-derived or preserved as mouse-derived sequences.

[0184] To screen the receptor framework region of the chimeric antibody CDR to be transplanted according to an embodiment of this disclosure, similar sequences of the chimeric antibody variable region heavy and light chains were searched using canine gene databases and the canine genome project. Screening was performed based on the overall sequence identity of the framework region, the consistency and compatibility of the contact points between the variable region heavy and light chains, and the standard structure of the CDR. Based on the analysis results, canine gonadal genes IGHV1-30*01 and IGHV3-23*01 were selected as suitable templates in the variable region heavy chain. Canine gonadal genes IGK3-18*01 and IGKV2-10*01 were selected as suitable templates in the variable region light chain. Three versions of canine-derived sequences were designed for each selected template.

[0185] Table 13

[0186] canine antibody sequence

[0187]

[0188]

[0189]

[0190] Therefore, according to one embodiment of this disclosure, the antibodies to be prepared are screened based on the results of the canine antibody design as shown in Table 14.

[0191] Table 14

[0192] Designed nomenclature for canine antibodies

[0193]

[0194] Preparation of canine-derived antibodies

[0195] The inventors of this disclosure commissioned an external organization to attempt to prepare the screened canine antibodies (shown in Table 14). Canine antibodies Ab1 to Ab14 and Ab21 to Ab24 were successfully prepared, but unfortunately, Ab15 to Ab20 were not prepared. All prepared antibodies showed a purity of over 98% and an endotoxin level below 1 EU. The purity and endotoxin levels of Ab1 to Ab24 are shown in Table 15.

[0196] Table 15 Purity and endotoxin levels of Ab1 to Ab24

[0197]

[0198] Evaluation of canine antibodies

[0199] To verify the function of a canine antibody prepared according to an embodiment of this disclosure and to identify clinical candidates, the binding ability of the antibody to cIL-13, the thermostability of the antibody, and the inhibitory ability of TF-1 cell proliferation were evaluated.

[0200] 1. Antigen reactivity (EC50)

[0201] The conventional ELISA assay was used to assess the antigenic reactivity of canine-derived antibodies. In this invention, His-tag-binding cIL-13 was coated at a concentration of 1 μg / mL in 96-well nickel culture plates and incubated at room temperature for 1 hour. Each well was washed three times with 200 μL of wash buffer. 1×PBS containing 0.1% Tween 20 was used as the wash buffer. In one embodiment of this disclosure, 100 μL of diluted antibody was aliquoted into each well, and the plates were incubated again at room temperature for 1 hour. A solution of 25 mL of 1× casein solution and 25 mL of 1×PBS was used as the dilution buffer. In another embodiment of this disclosure, 50 μL of HRP-conjugated goat anti-human IgG was diluted in 10 mL of dilution buffer, 100 μL was aliquoted into each well, and the plates were incubated again at room temperature for 1 hour. Each well was washed three times with 200 μL of wash buffer. After adding 50 μL of TMB substrate to each well, the reaction was allowed to proceed for 10 minutes at room temperature. 50 μL of 1N sulfuric acid was then dispensed into each well as a stop solution, and the absorbance was measured at 450 nm using a spectrophotometer. The EC50 values ​​shown in Table 16 were calculated based on the results. It was confirmed that Ab1 to Ab5 exhibited superior antigen reactivity compared to the chimeric antibody, while Ab21 to Ab24 showed similar antigen reactivity to the chimeric antibody.

[0202] Table 16 EC50 for Ab1 to Ab24

[0203]

[0204]

[0205] 2. Thermal stability

[0206] In hot environments, antibody instability can lead to reduced production yield, loss or damage to efficacy, harmful immune responses, and related patient complications. Under extreme conditions, its function may be limited or impaired during handling or long-term storage. Therefore, antibody thermostability is evaluated as an indicator of potential variability in future use. According to one embodiment of this disclosure, 200 μL of test sample was stored at 60°C or 4°C for 1 hour. Then, 180 μL of sample was aliquoted into each well of an opaque 96-well culture plate. After aliquoting 20 μL of 0.2 mg / mL ANS solution into each well, the plate was incubated in the dark for 30 minutes. The culture plate was then read using a fluorescence analyzer. (Excitation: 375 nm, Emission: 470 nM)

[0207] To quantify the thermostability of antibodies, the following formula is used.

[0208] Relative instability = (thermal stress at 60℃ - no treatment) / no treatment × 100

[0209] The results of comparing the thermostability of the candidate antibodies are as follows: Figure 12 As shown in Table 17, the thermal stability of Ab21 to Ab24 is similar to or better than that of chimeric antibodies.

[0210] Table 17 Relative instabilities of Ab1 to Ab24

[0211]

[0212]

[0213] 1. TF-1 proliferation inhibitory activity

[0214] According to one embodiment of this disclosure, the effect of canine-derived antibodies on inhibiting cell proliferation was evaluated. One day prior to the experiment, the inventors, under GM-CSF-free conditions, aliquoted 50 μL of 2×10⁻⁶ canine-derived antibodies into each well, except for B2, B3, and B4. 4TF-1 cells were then cultured overnight at 37°C in a 5% CO2 incubator. Next, 90 μL of 300 μg / ml antibody diluent was added to wells B11, C11, D11, E11, F11, and G11 of a clear 96-well V-bottom culture plate. Using a multichannel pipette, 30 μL of the antibody diluent was transferred from wells B11, C11, D11, E11, F11, and G11 to wells B10, C10, D10, E10, F10, and G11, respectively, and then thoroughly mixed by pipetting several times. This process was repeated, removing 30 μL from wells B2, C2, D2, E2, F2, and G2 to adjust the final volume to 60 μL. In one embodiment of this disclosure, 60 μL of culture medium was added to wells A2 to A7 and H2 to H7. Add 60 μL of cIL-13 solution (prepared at a concentration of 100 ng / mL) to each well containing 60 μL of diluted antibody solution and IL-13-only solution. Add 120 μL of culture medium to each well containing only TF-1 cells and GM-CSF-only solution, and incubate at 37°C for 1 hour in a 5% CO2 incubator. Remove the overnight TF-1 cell culture plate and add 100 μL of the cIL-13 and antibody mixture to each well to a final volume of 150 μL. Incubate at 37°C for 72 hours in a CO2 incubator. Before the end of the culture, remove the matrix solution used for proliferation inhibition analysis. Mix 20 μL of matrix with 30 μL of culture medium in each well. Add 50 μL of the mechanism solution to each well of the culture plate after the culture is complete, and incubate for an additional 3 hours in a CO2 incubator at 37°C. After equilibration at room temperature for 15 minutes, measure the absorbance using a spectrophotometer. The following IC50 values ​​were calculated based on the measurement results. The cell proliferation inhibitory abilities of Ab3 to Ab5 and Ab21 to Ab24 antibodies, including chimeric antibodies, are as follows: Figures 13a to 13c As shown in the figure. The comparison results confirmed that Ab21 and Ab22 maintained TF-1 cell proliferation inhibition capacity at a similar level to chimeric antibody 32A10, but Ab3 to Ab5, as well as Ab23 and Ab24, did not maintain sufficient proliferation inhibition capacity.

[0215] Table 18 IC50 of Ab3 to Ab5 and Ab21 to Ab24

[0216] Antibody name IC50(nM) Ab3 52.67 Ab4 71.49 Ab5 34.20 Ab21 1.13 Ab22 0.98 Ab23 35.19 Ab24 4.09 chimeric antibodies 1.74

[0217] Table 19

[0218]

[0219]

[0220] in conclusion

[0221] A method according to an embodiment of this disclosure successfully prepared and produced a recombinant canine interleukin-13 histidine-tagged protein. The glycan-removed cIL-13 recombinant protein polypeptide has a molecular weight of approximately 13 kDa and is expressed as a glycoprotein with an over-linked N-glycan chain. cIL-13 exhibits biological activity that induces TF-1 cell proliferation.

[0222] The method of one embodiment of this disclosure subsequently successfully developed a monoclonal antibody, 32A10, that specifically recognizes canine interleukin-13. Of the three subclones, clone "32A10-#20" showed the highest antigen affinity, with an EC50 of 463 pM for antigen binding. The 32A10 monoclonal antibody was confirmed to inhibit interleukin-13-dependent TF-1 cell proliferation, with an IC50 value ranging from 2.9 to 4.2 nM. The 32A10 monoclonal antibody was confirmed to detect the antigen and recognize recombinant protein peptides independently of N- and O-glycan chains.

[0223] One embodiment of this disclosure describes a method for obtaining two variable region sequences each from the heavy and light chains of a 32A10 monoclonal antibody and preparing mouse / dog chimeric antibodies. Of the four combinations, the FO-type chimeric antibody exhibited antigen-binding affinity and TF-1 proliferation-inhibiting activity similar to the parent clone. Furthermore, the FO-type chimeric antibody demonstrated an effect of blocking STAT-6 phosphorylation induced by interleukin-13. Therefore, it was ultimately confirmed that the heavy chain variable region sequence of 32A10 is type F, and the light chain variable region sequence is type O.

[0224] The 32A10 monoclonal antibody effectively inhibited the proliferation of TF-1 cell lines within an IC50 range of 2.2–3.3 nM by neutralizing cIL-13. The outstanding cIL-13 neutralizing activity exhibited by the 32A10 monoclonal antibody at low concentrations suggests that it can be developed as a therapeutic agent for canine atopic dermatitis. The mouse / canine chimeric antibody with 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 very high purity of the 32A10 chimeric antibody can be interpreted as meeting the stable physical properties required for development.

[0225] One embodiment of this disclosure describes the canine derivatization of a 32A10 chimeric antibody. During the canine derivatization design, the mouse-derived complementarity-determining region (CDR) of the chimeric antibody is transplanted into the canine gonad-derived framework region using CDR grafting technology. The method of this embodiment first predicts the tertiary structure of the chimeric antibody using a homology model. Considering the possibility of Vernier region and post-translational modifications, the chimeric antibody sequence is either retained or replaced with a canine-derived sequence. The method of this embodiment uses canine gene databases to search and screen canine gonad-derived IGHV1-30*01 and IGHV3-23*01 as heavy chain templates for the variable region, and IGK3-18*01 and IGKV2-10*01 as light chain templates for the variable region. Three versions of the canine-derived sequence are designed according to each different template.

[0226] A total of 36 sequences were derived through combinations of heavy and light chain sequences. Among the 24 named antibodies, Ab1 to Ab14 and Ab21 to Ab24 were successfully obtained. An embodiment of this disclosure tested and evaluated the antigen reactivity, thermostability, and TF-1 cell proliferation inhibition ability of the antibodies. As a result, canine antibodies Ab3 to Ab5 and Ab21 to Ab24 were derived as candidate materials after considering productivity, biological activity, and other factors.

[0227] On the other hand, the antibody may be a chimeric antibody, a humanized antibody, a caninized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, a pseudo-antibody, a diabody, a triabody, a tetrabody, or a fragment thereof, but is not limited thereto.

[0228] In this disclosure, the "chimeric antibody" is an antibody that combines the variable region of a recombinant mouse antibody with the constant region of a canine antibody, and compared to the mouse antibody, it is an antibody that significantly improves the immune response.

[0229] In this disclosure, "humanized antibody" refers to an antibody whose protein sequence is modified in a manner similar to naturally occurring antibody variants in humans. For example, the humanized antibody can be prepared by recombining a mouse-derived CDR with a human-derived FR to create a humanized variable region, and then recombining that with a preferred human antibody's constant region.

[0230] In this disclosure, the antibodies also apply to veterinary medicine. For example, the term "caninized antibody" refers to an antibody whose protein sequence, derived from a non-canine antibody, is modified in a manner similar to naturally occurring antibody variants in dogs.

[0231] Furthermore, this disclosure can provide pharmaceutical compositions for treating immune diseases or cancer that include the said antibody and a pharmaceutically acceptable carrier.

[0232] In this disclosure, the antibody may be in the form of an antibody-drug conjugate. The term "antibody-drug conjugate (ADC)" refers to a form in which the drug and antibody are chemically linked without reducing the biological activity of the antibody and drug.

[0233] In this disclosure, the chemical linkage can be a covalent bond. In this disclosure, the antibody-drug conjugate refers to a form in which the N-terminal amino acid residues of the antibody's heavy and / or light chains are bound to a drug; specifically, it refers to a form in which the N-terminus and α-amino group of the antibody's heavy and / or light chains are bound to a drug.

[0234] In this disclosure, the pharmaceutical composition may be in injectable form, and the pharmaceutical composition may be intended for use in at least one of humans or animals.

[0235] In this disclosure, the routes of administration of the pharmaceutical composition include, but are not limited to, oral, intravenous, intramuscular, intravenous, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, lesion, sublingual, or rectal administration, with parenteral administration being preferred.

[0236] In this disclosure, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this disclosure can also be administered in the form of suppositories for rectal administration.

[0237] The pharmaceutical compositions disclosed herein can vary widely depending on factors including the activity of the specific compound used, age, weight, general health condition, sex, diet, time of administration, route of administration, metabolic rate, drug combination, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition can vary depending on the patient's condition, weight, degree of disease, drug form, route of administration, and duration, which can be appropriately selected by the relevant practitioner.

[0238] In this disclosure, "immune-related diseases" are diseases induced by the overexpression of various immune cells and inflammatory cells, such as autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, specific diseases, or dosage form or chronic inflammatory diseases, but are not limited thereto.

[0239] Furthermore, in this disclosure, the term "autoimmune disease" can be one or more selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa, but is not limited thereto.

[0240] In this disclosure, "cancer" refers to a disease characterized by the rapid and uncontrollable growth of mutant cells, and may be one or more selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, leukemia, thyroid cancer, endocrine gland cancer, oral cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid carcinoma, uterine cancer, colon cancer, bladder cancer, ureter 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.

[0241] Furthermore, this disclosure can provide host cells for generating the antibodies.

[0242] The host cells used in the preparation of antibody drugs can vary depending on the specific antibody and the desired application. Typically, to produce antibody proteins, antibody drugs utilize living cells such as bacteria, yeast, or mammalian cells. The choice of host cell affects the yield, quality, and glycosylation pattern of the final antibody product. For example, the host cells can be bacteria such as E. coli, or mammalian cells such as yeast, CHO cells, or HEK cells.

[0243] On the other hand, besides the selection of host cells, the production of antibody drugs can involve genetic engineering of host cells to improve antibody expression by several decimeters. For example, inserting genes encoding chaperone proteins or glycosylation enzymes can improve the yield and quality of the final antibody product.

[0244] Furthermore, this disclosure may provide a vector containing a nucleic acid encoding the antibody.

[0245] A vector is a DNA molecule used in the process of delivering exogenous 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 can vary depending on the specific host cell and the intended use of the recombinant protein.

[0246] The description of the proposed embodiments is provided to enable those skilled in the art to utilize or implement this disclosure. Those skilled in the art will understand that various modifications to these embodiments are possible, and the general principles defined herein apply to other embodiments without departing from the scope of this disclosure. Therefore, this disclosure is not limited to the embodiments presented herein, but should be interpreted within the broadest scope consistent with the principles set forth herein and with new features.

[0247] Best practice

[0248] The relevant content is described in the above-described specific implementation method.

[0249] Industrial availability

[0250] It can be used to treat atopic dermatitis in individuals, including dogs.

[0251] Preface text of sequence list

[0252] SEQ ID NO: 5 in the sequence listing of this application is a sequence of fewer than 4 amino acids, which is described separately below.

[0253] SEQ ID NO: 5.

[0254] Leu Ala Ser

Claims

1. An anti-canine interleukin-13 antibody or its antigen-binding fragment, characterized in that, It specifically recognizes and neutralizes canine interleukin-13.

2. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment comprises a monoclonal antibody.

3. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment includes mouse antibodies, chimeric antibodies, or canine-derived antibodies.

4. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment contains a heavy chain constant region derived from type A, B, C, or D of canine immunoglobulin G.

5. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment contains a light chain constant region derived from the canine κ or λ chain.

6. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment comprises a light chain variable region having at least one amino acid sequence selected from sequence listings 12, 23, 24, 25, 26, 27 and 28.

7. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 6, characterized in that, The light chain of the anti-canine interleukin-13 antibody or its antigen-binding fragment has at least one amino acid sequence selected from sequence listings 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 its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment comprises a heavy chain variable region having at least one amino acid sequence selected from sequence listings 10, 17, 18, 19, 20, 21 and 22.

9. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain of the anti-canine interleukin-13 antibody or its antigen-binding fragment has at least one amino acid sequence selected from sequence listings 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 its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment has at least one of the characteristics of inhibiting the proliferation of TF-1 cell lines or inhibiting STAT-6 phosphorylation.

11. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment comprises at least one of the following: Heavy chains or variants thereof having the amino acid sequence of sequence listing 69 or 71; and A light chain or a variant thereof having an amino acid sequence of sequence listing 70 or 72.

12. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment comprises an antibody consisting of two light chains and two heavy chains.

13. The anti-canine interleukin-13 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-canine interleukin-13 antibody or its antigen-binding fragment includes a Fab fragment, an Fv fragment, a single-chain variable region fragment, a double-chain antibody, a triple-chain antibody, a quadruple-chain antibody, or at least one of these fragments.

14. A method, characterized in that, Use the anti-canine interleukin-13 antibody or its antigen-binding fragment as described in claim 1 to prevent or treat diseases in animals.

15. The method according to claim 14, characterized in that, The method is applied to the animal to treat or prevent at least one of the following diseases: inflammatory diseases, immune diseases, autoimmune diseases, and cancer.

16. The method according to claim 15, characterized in that, The diseases mentioned include atopic dermatitis.

17. The method according to claim 14, characterized in that, The animals mentioned include dogs.

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

19. The pharmaceutical composition according to claim 18, characterized in that, The pharmaceutical composition is formulated as an injectable dosage form.

20. A host cell, characterized in that, This is used to prepare anti-canine interleukin-13 antibodies or antigen-binding fragments thereof that specifically recognize and neutralize canine interleukin-13.

21. The host cell according to claim 20, characterized in that, The host cells were selected from a group consisting of bacteria including Escherichia coli, yeast, CHO cells, and HEK cells.

22. The host cell according to claim 20, characterized in that, The host cell, as a result of genetic engineering operations to enhance antibody expression and secretion, contains genes encoding chaperone proteins or glycosylation enzymes.

23. A carrier, characterized in that, The antibody is applied to the host cells of claim 20 to prepare the anti-canine interleukin-13 antibody or its antigen-binding fragment.

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

Images