Monoclonal antibody that specifically binds to nectin-4 and use thereof

Abstract

One embodiment of the present invention provides a monoclonal antibody that specifically binds to nectin-4 and uses thereof, and more specifically, provides a monoclonal antibody that specifically binds to nectin-4, a nucleic acid molecule encoding said monoclonal antibody, an expression vector comprising said nucleic acid molecule, a transformant comprising said expression vector, and a pharmaceutical composition for the prevention or treatment of atopic dermatitis or asthma comprising said monoclonal antibody.

Description

Technology Field

[0001] The present invention relates to a monoclonal antibody that specifically binds to nectin-4 and its uses, and more specifically, to a monoclonal antibody that specifically binds to nectin-4, a nucleic acid molecule encoding said monoclonal antibody, an expression vector comprising said nucleic acid molecule, a transformant comprising said expression vector, and a pharmaceutical composition for the prevention or treatment of atopic dermatitis or asthma comprising said monoclonal antibody. Background Technology

[0003] Atopic dermatitis is a non-contagious, inflammatory skin disease accompanied by pruritus that chronically alternates between periods of remission and relapse. As a type of eczema, it is a representative allergic disease alongside asthma and allergic rhinitis, and its incidence is steadily increasing. While atopic dermatitis can be significantly improved through proper skin care or lifestyle changes, patients with severe conditions exhibit a refractory nature that is not adequately treated by currently available therapies.

[0005] Generally, these symptoms can be alleviated through treatment with steroids or immunomodulators such as tacrolimus and pimecrolimus ointments. However, there are side effects associated with long-term use, and there are limitations to complete cure. Accordingly, there is a need for the development of new, effective treatments with guaranteed safety.

[0007] The asthma treatment market is divided among drugs intended for the management of asthma symptoms, such as inhaled corticosteroids, short-acting or long-acting beta-agonists, and combination drugs. Currently, biological treatments, such as antibody therapies, account for about 2% of the total asthma treatment market, so there is a need for the development of effective treatments related to this. Prior art literature

[0009] Republic of Korea Registered Patent No. 10-1535341 The problem to be solved

[0010] The technical problem that the present invention aims to solve is to provide a monoclonal antibody that specifically binds to nectin-4.

[0012] In addition, the invention provides a monoclonal antibody capable of reducing side effects by minimizing toxicity to the human body in the treatment of atopic dermatitis or asthma, and a pharmaceutical composition utilizing the same.

[0014] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0016] To achieve the above technical problem, one embodiment of the present invention provides a monoclonal antibody that specifically binds to nectin-4.

[0018] In an embodiment of the present invention, the monoclonal antibody may be a monoclonal antibody comprising a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 1; a heavy chain CDR2 described as SEQ ID NO. 2; and a heavy chain CDR3 described as SEQ ID NO. 3, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 4; a light chain CDR2 described as SEQ ID NO. 5; and a light chain CDR3 described as SEQ ID NO. 6.

[0020] In an embodiment of the present invention, the monoclonal antibody may be a monoclonal antibody comprising a heavy chain variable region amino acid sequence described as SEQ ID NO. 7 and a light chain variable region amino acid sequence described as SEQ ID NO. 8.

[0022] In an embodiment of the present invention, the monoclonal antibody may be a monoclonal antibody comprising a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 9; a heavy chain CDR2 described as SEQ ID NO. 10; and a heavy chain CDR3 described as SEQ ID NO. 11, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 12; a light chain CDR2 described as SEQ ID NO. 13; and a light chain CDR3 described as SEQ ID NO. 14.

[0024] In an embodiment of the present invention, the monoclonal antibody may be a monoclonal antibody comprising a heavy chain variable region amino acid sequence described as SEQ ID NO. 15 and a light chain variable region amino acid sequence described as SEQ ID NO. 16.

[0026] To achieve the above technical problem, another embodiment of the present invention provides a nucleic acid molecule encoding the monoclonal antibody.

[0028] To achieve the above technical problem, another embodiment of the present invention provides an expression vector comprising the nucleic acid molecule.

[0030] To achieve the above technical problem, another embodiment of the present invention provides a transformant comprising the expression vector.

[0032] To achieve the above technical objective, another embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising the monoclonal antibody.

[0034] To achieve the above technical objective, another embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of asthma comprising the monoclonal antibody. Effects of the invention

[0036] According to an embodiment of the present invention, the monoclonal antibody of the present invention is manufactured based on a gene present in vivo, and can efficiently neutralize and control a target gene without toxicity to the human body.

[0038] In addition, the manufactured monoclonal antibody can treat patients with atopic dermatitis or asthma through specific binding to Nectin-4 and can reduce side effects caused by existing synthetic drugs. Brief explanation of the drawing

[0040] Figure 1 is an experimental design regarding an atopic dermatitis model. Figure 2 shows changes in mouse skin after treatment with a monoclonal antibody. Figure 3 is a photograph showing the H&E staining results. Figure 4 is a graph showing the change in thickness of the epidermal and dermal layers after treatment with a monoclonal antibody. Figure 5 is a photograph showing the result of toluidine blue staining. Figure 6 is a photograph showing the results of Mason trichrome staining. Figure 7 is a graph showing the change in collagen amount after treatment with a monoclonal antibody. Figure 8 shows an experimental design regarding an asthma model. Figure 9 is a graph showing the results of measuring airway resistance. Figure 10 is a graph showing the results of observing the number of inflammatory cells. Figure 11 is a photograph showing the H&E staining results. Figure 12 is a graph showing the results regarding the degree of inflammation. Figure 13 is a photograph showing the results of PAS staining. Figure 14 is a graph showing the results of PAS staining. Figure 15 is a photograph showing the results of the degree of fibrosis according to Masson's Trichrome staining. Figure 16 is a graph showing the results of the degree of fibrosis according to Masson's Trichrome staining. Specific details for implementing the invention

[0041] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0043] Furthermore, the terms used in this specification are used to appropriately describe preferred embodiments of the present invention, and these may vary depending on the intent of the user or operator, or the customs of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0045] One embodiment of the present invention provides a monoclonal antibody that specifically binds to Nectin-4.

[0047] The term "specifically binds" as used in the present invention has the same meaning as commonly known to those skilled in the art, and refers to an immunological reaction in which an antigen and an antibody specifically interact.

[0049] Preferably, the monoclonal antibody is a monoclonal antibody comprising a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 1; a heavy chain CDR2 described as SEQ ID NO. 2; and a heavy chain CDR3 described as SEQ ID NO. 3, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 4; a light chain CDR2 described as SEQ ID NO. 5; and a light chain CDR3 described as SEQ ID NO. 6.

[0051] Preferably, the monoclonal antibody is a monoclonal antibody comprising the heavy chain variable region amino acid sequence described as SEQ ID NO. 7 and the light chain variable region amino acid sequence described as SEQ ID NO. 8.

[0053] Additionally, preferably, the monoclonal antibody is a monoclonal antibody comprising a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 9; a heavy chain CDR2 described as SEQ ID NO. 10; and a heavy chain CDR3 described as SEQ ID NO. 11, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 12; a light chain CDR2 described as SEQ ID NO. 13; and a light chain CDR3 described as SEQ ID NO. 14.

[0055] Preferably, the monoclonal antibody is a monoclonal antibody comprising the heavy chain variable region amino acid sequence described in SEQ ID NO. 15 and the light chain variable region amino acid sequence described in SEQ ID NO. 16.

[0057] The term "antibody" as used in the present invention refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes forms produced by genetic engineering such as chimeric antibodies, humanized antibodies, human antibodies, and heterojunction antibodies (e.g., bispecific antibodies). Furthermore, the antibody includes not only the whole antibody form but also functional fragments of the antibody molecule.

[0059] Generally, antibodies have a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. In the present invention, the antibody may include a functional fragment of an antibody molecule having an antigen-binding function.

[0061] The term "heavy chain" as used in the present invention refers to a variable region domain V comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. H and 3 invariant domains C H1 ,C H2and C H3 It is interpreted to include both the full-length heavy chain containing the hinge and its fragments. Additionally, the term "light chain" refers to a variable region domain V containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. L and invariant domain C L It is interpreted to mean that it includes all of the battlefield light chains and fragments thereof.

[0063] The term "variable region" or "variable domain" used in the present invention refers to a part of an antibody molecule that exhibits many sequence variations while performing the function of specifically binding to an antigen, and the variable region contains complementary determining regions CDR1, CDR2, and CDR3. Between the CDRs, there exists a framework region (FR) that serves to support the CDR loop. The "complementary determining region" is a loop-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

[0065] The antibodies of the present invention can be prepared by general methods known in the art, for example, by recombinant methods, and preferably, the method for preparing monoclonal antibodies can be carried out by using phage display technology, but is not limited thereto. An antibody library using phage display technology is a method of obtaining antibody genes directly from B lymphocytes without producing hybridomas and expressing antibodies on the surface of phages. Using phage display technology can overcome many existing difficulties associated with generating monoclonal antibodies through B-cell immortalization. Generally, phage display technology comprises: 1) a step of inserting an oligonucleotide of a random sequence into a gene region corresponding to the N-terminus of the phage's coat protein pIII (or pIV); 2) a step of expressing a fusion protein of a portion of the natural coat protein and a polypeptide encoded by the oligonucleotide of the random sequence; and 3) a step of treating with a receptor substance capable of binding to the polypeptide encoded by the oligonucleotide. 4) a step of eluting peptide-phage particles bound to a receptor using a low pH or a binding competitive molecule; 5) a step of amplifying the eluted phage within a host cell by panning; 6) a step of repeating the above method to obtain a desired amount; and 7) a step of determining the sequence of an active peptide from the DNA sequences of phage clones selected by panning.

[0067] In another aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, an expression vector encoding the nucleic acid molecule, and a transformant comprising the expression vector.

[0069] The nucleic acid molecule encoding the monoclonal antibody may be a DNA sequence of either SEQ ID NO. 17 or SEQ ID NO. 18.

[0071] The expression vector comprising the nucleic acid molecule encoding the monoclonal antibody provided in the present invention is not particularly limited thereto, but may be an expression vector capable of replicating and / or expressing said nucleic acid molecule in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., E. coli, etc.), and preferably may be an expression vector comprising at least one selection marker that is operably linked to a suitable promoter so that said nucleic acid molecule can be expressed in a host cell. Examples include a form in which said nucleic acid molecule is introduced into a plasmid, a cosmid, a virus, etc.

[0073] An expression vector comprising a nucleic acid molecule encoding the monoclonal antibody may be an expression vector comprising a nucleic acid molecule encoding the heavy chain or light chain of the monoclonal antibody, respectively, or an expression vector comprising both nucleic acid molecules encoding the heavy chain and the light chain.

[0075] The transformant to which the expression vector provided in the present invention has been introduced is not particularly limited thereto, but may be a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella typhimurium; a yeast cell; a fungal cell such as Pichia pastoris; an insect cell such as Drozophylla or Spodoptera Sf9 cell; an animal cell such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cell, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells); or a plant cell.

[0077] The term "introduction" as used in the present invention refers to a method of delivering an expression vector containing a nucleic acid molecule encoding the monoclonal antibody to a host cell. Such introduction may be carried out by various methods known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Furthermore, introduction refers to delivering a target into a cell using viral particles by means of infection. In addition, the expression vector may be introduced into a host cell by means of gene bombardment, etc. In the present invention, introduction may be used interchangeably with transformation.

[0079] The present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising the monoclonal antibody.

[0081] The present invention provides a pharmaceutical composition for the prevention or treatment of asthma comprising the monoclonal antibody.

[0083] As used in the present invention, the term "prevention" refers to any act of suppressing or delaying the onset of cancer by administering the above-mentioned composition, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of cancer by administering the composition.

[0085] A pharmaceutical composition comprising the antibody of the present invention may be formulated by further including a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a carrier or diluent that does not significantly irritate the organism and does not impair the biological activity and properties of the administered compound. The pharmaceutically acceptable carrier may be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, by additionally adding diluents, dispersants, surfactants, binders, and lubricants, the composition may be formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, or tablets.

[0087] In addition, the pharmaceutical composition of the present invention may further include fillers, excipients, disintegrants, binders, and lubricants. Furthermore, the pharmaceutical composition of the present invention may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to mammals. The formulation may be in the form of powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable drugs, or sterile powders, but is not limited thereto.

[0089] The pharmaceutical composition of the present invention may be administered to an individual or patient through an appropriate method, and the route of administration of the composition of the present invention may be administered via various oral or parenteral routes as long as it can reach the target tissue. The therapeutic method of the present invention comprises administering the monoclonal antibody at a pharmaceutically effective amount. Preferably, the dosage of the pharmaceutical composition may be administered at a dose of 1.5 mg to 10 mg per kg and may be administered in a single dose or in multiple doses, but is not limited thereto. For the purposes of the present invention, it is desirable to apply a specific therapeutically effective amount for a specific patient differently depending on various factors and similar factors well known in the pharmaceutical field, including the type and degree of response to be achieved, the specific composition including whether other preparations are used in some cases, the patient's age, weight, general health condition, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and drugs used together or concurrently with the specific composition.

[0091] In another aspect, the present invention provides a method for treating an atopic skin disease using the monoclonal antibody. The monoclonal antibody may be a method for treating atopic dermatitis comprising the step of administering a pharmaceutical composition further comprising a pharmaceutically acceptable carrier to an individual who has developed or is suspected of having developed an atopic skin disease.

[0093] In another aspect, the present invention provides a method for treating asthma using the monoclonal antibody. The monoclonal antibody may be a method for treating asthma comprising the step of administering a pharmaceutical composition further comprising a pharmaceutically acceptable carrier to an individual who has developed or is suspected of having developed asthma.

[0095] The present invention will be explained in more detail below through examples. These examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0097] Example. Preparation of Nectin-4 Monoclonal Antibodies N1 and N2

[0098] Recombinant human Nectin-4 solution was added to an immunotube and the protein was adsorbed onto the tube surface overnight at 4°C; then, a 1% BSA (Bovine serum albumin) solution was added to the tube to protect the surface where Nectin-4 was not adsorbed. After emptying the tube, 10 dispersed in the 1% BSA solution 15 A CFU antibody phage library was added and bound to the antigen. Non-specifically bound phages were washed five times with PBS-T (Phosphate buffered saline-0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were recovered using 100 mM triethylamine solution. The recovered phages were neutralized with 1 M Tris buffer (pH 7.4), E. coli were infected at 37°C for 1 hour, and the infected E. coli were plated onto LB (Luria-Bertani) agar medium containing ampicillin and incubated overnight at 37°C. The next day, cultured E. coli was suspended in 4 ml of SB (superbroth)-ampicillin culture medium, and 15% glycerol was added. A portion was stored at -80°C, and 50 µl of the remainder was added to 20 ml of SB-ampicillin culture medium with 2% glucose solution and cultured at 37°C. When the absorbance of the culture medium reached 0.6 at 600 nm, the culture medium was centrifuged to remove the medium, which was then resuspended in 20 ml of SB-ampicillin culture medium. VCSM13 helper phage was added, and the mixture was cultured at 37°C while stirring slowly. The next day, the culture medium was centrifuged, and only the medium was taken. 4% polyethylene glycol 8000 (PEG8000) and 3% sodium chloride (NaCl) were added, and the mixture was precipitated at 4°C for 30 minutes before centrifugation. The supernatant was removed, and the precipitated phages were suspended in 1 ml of PBS. Using this as a library, the above panning process was repeated to amplify / concentrate antigen-specific clones.

[0100] Panning was performed up to three rounds to select antibodies binding to human Nectin-4 protein. Subsequently, single colonies were obtained by plating and incubating them on LB-ampicillin agar medium containing antibody genes. These colonies were then inoculated into 100 µl of SB-ampicillin culture medium and cultured, after which scFv-type proteins were expressed in the periplasm of E. coli by induction with IPTG. E. coli was suspended in a TES solution (Tris, EDTA, sucrose), incubated at 4°C for 1 hour, and then centrifuged to extract the periplasm. This periplasm was then used to confirm the binding of the recombinant human Nectin-4 antigen to scFv using the ELISA technique. The degree of binding reaction was assessed on plates coated with the human Nectin-4 protein epitope as the antigen and on a control plate (Background) without the antigen coating. Subsequently, clones were selected by deriving the final OD value (Delta) from the difference between the obtained OD values. The antigen-specific antibody clones identified therefrom were analyzed using DNA sequencing.

[0102] Through the above method, Nectin-4 monoclonal antibodies N1 (Example 1) and N2 (Example 2), capable of specifically binding to Nectin-4, were prepared, respectively, and each included a monoclonal antibody N1 comprising a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 1; a heavy chain CDR2 described as SEQ ID NO. 2; and a heavy chain CDR3 described as SEQ ID NO. 3, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 4; a light chain CDR2 described as SEQ ID NO. 5; and a light chain CDR3 described as SEQ ID NO. 6; a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 9; a heavy chain CDR2 described as SEQ ID NO. 10; and a heavy chain CDR3 described as SEQ ID NO. 11, and a light chain CDR1 described as SEQ ID NO. 12; and a light chain CDR2 described as SEQ ID NO. 13; and identified a monoclonal antibody N2 containing a light chain variable region including the light chain CDR3 described in SEQ ID NO. 14.

[0104] antibodies Sequence number designation order N1 Sequence No. 1 HCDR1 GFTFEDYA Sequence No. 2 HCDR2 VTWNSGII Sequence No. 3 HCDR3 ARDRNDFWSGYWYYYGMDV Sequence No. 4 LCDR1 SSTFNADR Sequence No. 5 LCDR2 GTN Sequence number 6 LCDR3 SSWDEMFKTV Sequence number 7 HCDR QITLKESGGGLVQPGRSLRLSCAASGFTFEDYAMHWVRQAPGKGLEWVSGVTWNSGIIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRNDFWSGYWYYYGMDVWGQGTTVTVSSAPTKAPDVTS Sequence number 8 LCDR VVTQPPSVSGSLGQKVIISCTGNSSTFNADRVGWYQQFPGMAPKTIIYGTNYRPTGVPDRFFASKSGTSATLTISNLQPEDEADYYCSSWDEMFKTVFGGGTQLTVLG N2 Sequence number 9 HCDR1 GYTFTSYG Sequence number 10 HCDR2 ISAYNGNT Sequence number 11 HCDR3 ARDWGIAAAGLDY Sequence No. 12 LCDR1 SSNIGDNF Sequence No. 13 LCDR2 RNN Sequence No. 14 LCDR3 AAWDDSISGPV Sequence number 15 HCDR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDWGIAAAGLDYWGQGTLVTVSSAPTKAPDVTS Sequence number 16 LCDR VLTQPPSASGTPGQRVSISCSGSSSSNIGDNFVYWYRQLPGTAPKVFIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSISGPVFGGGTKVTVLG

[0106] Meanwhile, the sequences of the nucleic acid molecules encoding the above N1 and N2 monoclonal antibodies are as shown in Table 2 below.

[0108] antibodies Sequence number order N1 Sequence number 17 GAGCTCGTGGTGACGCAGCCGCCCTCAGTGTCTGGGTCCCTGGGCCAGAAGGTCATCATCTCCTGCACTGGAAACAGTTCCACCTTCAATGCTGATCGTGTGGGTTGGTATCAACAGTTTCCCGGGATGGCCCCTAAGACCATCATTTACGGTACTAATTACAGACCTACAGGGGTCCCCGATCGATTCTTTGCCTCCAAGTCCGGCACTTCAGCCACCCTGACCATCTCTAATCTCCAACCTGAGGACGAGGCTGACTATTACTGCTCCTCTTGGGACGAGATGTTCAAGACTGTGTTCGGCGGAGGCACCCAGCTGACCGTCCTCGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGATCACCTTGAAGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGAAGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTGTTACTTGGAATAGTGGTATCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGCAACGATTTTTGGAGTGGTTATTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCACCCACCAAGGCTCCGGATGTGACTAGT N2 서열번호 18 GAGCTCGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCTCCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGATAATTTTGTGTACTGGTACCGGCAGCTCCCAGGGACGGCCCCCAAAGTCTTCATTTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGG CTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCGGAAGATGAGGCAGATTATTACTGTGCAGCATGGGATGACAGCATAAGTGGTCCGGTGTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGC AGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTACGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAG GGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTGGGGTATAGCAGCAGCTGGCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCACCCACCAAGGCTCCGGATGTGACTAGTAGT

[0109] Test Example 1. Atopic Dermatitis Experiment Design

[0110] To confirm the effect of monoclonal antibodies on atopic dermatitis, an animal model of atopic dermatitis was prepared.

[0111] Specifically, 1% dinitrochlorobenzene (DNCB) was administered on day 1 after depilating the backs of 6-week-old C57BL / 6 female mice. Subsequently, an atopic dermatitis mouse model was prepared by administering 0.5% DNCB 2 to 3 times a week for 3 weeks. From day 31 to day 33, Nectin-4 monoclonal antibodies N1 and N2 were administered at different concentrations. The day after administration, collagen was extracted from the skin tissue and the amount of collagen was measured. A portion of the skin tissue was fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, and fractionated into sections (4 µm). Hematoxylin-eosin (H&E), toluidine blue, and Mason's Trichrome staining were performed, respectively.

[0113] Test Example 2. Confirmation of mouse skin lesions

[0114] Compared to the normal control, increased edema, boils, dryness, and oozing were observed in the skin of the atopy group. On the other hand, when treated with the monoclonal antibodies N1 and N2 of Examples 1 and 2, it was found that the symptoms were significantly improved compared to the atopy group, as shown in Figure 2.

[0116] Test Example 3. Confirmation of Histological Changes

[0117] 1) Hematoxylin-Eosin (H&E) staining

[0118] H&E staining was performed to observe changes in skin tissue, and the results are shown in Figures 3 and 4. As shown in Figures 3 and 4, it can be confirmed that the thickness of the epidermal and dermal layers increased significantly in the atopic group compared to the control group. In contrast, when treated with monoclonal antibodies N1 and N2, it can be seen that the thickness of the dermal layer decreased by 40–60% compared to the atopic group.

[0120] 2) Toluidine staining

[0121] Toluidine blue staining was performed to observe the infiltration of mast cells in skin tissue, and the results are shown in Figure 5. It can be seen that the number of mast cells in the atopic group increased significantly compared to the control group. On the other hand, when the monoclonal antibodies N1 and N2 of the present invention were injected at 10 mg / kg each, the number of mast cells decreased, confirming that it is effective in improving the symptoms of atopic dermatitis.

[0123] 3) Mason's Trichrome Staining

[0124] The degree of fibrosis in each tissue was determined by staining with Masson's Trichrome, which stains fibrous tissue blue.

[0126] As shown in Figure 6, it was confirmed that tissue fibrosis in the dermis and subcutaneous fat was significantly increased in the atopic group, and it was confirmed that when the monoclonal antibodies N1 and N2 of the present invention were injected, the thickness of the dermal layer decreased and the degree of fibrosis decreased.

[0128] Test Example 4. Changes in collagen within skin tissue

[0129] A Sircol Collagen Assay was performed to measure the amount of collagen contained in the skin tissues of all groups.

[0131] As shown in Figure 7, the amount of collagen in the control group was 360 μg / ml, and the amount of collagen in the atopic group was 450 μg / ml, showing an increase of about 20% compared to the control group. This change in collagen is known to be due to the phenomenon of increased activity caused by stimulation of fibroblasts resulting from skin barrier damage.

[0133] However, when N1 and N2 antibodies were treated respectively, it was confirmed that the levels of these collagens decreased by as little as 10% to as much as 20% compared to the atopic group. Through this, it can be seen that the monoclonal antibody of the present invention can restore skin barrier damage caused by atopic dermatitis.

[0135] Test Example 5. Asthma Experiment Design

[0136] To confirm the effect of monoclonal antibodies on asthma, an asthma animal model was prepared (Fig. 8).

[0138] Specifically, 100 μl of D-PBS containing 50 μg of Grade V chicken egg ovalbumin OVA (Sigma-Aldrich, St Louis, MO) emulsified in 10 mg aluminum hydroxide was injected intraperitoneally into female 6-week-old BALB / c mice on days 0 and 14.

[0140] From the 18th to the 23rd, Nectin-4 antibodies N1 and N2 were administered intratracheally to anesthetized mice at a dose of 10 mg / kg each, while positive control mice were administered saline (PBS). Three hours later, 5% Grade III OVA (Sigma-Aldrich) was administered aerosolically. Control mice were administered saline (PBS).

[0142] On the 24th, airway resistance was evaluated after administering 0, 6.25, 12.5, 25, and 50 mg / ml of methacholine (Sigma-Aldrich). Bronchoalveolar lavage fluid (BALF) was collected, the supernatant was stored, and the precipitate was suspended in 1.05X PBS for cell counting and cytospin slides were prepared for cell differential counting (500 cells / mouse, Diff-Quick staining). A portion of the lung was fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, and fractionated into sections (4 μm), which were stained with H&E, PAS, and Trichrome.

[0144] Test Example 6. Observation of airway resistance

[0145] Compared to the control group (Normal control), the asthma group (OVA) and the asthma-positive control group (OVA+PBS) showed increased airway resistance, but when treated with the monoclonal antibody N1 or N2 of the present invention, it was found that the airway resistance decreased compared to the asthma group (OVA) and the asthma-positive control group (OVA+PBS) (Fig. 9).

[0147] Test Example 7. Observation of inflammatory cell count

[0148] Compared to the control group (Normal control), the number of inflammatory cells (Total, macrophages, eosinophils, neutrophils, lymphocytes) increased in the asthma group (OVA) and asthma-positive control group (OVA+PBS), but it can be seen that when treated with the monoclonal antibody N1 or N2 of the present invention, the number of inflammatory cells decreased compared to the asthma group (OVA) and asthma-positive control group (OVA+PBS) (Fig. 10).

[0150] Test Example 8. Observation of Histological Changes

[0151] As a result of performing H&E (Figs. 11 and 12), PAS (Figs. 13 and 14), and Trichrome staining (Figs. 15 and 16) on mouse lung tissue, compared to the control group (Normal control), the asthma group (OVA) and the asthma positive control group (OVA+PBS) showed increased levels of inflammation, the number of goblet cells, and fibrosis, respectively; however, when treated with the monoclonal antibody N1 or N2 of the present invention, it was confirmed that the levels of inflammation, the number of goblet cells, and the degree of fibrosis, respectively, tended to decrease compared to the asthma group (OVA) and the asthma positive control group (OVA+PBS).

[0153] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0155] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A monoclonal antibody that specifically binds to Nectin-4, wherein the monoclonal antibody comprises a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 1; a heavy chain CDR2 described as SEQ ID NO. 2; and a heavy chain CDR3 described as SEQ ID NO. 3, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 4; a light chain CDR2 described as SEQ ID NO. 5; and a light chain CDR3 described as SEQ ID NO. 6, or a heavy chain variable region including a heavy chain CDR1 described as SEQ ID NO. 9; a heavy chain CDR2 described as SEQ ID NO. 10; and a heavy chain CDR3 described as SEQ ID NO. 11, and a light chain variable region including a light chain CDR1 described as SEQ ID NO. 12; a light chain CDR2 described as SEQ ID NO. 13; and a light chain CDR3 described as SEQ ID NO.

14. Claim 2 delete Claim 3 In claim 1, the monoclonal antibody comprises a heavy chain variable region amino acid sequence described as SEQ ID NO. 7 and a light chain variable region amino acid sequence described as SEQ ID NO.

8. Claim 4 delete Claim 5 In claim 1, the monoclonal antibody comprises a heavy chain variable region amino acid sequence described as SEQ ID NO. 15 and a light chain variable region amino acid sequence described as SEQ ID NO.

16. Claim 6 A nucleic acid molecule encoding the monoclonal antibody of claim 1. Claim 7 An expression vector comprising the nucleic acid molecule of claim 6. Claim 8 delete Claim 9 A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising the monoclonal antibody of claim 1. Claim 10 A pharmaceutical composition for the prevention or treatment of asthma comprising the monoclonal antibody of claim 1.

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