Human monoclonal antibody targeting YKL-40

Abstract

The present invention relates to a monoclonal antibody that specifically binds to YKL-40. When the expression of the above-mentioned YKL-40 is high, it is related to the proliferation and metastasis of cancer cells. The monoclonal antibody against YKL-40 has the effect of inhibiting the proliferation and metastasis of cancer cells. Therefore, the monoclonal antibody of the present invention can be effectively used for diagnosing, preventing or treating cancer.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody specifically binding to YKL-40, a nucleic acid molecule encoding the same, a pharmaceutical composition for preventing or treating cancer comprising the monoclonal antibody, and a composition for diagnosing cancer comprising the monoclonal antibody. Background Art

[0002] Antibodies are immune proteins that bind to specific antigens. In most mammals, including humans and mice, antibodies are formed by pairing heavy and light chain polypeptides. Each chain is formed by two regions called variable regions (Fv) and constant regions (Fc). The light and heavy chain variable regions contain the antigen binding determinants of the molecule and participate in binding to the target antigen. The constant region defines the group (or reference sample type) of antibodies (e.g., human immunoglobulin G (IgG)), confers a series of important functional characteristics called effector functions, and participates in binding to most Fc receptors and Fc ligands. For example, antibodies have some important characteristics such as target specificity, the ability to mediate immune effector mechanisms, and a long serum half-life, and are therefore used as powerful therapeutic agents.

[0003] Phage display technology was first developed by the Medical Research Council in the UK in 1990. It is a technology that prepares a human antibody library and expresses it on the surface of phage in the form of antibody fragments (Fab, ScFv) to screen antibody clones for specific antigens. It is proposed that all types of human recombinant monoclonal antibodies that react specifically with antigens can be screened from the pot antibody library system. Therefore, when using phage display antibody technology, a variety of antibody fragments (Fab or ScFv form) that can be used for in vivo diagnosis or treatment can be obtained.

[0004] The relevant prior art document is U.S. Patent No. 7,670,599. Summary of the invention

[0005] Technical issues

[0006] An object of the present invention is to provide a monoclonal antibody that specifically binds to YKL-40.

[0007] Another object of the present invention is to provide a nucleic acid molecule encoding the above monoclonal antibody.

[0008] Another object of the present invention is to provide a recombinant expression vector comprising the above nucleic acid molecule.

[0009] Another object of the present invention is to provide a transformant transformed by the above recombinant expression vector.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the above-mentioned monoclonal antibody as an active ingredient.

[0011] Another object of the present invention is to provide a composition for diagnosing cancer, comprising the above-mentioned monoclonal antibody as an active ingredient.

[0012] Technical Solution

[0013] In order to achieve the above object, the present invention provides a monoclonal antibody that specifically binds to YKL-40.

[0014] In one embodiment of the present invention, the above-mentioned monoclonal antibody may comprise: (a) a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence of sequence 5, a CDR2 consisting of an amino acid sequence of sequence 6, and a CDR3 consisting of an amino acid sequence of sequence 7; and (b) a light chain variable region comprising a CDR1 consisting of an amino acid sequence of sequence 11, a CDR2 consisting of an amino acid sequence of sequence 12, and a CDR3 consisting of an amino acid sequence of sequence 13, or may comprise: (a) a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence of sequence 8, a CDR2 consisting of an amino acid sequence of sequence 9, and a CDR3 consisting of an amino acid sequence of sequence 10; and (b) a light chain variable region comprising a CDR1 consisting of an amino acid sequence of sequence 14, a CDR2 consisting of an amino acid sequence of sequence 15, and a CDR3 consisting of an amino acid sequence of sequence 16.

[0015] In one embodiment of the present invention, the above-mentioned monoclonal antibody may comprise: (a) a heavy chain variable region consisting of the amino acid sequence of sequence 1; and (b) a light chain variable region consisting of the amino acid sequence of sequence 3, or may comprise: (a) a heavy chain variable region consisting of the amino acid sequence of sequence 2; and (b) a light chain variable region consisting of the amino acid sequence of sequence 4.

[0016] In one embodiment of the present invention, the monoclonal antibody may be a humanized antibody.

[0017] Furthermore, the present invention provides a nucleic acid molecule encoding the above monoclonal antibody.

[0018] In one embodiment of the present invention, the above-mentioned nucleic acid molecule may include: (a) a heavy chain variable region, comprising a CDR1 composed of a base sequence of sequence 21, a CDR2 composed of a base sequence of sequence 22, and a CDR3 composed of a base sequence of sequence 23; and (b) a light chain variable region, comprising a CDR1 composed of a base sequence of sequence 27, a CDR2 composed of a base sequence of sequence 28, and a CDR3 composed of a base sequence of sequence 29, or may include: (a) a heavy chain variable region, comprising a CDR1 composed of a base sequence of sequence 24, a CDR2 composed of a base sequence of sequence 25, and a CDR3 composed of a base sequence of sequence 26; and (b) a light chain variable region, comprising a CDR1 composed of a base sequence of sequence 30, a CDR2 composed of a base sequence of sequence 31, and a CDR3 composed of a base sequence of sequence 32.

[0019] In one embodiment of the present invention, the above-mentioned nucleic acid molecule may include: (a) a heavy chain variable region, consisting of a base sequence of sequence 17; and (b) a light chain variable region, consisting of a base sequence of sequence 19, or may include: (a) a heavy chain variable region, consisting of a base sequence of sequence 18; and (b) a light chain variable region, consisting of a base sequence of sequence 20.

[0020] Furthermore, the present invention provides a recombinant expression vector comprising the above nucleic acid molecule.

[0021] Furthermore, the present invention provides a transformant transformed with the above-mentioned recombinant expression vector.

[0022] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above monoclonal antibody as an active ingredient.

[0023] In one embodiment of the present invention, the above-mentioned cancer can be one selected from the group consisting of breast cancer, colorectal cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, head and neck cancer, colon cancer, ovarian cancer, rectal cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer.

[0024] Furthermore, the present invention provides a composition for diagnosing cancer, comprising the above-mentioned monoclonal antibody as an active ingredient.

[0025] In one embodiment of the present invention, the above-mentioned cancer can be one selected from the group consisting of breast cancer, colorectal cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, head and neck cancer, colon cancer, ovarian cancer, rectal cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer.

[0026] Effects of the Invention

[0027] YKL-40 of the present invention is related to the proliferation and metastasis of cancer cells. The monoclonal antibody specifically binding to YKL-40 has the effect of inhibiting the proliferation and metastasis of cancer cells. Therefore, the monoclonal antibody of the present invention can be effectively used for diagnosing, preventing or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1a Schematic diagram showing the process of screening antibodies showing significant binding ability to human YKL-40 by phage display, Figure 1b to Figure 1d The panning titration results for Fab-I, Fab-II and scFv are shown respectively.

[0029] Figure 2a and Figure 2b Seven antibodies to human YKL-40 screened by phage display are shown.

[0030] Figure 3a Schematic diagram showing the process of screening antibodies showing significant binding ability to mouse YKL-40 by phage display, Figure 3b to Figure 3d The panning titration results for Fab-I, Fab-II and scFv are shown respectively.

[0031] Figure 4 Six antibodies to mouse YKL-40 screened by phage display are shown.

[0032] Figure 5a to Figure 5c The figures show the results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) performed to confirm the production of immunoglobulin G (IgG) from six clones (H1, H2, H4, H5, H6, and H7) having human YKL-40 antibodies and the results of detecting the production amount of immunoglobulin G.

[0033] Figure 6a and Figure 6b The results of size exclusion chromatography (SEC) analysis of six human YKL-40 antibodies are shown.

[0034] Figure 7 Shown are the results of measuring EC50 values ​​by enzyme-linked immunosorbent assay (ELISA) for 6 human YKL-40 antibodies.

[0035] Figure 8a and Figure 8b The figures show the results of SDS polyacrylamide gel electrophoresis to confirm the production of immunoglobulin G from four clones (M1, M3, M4, and M5) having mouse YKL-40 antibodies and the results of detecting the production amounts of immunoglobulin G.

[0036] Figure 9a and Figure 9b The results of size exclusion chromatography analysis of four mouse YKL-40 antibodies are shown.

[0037] Fig.10 The results of measuring EC50 values ​​by enzyme-linked immunosorbent assay for 4 mouse YKL-40 antibodies are shown.

[0038] Fig.11a and Fig.11b The results were obtained by ELISA to confirm whether human YKL-40 antibody 3A10-F1 (H1) and mouse YKL-40 antibody 4E3-F2 (M3) clones cross-bind to human YKL-40 antigen and mouse YKL-40 antigen.

[0039] Figures 12a to 12c The graph shows the results of testing the antigen affinity of human YKL-40 antibody 3A10-F1 (H1) and mouse YKL-40 antibody 4E3-F2 (M3) for human YKL-40 antigen and mouse YKL-40 antigen.

[0040] Figures 13a to 13d The graph shows the results of measuring the number of migrated cells after treating A549 cell line and H460 cell line with human YKL-40 antibodies H1, H2, H4, H5, H6 and H7 as candidate antibodies.

[0041] Figures 14a to 14d The graph shows the results of measuring the number of migrated cells after treating A549 cell line and H460 cell line with mouse YKL-40 antibodies M1, M3, M4 and M5 as candidate antibodies.

[0042] Fig.15a and Fig.15b The results of measuring the tumor area on the lung surface after treating a lung metastasis animal model with human YKL-40 antibodies H1, H2 and H4 and mouse YKL-40 antibodies M2 and M3 as candidate antibodies are shown (n=3).

[0043] Figures 16a to 16c The figures show the results of detecting the number of tumor nodules in lung tissues after treating lung metastasis animal models with human YKL-40 antibody H1 and mouse YKL-40 antibody M3 (n=8). DETAILED DESCRIPTION

[0044] The term "antibody" in the present invention is a concept that includes immunoglobulins that are immunologically reactive with specific antigens and includes all polyclonal antibodies and monoclonal antibodies. In addition, the above term includes forms produced by genetic engineering such as chimeric antibodies (e.g., humanized mouse antibodies) and hybrid antibodies (e.g., bispecific antibodies).

[0045] The term "monoclonal antibody" in the present invention is a well-known term in the technical field to which the present invention belongs, and refers to an antibody with high specificity for a single antigenic site. Generally, unlike polyclonal antibodies, which contain multiple different antibodies against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies have the advantages of improving the selectivity and specificity of diagnostic and analytical analysis methods using antigen-antibody binding, and, because they are synthesized by hybridoma culture, they also have the advantage of not being contaminated by other immunoglobulins.

[0046] Typically, an immunoglobulin has a heavy chain and a light chain, each of which contains a constant region and a variable region (the above-mentioned parts are also referred to as "domains"). The light chain variable region and the heavy chain variable region contain three variable regions called "complementarity determining regions" (hereinafter referred to as "CDRs") and four "framework regions". The above-mentioned CDRs mainly play a role in binding to the epitope of the antigen. Each chain is typically called CDR1, CDR2, and CDR3 in order from the N-terminus, and is identified by the chain in which the specific CDR is located.

[0047] The term "heavy chain" as used herein refers to all full-length heavy chains and fragments thereof, including a variable region domain V having an amino acid sequence sufficient to confer specificity to an antigen. H and 3 constant region domains C H1 , C H2 and C H3 The term "light chain" as used herein refers to all full-length light chains and fragments thereof, comprising a variable region domain V having an amino acid sequence sufficient to confer specificity to an antigen. L and constant region domain C L .

[0048] The term "variable" in the present invention means that the sequence of a specific region between antibodies is very different and shows the binding specificity of each specific antibody to a specific antigen. The variability of the antibody is not evenly distributed throughout the variable domain of the antibody, but is concentrated in the CDR. The heavy chain and light chain of a monoclonal antibody each have three CDRs, and these regions form an antigen-antibody complex by recognizing the surface antigen of Propionibacterium acnes. This CDR has a characteristic sequence in each monoclonal antibody, and a monoclonal antibody can interact with some or all of these six CDRs in order to recognize a specific epitope.

[0049] The term "phage display technology" in the present invention refers to a technology for screening antibodies that show binding ability to antigens by screening only phages that show significant binding ability to target antigens from a phage library. In the above technology, "panning" refers to the process of screening only phages that express peptides on the surface that have the property of binding to target molecules (antibodies, enzymes, cell surface receptors, etc.) from a phage library that expresses (displays) peptides on the coat of the phage. Antibodies that show significant binding ability to the target antigen can be screened by repeating this process 3 to 10 times, and the screened antibodies can be prepared as humanized monoclonal antibodies.

[0050] The monoclonal antibodies of the present invention may include variants of the amino acid sequences described in the attached sequence table within the scope of specific binding to YKL-40. For example, the amino acid sequence of the monoclonal antibody may be changed in order to improve the binding affinity and / or other biological properties of the monoclonal antibody. Such variations may include, for example, deletion, insertion and / or substitution of residues in the amino acid sequence of the monoclonal antibody. Such amino acid variations may be achieved based on relative similarity with amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutes shows that arginine, lysine and histidine are all residues with positive charges; alanine, glycine and serine have similar sizes; phenylalanine, tryptophan and tyrosine have similar morphological shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine can be regarded as biological functional equivalents. In terms of introducing variations, the hydrophobicity index (hydropathy index) of amino acids can be considered. Each amino acid is assigned the following hydrophobicity index based on hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobicity amino acid index is very important in assigning interactive biological functions to proteins. It is a well-known fact that biological activity can only be retained by substitution with amino acids having similar hydrophobicity indices. When introducing mutations with reference to the hydrophobicity index, it is preferred to substitute between amino acids with hydrophobicity indices that differ by ±2, more preferably, between amino acids with hydrophobicity indices that differ by ±1, and even more preferably, between amino acids with hydrophobicity indices that differ by ±0.5.

[0051] On the other hand, it is well known that substitutions between amino acids having similar hydrophilicity values ​​result in proteins having equivalent biological activities. As disclosed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.01); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In the case of introducing variation with reference to the hydrophilicity index, it is preferred to substitute between amino acids with hydrophilicity values ​​within ±2, more preferably, between amino acids with hydrophobicity values ​​within ±1, and even more preferably, between amino acids with hydrophobicity values ​​within ±0.5. Amino acid exchange in protein does not substantially change the activity of the molecule, which is well known in the art to which the present invention belongs (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges between amino acid residues are exchanges between Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. If the above-mentioned variations with biologically equivalent activity are taken into account, the monoclonal antibodies of the present invention or nucleic acid molecules encoding the same should be interpreted as also including sequences that show substantial identity with the sequences recorded in the sequence table. The above-mentioned substantial identity refers to sequences that show at least 61% homology, more preferably, sequences that show at least 70% homology, more preferably, sequences that show at least 80% homology, and most preferably, sequences that show at least 90% homology when the aligned sequences are analyzed using algorithms commonly used in the technical field to which the present invention belongs, after aligning any other sequence with the sequence of the present invention in a manner of maximum correspondence.

[0052] The term "nucleic acid molecule" in the present invention has a comprehensive meaning including deoxyribonucleic acid (DNA (genomic deoxyribonucleic acid (gDNA) and complementary deoxyribonucleic acid (cDNA)) and ribonucleic acid (RNA) molecules. The nucleotides as the basic component units in the nucleic acid molecules include not only natural nucleotides, but also analogs (analogues) with modified sugar or base sites (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)). The base sequence of the nucleic acid molecules encoding the heavy chain variable region and light chain variable region of the monoclonal antibody of the present invention can be modified. The above-mentioned modification includes the addition, deletion or non-conservative substitution or conservative substitution of nucleotides.

[0053] In the present invention, the prepared vector is constructed for expressing a desired gene in a host cell. Usually, an operably linked promoter and terminator are located upstream and downstream of the above-mentioned vector, respectively.

[0054] The term "promoter" in the present invention refers to a deoxyribonucleic acid sequence that regulates the expression of a coding sequence or a functional ribonucleic acid. In the recombinant vector of the present invention, the target base sequence is operably linked to the above promoter.

[0055] The term "operatively linked" in the present invention refers to the functional combination between a nucleic acid expression regulatory sequence (such as a promoter sequence, a signal sequence or an array of transcriptional regulatory factor binding sites) and other nucleic acid sequences, wherein the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequences in this way.

[0056] The vector system of the present invention can be constructed by various methods known in the art to which the present invention belongs, and the specific methods are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). The vector of the present invention can be constructed as a typical vector for expression.

[0057] The method for transferring the vector of the present invention into the host cell as a transformant can be carried out by a heat shock method, a CaCl2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), a Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973) and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and an electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like.

[0058] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means that it is not toxic to cells or humans exposed to the above composition. The above carrier may be a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc., which are known in the technical field of the present invention without limitation.

[0059] And, pharmaceutical composition of the present invention can be formulated into the form of oral dosage forms, external preparations, suppositories and sterile injection solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays according to various conventional methods to use. And then, it can be used in the form of skin external preparations in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels or gel forms. The carrier, excipient and diluent included in the composition of the present invention can be lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of dosage form, diluents or excipients such as commonly used fillers, extenders, bonding agents, wetting agents, disintegrants, surfactants can be used to prepare.

[0060] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations need to be prepared by mixing at least one excipient in the above-mentioned nine-section extract, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, internal liquid preparations, emulsions, syrups, etc., in addition to widely used simple diluents such as water and liquid paraffin, they can also contain a variety of excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. As the base of the suppository, witepsol, polyethylene glycol, Tween 61, cocoa butter, laurel butter, glycerin gelatin and the like can be used.

[0061] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention refers to the introduction of a prescribed substance into an individual by an appropriate method, and the administration route of the above-mentioned composition can be administered by any common route that can reach the target tissue. It can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, and intrarectally, but is not limited thereto.

[0062] The term "individual" refers to all animals including humans, such as rats, mice, livestock, etc. Preferably, it can be a mammal including humans.

[0063] The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment without side effects. The level of the effective dose can be easily determined by a person skilled in the art of the present invention according to the patient's gender, age, weight, health status, type of disease, severity of disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route, factors including metabolic ratio, treatment period, drugs used in combination or simultaneously, and other factors well known in the medical field. The above-mentioned recommended dosage can be administered once a day or divided into several doses.

[0064] The present invention will be described in detail below by way of examples. These examples are only used to more specifically describe the present invention, and the scope of the present invention is not limited to these examples.

[0065] Example 1. Phage display library panning

[0066] YKL-40 (Chitinase-3-like protein 1) is a secreted glycoprotein of about 40 kDa encoded by the CHI3L1 gene, and is known to be expressed and secreted in a variety of cells such as macrophages. In addition, according to recent studies, it is inferred that YKL-40 plays an important role in the proliferation, survival and metastasis of cancer cells. In addition, in previous studies, it was confirmed that the expression of YKL-40 is inhibited in 38 of 49 diseases related to cancer, and 8 of them are related to lung cancer. Therefore, the present inventors conducted experiments to discover antibodies that can inhibit the expression of YKL-40.

[0067] The antibodies that showed significant binding ability to human YKL-40 (hYK L-40) were screened by phage display technology as antibodies against YKL-40 ( Figure 1a to Figure 1d ). The binding ability to hYKL-40 was confirmed by enzyme-linked immunosorbent assay of hYKL-40 phage. Briefly, 30 ng of hYKL-40 antigen was fixed in each well, and after blocking the wells with the antigen fixed with MPBS (phosphate buffer solution containing 5% skim milk (5% skim milk in PBS)), hYKL-40 phage and mYKL-40 phage were added for reaction. In order to detect the bound phage, after washing 4 times with PBST (phosphate buffer solution containing 0.05% Tween 20 (0.05% Tween20 in PBS)), Anti-M13-HRP (1:5000) was added for reaction. Finally, after washing four times with PBST, 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to induce color reaction and 2N H2SO4 was added to terminate the reaction, and the absorbance of each well was measured at a wavelength of 450 nm.

[0068] As a result, 7 clones with antibodies against hYKL-40 were screened in the Fab-I library and the Fab-II library, and no clones were formed in the scFv library ( Figure 2a and Figure 2b). The two clones recorded in the Fab-I library are 3A10-F1 (H1) and 3B12-F1 (H2), and the five clones recorded in the KFab-II library are 3A11-F2 (H3), 3B7-F2 (H4), 3D12-F2 (H5), 3E12-F2 (H6) and 3G7-F2 (H7).

[0069] Furthermore, antibodies showing significant binding ability to mouse YKL-40 (mYKL-40) were screened ( Figure 3a to Figure 3d ), the binding ability to mYKL-40 was confirmed by enzyme-linked immunosorbent assay of mYKL-40 phage. Briefly, 30 ng of mYKL-40 antigen was fixed in each well, and the wells fixed with antigen were blocked with MPBS (phosphate buffer solution containing 5% skim milk), and then hYKL-40 phage and mYKL-40 phage were added for reaction. In order to detect the bound phage, PBST (phosphate buffer solution containing 0.05% Tween 20) was used for washing 4 times, and Anti-M13-HRP (1:5000) was added for reaction. Finally, after washing 4 times with PBST, 3,3',5,5'-tetramethylbenzidine substrate was added to induce color reaction and 2N H2SO4 was added to stop the reaction, and the absorbance of each well was detected at a wavelength of 450nm. As a result, 6 antibodies were screened in the Fab-II library, and no antibodies were generated in the Fab-I or scFv libraries ( Figure 4 ). The six clones recorded in the Fab-II library are 4A12-F2 (M1), 4E12-F2 (M2), 4E3-F2 (M3), 4D10-F2 (M4), 4A7-F2 (M5) and 4A1-F2 (M6).

[0070] Example 2. Production of Immunoglobulin G and Analysis of Properties

[0071] The present inventors converted the screened YKL-40 phage into the immunoglobulin G form of human antibodies and conducted experiments to verify it. In short, the screened YKL-40 phage was transfected into the Expi293 cell line and then cultured, and the immunoglobulin G was purified from 300 ml of the cell culture fluid of the cultured Expi293 cell line. The molecular weight and separation purity of the purified immunoglobulin G were confirmed by SDS polyacrylamide gel electrophoresis.

[0072] As a result, immunoglobulin G was produced in 6 clones having hYKL-40 antibody, among which 0.237 mg of immunoglobulin G was finally produced in 3A10-F1 (H1), 1.84 mg of immunoglobulin G was finally produced in 3B12-F1 (H2), 6.5 mg of immunoglobulin G was finally produced in 3B7-F2 (H4), 0.3 mg of immunoglobulin G was finally produced in 3D12-F2 (H5), 0.8 mg of immunoglobulin G was finally produced in 3E12-F2 (H6), and 5.3 mg of immunoglobulin G was finally produced in 3G7-F2 (H7). Figure 5a to Figure 5c ).

[0073] In addition, the present inventors confirmed whether they were single antibodies by size exclusion chromatography (SEC), and conducted experiments to detect antigen affinity by EC50 values ​​of each antibody by enzyme-linked immunosorbent assay. As a result, 3A10-F1 (H1), 3B12-F1 (H2), 3B7-F2 (H4), 3D12-F2 (H5), 3E12-F2 (H6), and 3G7-F2 (H7) all showed single peaks in size exclusion chromatography, thus confirming that they were all single antibodies ( Figure 6a and Figure 6b ). The results of the ELISA assay confirmed that the EC50 value of 3A10-F1(H1) was 582pM (0.582nM), which was the lowest EC50 value ( Figure 7 ).

[0074] The present inventors also conducted the same experiment on the screened mouse YKL-40 phage. As a result, 1.6 mg of immunoglobulin G was finally produced in 4E12-F2 (M2), 3.6 mg of immunoglobulin G was finally produced in 4E3-F2 (M3), 6.3 mg of immunoglobulin G was finally produced in 4D10-F2 (M4), and 0.7 mg of immunoglobulin G was finally produced in 4A7-F2 (M5). Figure 8a and Figure 8b ).

[0075] Furthermore, in the size exclusion chromatography analysis (SEC) results, 4E12-F2 (M2), 4E3-F2 (M3), 4D10-F2 (M4) and 4A7-F2 (M5) all showed single peaks, thus confirming that they were all single antibodies ( Figure 9a and Figure 9b ). In the enzyme-linked immunosorbent assay, it was confirmed that the EC50 value of 4E3-F2(M3) was 67pM (0.067nM), which was the lowest EC50 value ( Fig.10 ).

[0076] Furthermore, in order to confirm whether the 3A10-F1 (H1) and 4E3-F2 (M3) clones showing the lowest EC50 values ​​for hYKL-40 and mYKL-40, respectively, cross-bind to hYKL-40 and mYKL-40, the present inventors performed an ELISA using the Fab proteins of each antibody. The results confirmed that 3A10-F1 (H1) only binds to hYKL-40, and 4E3-F2 (M3) cross-binds to hYKL-40 and mYKL-40 ( Fig.11a and Fig.11b ).

[0077] To reconfirm the EC50 values ​​obtained by ELISA, the antigen affinity tests for 4E3-F2 (M3) and 3A10-F1 (H1) were performed using the Octet instrument. Cross-binding experiments were performed for both antibodies using hYKL-40 and mYKL-40. nd The antigen proteins were fixed to the sensor of Generation 1 and the direct immobilization method was used to bind the antibodies at different concentrations (0 μM to 1 μM). As a result, 6.7×10 -8 M and 5.7×10- 8 M of K D The values ​​were 5.0×10, confirming that both mouse and human antigen proteins bound at similar levels. In the case of 3A10-F1(H1), no binding was observed with mYKL-40, but 5.0×10 -11 M of K D value( Figures 12a to 12c ).

[0078] Example 3. Analysis of the anticancer efficacy of candidate antibodies in vitro

[0079] The present inventors conducted an experiment to confirm whether the candidate antibodies against YKL-40 have the effect of inhibiting cancer cell metastasis when treating lung cancer cell lines. Briefly, the migration of lung cancer cell lines A549 and H460 was quantitatively measured in permeable inserts (8 μm pore trans-well; Corning Inc.). Lung cancer cell lines A549 and H460 were treated with hYKL-40 antibodies 3A10-F1 (H1), 3B12-F1 (H2), 3B7-F2 (H4), 3D12-F2 (H5), 3E12-F2 (H6), and 3G7-F2 (H7) and mYKL-40 antibodies 4E12-F2 (M2), 4E3-F2 (M3), 4D10-F2 (M4), and 4A7-F2 (M5) at a concentration of 1 μg / ml, respectively, and then A549 and H460 cells treated with the candidate antibodies were plated at 2.0×10 per well. 4 The number of cells was placed in the wells and cultured for 17 hours in a humidified incubator at 37°C and 5% CO2. After culture, the cells were fixed with 3.7% formalin solution for 2 minutes and washed twice with 1×PBS. Then, the cells were infiltrated with 100% methanol for 15 minutes and stained with trypan blue for 20 minutes. Cotton swabs were used to remove cells that did not migrate inside the wells, and images taken with an optical microscope (Olympus) at a magnification of ×200 were analyzed using NIH ImageJ software.

[0080] As a result, when the six antibodies against hYKL-40 were treated, the number of migrated cells was confirmed to be reduced in both the A549 cell line and the H460 cell line compared with the control group. In particular, when the 3A10-F1 (H1), 3B12-F1 (H2), and 3B7-F2 (H4) antibodies were treated, the number of migrated cells was confirmed to be significantly reduced ( Figures 13a to 13d ). Similarly, when the four antibodies against mYKL-40 were treated, the number of migrated cells was confirmed to be reduced in both A549 cell line and H460 cell line compared with the control group. In particular, when the 4E12-F2 (M2) and 4E3-F2 (M3) antibodies were treated, the number of migrated cells was confirmed to be significantly reduced ( Figures 14a to 14d ).

[0081] The above results confirmed that the screened antibodies 3A10-F1(H1), 3B12-F1(H2), 3B7-F2(H4), 4E12-F2(M2) and 4E3-F2(M3) have the effect of inhibiting lung cancer cell metastasis by inhibiting YKL-40 protein.

[0082] Example 4. Analysis of the anticancer efficacy of candidate antibodies in animal experiments (in vivo)

[0083] The present inventors conducted an experiment to confirm whether the five candidate antibodies screened from the cell line experiment had the effect of inhibiting cancer cell metastasis when used to treat lung metastasis animal models. Briefly, B16F10 mouse melanoma cells (3.75×10 4 After inducing lung metastasis by inducing lung metastasis, hYKL-40 antibodies 3A10-F1 (H1), 3B12-F1 (H2) and 3B7-F2 (H4) and mYKL-40 antibodies 4E12-F2 (M2) and 4E3-F2 (M3) were administered to mice at 0.5 mg / kg once a week for 3 weeks. 3 weeks after administration of melanoma cells, lungs were removed to measure the tumor area on the lung surface.

[0084] The results confirmed that the area of ​​lung surface tumors was significantly reduced when treated with 3A10-F1 (H1) of the hYKL-40 antibody and 4E3-F2 (M3) of the mYKL-40 antibody compared to the control group ( Fig.15a and Fig.15b ). This result is consistent with the EC50 value measured in the cell line, thus confirming that H1 and M3 antibodies have the effect of inhibiting lung cancer cell metastasis.

[0085] In addition, the present inventors increased the number of experimental animals to verify the YKL-40 antibodies 3A10-F1 (H1) and 4E3-F2 (M3) selected from the above results and conducted an experiment to confirm whether they have the effect of inhibiting cancer cell metastasis. As a result, compared with the control group, the number of tumor nodules on the surface of lung tissue in 3A10-F1 (H1) and 4E3-F2 (M3) decreased, thereby confirming the effect of inhibiting lung cancer cell metastasis. In particular, it was confirmed that the 3A10-F1 (H1) antibody had the most significant effect of inhibiting the number of tumor nodules ( Figures 16a to 16c ).

[0086] The above results confirmed that the 3A10-F1(H1) antibody against YKL-40 has a significant effect in inhibiting lung cancer.

[0087] Example 5. Analysis of antibody sequences

[0088] The present inventors analyzed the sequences of the 3A10-F1(H1) antibody and the 4E3-F2(M3) antibody, which were confirmed to have cancer inhibitory effects by the above results. The amino acid sequences of the heavy chain variable regions of the 3A10-F1(H1) antibody and the 4E3-F2(M3) antibody are shown in Table 1 below, and the amino acid sequences of the light chain variable regions are shown in Table 2. The amino acid sequences of CDR1 to CDR3 in the heavy chain variable region are shown in Table 3 below, and the amino acid sequences of CDR1 to CDR3 in the light chain variable region are shown in Table 4 below.

[0089] In addition, the base sequence of the heavy chain variable region of the nucleic acid molecule encoding the above antibody is shown in the following Table 5, and the base sequence of the light chain variable region is shown in the following Table 6. The base sequence encoding CDR1 to CDR3 in the heavy chain variable region is shown in the following Table 7, and the base sequence encoding CDR1 to CDR3 in the light chain variable region is shown in Table 8.

[0090] Table 1

[0091]

[0092] Table 2

[0093]

[0094]

[0095] Table 3

[0096]

[0097] Table 4

[0098]

[0099] Table 5

[0100]

[0101]

[0102] Table 6

[0103]

[0104] Table 7

[0105]

[0106] Table 8

[0107]

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, characterized in that: Specific binding to YKL-40, The above monoclonal antibodies include: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; as well as (b) a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 11, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, or The above monoclonal antibodies include: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; as well as (b) A light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 15, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:

16.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The above monoclonal antibodies include: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1; and (b) The light chain variable region consists of the amino acid sequence of SEQ ID NO:

3.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The above monoclonal antibodies include: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; and (b) The light chain variable region consists of the amino acid sequence of SEQ ID NO:

4.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The above monoclonal antibodies are humanized antibodies.

5. A nucleic acid molecule, characterized in that Encodes the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule comprises: (a) a heavy chain variable region comprising a CDR1 consisting of the base sequence of SEQ ID NO: 21, a CDR2 consisting of the base sequence of SEQ ID NO: 22, and a CDR3 consisting of the base sequence of SEQ ID NO: 23; as well as (b) A light chain variable region comprising a CDR1 consisting of the base sequence of SEQ ID NO:27, a CDR2 consisting of the base sequence of SEQ ID NO:28, and a CDR3 consisting of the base sequence of SEQ ID NO:

29.

7. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule comprises: (a) a heavy chain variable region comprising a CDR1 consisting of the base sequence of SEQ ID NO: 24, a CDR2 consisting of the base sequence of SEQ ID NO: 25, and a CDR3 consisting of the base sequence of SEQ ID NO: 26; as well as (b) A light chain variable region comprising a CDR1 consisting of the base sequence of SEQ ID NO:30, a CDR2 consisting of the base sequence of SEQ ID NO:31, and a CDR3 consisting of the base sequence of SEQ ID NO:

32.

8. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule comprises: (a) a heavy chain variable region consisting of the base sequence of SEQ ID NO: 17; and (b) The light chain variable region consists of the base sequence of sequence 19.

9. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule comprises: (a) a heavy chain variable region consisting of the base sequence of SEQ ID NO: 18; and (b) The light chain variable region consists of the base sequence of sequence 20.

10. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule of claim 5.

11. A transformant, characterized in that: Transformed by the recombinant expression vector according to claim 10.

12. A pharmaceutical composition for preventing or treating cancer, characterized in that: A monoclonal antibody or antigen-binding fragment thereof according to claim 1, a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof, or a vector comprising a polynucleotide thereof as an active ingredient.

13. A composition for diagnosing cancer, characterized in that A monoclonal antibody or antigen-binding fragment thereof according to claim 1, a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof, or a vector comprising a polynucleotide thereof as an active ingredient.

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