Novel antibodies specific to CTHRC1 and uses thereof

Abstract

The present invention relates to novel antibodies that bind to CTHRC1 and uses thereof.

Description

[Technical field]

[0001] The present invention relates to novel antibodies that bind to CTHRC1 and uses thereof. [Background technology]

[0002] Anticancer drugs currently used in clinical practice are classified into chemotherapy drugs and biotherapeutic drugs. Chemotherapeutic drugs that have been actively developed in the past are drugs that are toxic to cancer cells, but they are toxic not only to cancer cells but also to normal cells, and have the problem of causing resistance, so there are limitations to their development and use. Therefore, biotherapeutic drugs that reduce the activity of cancer cells by restoring or increasing the immune function of the human body have been actively developed as an alternative. Biotherapeutic drugs currently being used or developed include cytokines, recombinant antibodies such as monoclonal antibodies, nucleic acid molecular therapeutic agents, and angiogenesis inhibitors.

[0003] Among the above-mentioned biotherapeutic agents, therapeutic monoclonal antibodies are characterized by high reaction specificity to the target and low side effects. Antibodies exert their therapeutic effects through various mechanisms, and can specifically bind to the antigen to inhibit signal transduction, induce cell death by cross-linking, and activate the immune system in the body to exert their effects. Thus, monoclonal antibodies as anticancer drugs are established as the mainstream of cancer treatment because they can not only specifically track cancer cells and suppress their activity, but also induce an immune response to effectively remove cancer cells. In this regard, monoclonal antibodies such as ramucirumab, rituximab, and trastuzumab have been developed and are used to treat gastric cancer, breast cancer, liver cancer, and the like.

[0004] On the other hand, CTHRC1 (Collagen triple helix repeat containing-1) is known as a secreted protein involved in vascular remodeling, bone formation and developmental morphogenesis.

[0005] Recent studies have reported that CTHRC1 is overexpressed in various cancer cells, such as pancreatic cancer, ovarian cancer, breast cancer, and melanoma, and is involved in the proliferation and metastasis of cancer, suggesting that CTHRC1 plays an important role in aggressive tumors. It is also known that CTHRC1 not only induces the proliferation and metastasis of cancer cells themselves, but also stimulates endothelial cells in the tumor microenvironment to participate in neovascularization (Non-Patent Document 1). cDNA array analysis of human tumors confirmed that CTHRC1 is mainly expressed in human solid tumors, and transcriptome analysis confirmed that there is a difference in expression between breast lobular carcinoma and general pancreatic ductal cells and lobular cells. CTHRC1 was expressed in invasive primary melanomas and metastatic melanomas, but not in benign nevi or noninvasive samples. Silencing of CTHRC1 expression reduced migration of melanoma cell lines in vitro. CTHRC1 was identified in dermatofibrosarcoma protuberans, a locally aggressive neoplasm that frequently metastasizes, but not in dermatosarcomas, a common benign fibrohistiocytic tumor. CTHRC1 expression was significantly higher in breast cancer tissues compared with general tissues and precursor lesions, and was associated with the risk of bone metastasis. However, there is still a need to develop antibodies that have improved binding strength and affinity for the CTHRC1 protein and are highly effective in the diagnosis and treatment of various cancers. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Park et al, Carcinogenesis (2013) 34:694, Lee et al, ExpMolMed (2016) 48:e261 [Non-Patent Document 2] Koeher and Milstein, 1976, Nature, 256:495 [Non-Patent Document 3] METHODS: A Companion to Methods in Enzymology 2:119, 1991 [Non-Patent Document 4] J. Virol. 2001 Jul;75(14):6692-9 [Non-Patent Document 5] Ann. Rev. Immunol. 12:433, 1994 Summary of the Invention [Problem to be solved by the invention]

[0007] In the midst of a continuing need for the development of antibodies that specifically bind to CTHRC1 for the treatment of various cancers, including refractory cancers, the present inventors have developed a new antibody with high affinity and specificity, and confirmed that the antibody exhibits anti-cancer effects against various cancers, thereby completing the present invention. [Means for solving the problem]

[0008] An object of the present invention is to provide an antibody that specifically binds to CTHRC1 (collagen triple helix repeat containing-1) protein.

[0009] Another object of the present invention is to provide a polynucleotide encoding an antibody that specifically binds to the CTHRC1 protein, an expression vector containing the same, and a host cell containing the same.

[0010] Furthermore, the present invention aims to provide a detection composition and kit comprising an antibody that specifically binds to the CTHRC1 protein.

[0011] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody.

[0012] A further object of the present invention is to provide a method for treating cancer using the antibody.

[0013] A further object of the present invention is to provide a composition for diagnosing or predicting the prognosis of cancer, comprising the antibody.

[0014] Furthermore, the present invention aims to provide a method for providing information for diagnosing cancer, which comprises a step of using the antibody to detect CTHRC1 protein in an isolated biological sample from an individual suspected of having cancer through an antigen-antibody reaction.

[0015] Furthermore, the present invention aims to provide a method for providing information for predicting the prognosis of cancer, comprising a step of detecting CTHRC1 protein in a biological sample isolated from an individual suffering from cancer by antigen-antibody reaction using the antibody.

[0016] A further object of the present invention is to provide a kit for diagnosing or predicting the prognosis of cancer, comprising the composition for diagnosing or predicting the prognosis of cancer.

[0017] Furthermore, the present invention aims to provide a method for detecting CTHRC1 protein in a sample, which comprises the step of detecting a CTHRC1 antigen-antibody complex using an antibody that specifically binds to the CTHRC1 protein. Effect of the Invention

[0018] The novel antibody of the present invention is capable of detecting minute amounts of CTHRC1 and also of detecting CTHRC1 conjugate vaccines, and is therefore useful for quantifying CTHRC1 and CTHRC1 conjugate vaccines. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of Indirect ELISA for mouse antibodies 4H5 and 9E6, which have high binding affinity to CTHRC1 (Sensitivity: 4H5, 9E6 >> 1D1 > 6C1). [Diagram 2] FIG. 1 shows the results confirming that mouse antibodies 4H5 and 9E6 have different epitopes, suggesting that they exert their anti-cancer effects through different mechanisms. [Diagram 3] FIG. 1 shows the results confirming that cCMAb45 and cCMAb96 not only have high binding affinity to the CTHRC1 protein, but also bind specifically to it. [Figure 4] FIG. 1 shows the in vitro results of the inhibitory effects of cCMAb45 and cCMAb96 on pancreatic cancer migration. [Diagram 5] FIG. 1 shows the results of in vitro confirmation of the breast cancer migration-inhibitory effects of cCMAb45 and cCMAb96. [Figure 6] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on ovarian cancer migration. [Figure 7] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on bladder cancer migration. [Figure 8] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on pancreatic cancer invasion. [Figure 9] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on the invasion of patient-derived pancreatic cancer cells. [Figure 10] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on breast cancer invasion. [Figure 11] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on ovarian cancer invasion. [Figure 12] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of cCMAb45 and cCMAb96 on bladder cancer invasion. [Figure 13] FIG. 1 shows the production results of 4H5 and 9E6 humanized candidate antibodies. [Figure 14] FIG. 1 shows the binding affinity measurements of the 4H5 and 9E6 humanized candidate antibodies. [Figure 15] FIG. 1 shows the results of verifying the efficacy of the 4H5 and 9E6 humanized candidate antibodies using the pancreatic cancer cell line Panc-1. [Figure 16] FIG. 1 shows the results of re-measurement of the binding affinities of the primarily selected 4H5 and 9E6 humanized candidate antibodies. [Figure 17] FIG. 1 shows the results of re-examining the efficacy of the initially selected 4H5 and 9E6 humanized candidate antibodies. [Figure 18] FIG. 1 shows the results confirming that hCMAb45 and hCMAb96 specifically bind to CTHRC1 protein. [Figure 19] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on pancreatic cancer migration. [Figure 20] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on ovarian cancer migration. [Figure 21] FIG. 1 shows the results of in vitro confirmation of the breast cancer migration-inhibitory effects of hCMAb45 and hCMAb96. [Figure 22] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on the migration of bladder cancer, lung cancer, and melanoma. [Diagram 23] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on pancreatic cancer invasion. [Figure 24] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on ovarian cancer invasion. [Diagram 25] FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on breast cancer invasion. [Figure 26]FIG. 1 shows the results of in vitro confirmation of the inhibitory effects of hCMAb45 and hCMAb96 on the invasion of bladder cancer, lung cancer, and melanoma. [Figure 27] 1 shows the results of confirming the in vivo anti-cancer effects of hCMAb45 and hCMAb96. In a model using the pancreatic cancer cell line BxPC-3, the tumor volume was reduced compared to the control group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] These will be described in detail below. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0021] Throughout this specification the conventional one-letter and three-letter codes for naturally occurring amino acids are used, and the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system. Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid Asp,D Cysteine ​​Cys,C Glutamic acid Glu,E Glutamine Gln,Q Glycine Gly,G Histidine Isoleucine Ile,I Leucine Leu,L Lysine Lys,K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin,V

[0022] One aspect of the present invention provides an antibody that specifically binds to CTHRC1 (Collagen triple helix repeat containing-1) protein.

[0023] The "CTHRC1 (Collagen triple helix repeat containing-1)" in the present invention is a water-soluble protein that acts on vascular remodeling, bone formation and morphogenesis, and is known to be expressed in damaged arteries, promote cell migration, inhibit collagen synthesis in fibroblasts and smooth muscle cells, and have the function of remodeling damaged aortas. It is known that the expression of CTHRC1 in endothelial cells inhibits the expression of transforming growth factor-β (TGF-β), including collagen, by regulating TGF-β. Furthermore, transgenic mice with CTHRC1 gene increased osteoblastic bone formation, differentiation and mineralization of osteoprogenitor cells, and also bound to Wnt5a to activate the planar cell polarity pathway, confirming the role of CTHRC1 in regulating morphogenesis during development.

[0024] The CTHRC1 protein of the present invention may be recombinantly obtained by a conventional method or purchased from a conventional commercial source. The CTHRC1 of the present invention may be in the form of an isolated protein or bound to other proteins or polysaccharides, specifically, bound to a bacterial exotoxin, but is not limited thereto.

[0025] Antibodies that specifically recognize the CTHRC1 protein are used in the diagnosis, prevention, or treatment of diseases such as cancer in which CTHRC1 is overexpressed, and the present inventors have developed antibodies that bind with high affinity to human and mouse CTHRC1 proteins. The antibodies of the present invention bind with high affinity to all human CTHRC1 proteins, and are therefore useful not only in the field of diagnosis of diseases in which CTHRC1 protein is overexpressed, but also in the field of cancer prevention or treatment.

[0026] The term "antibody" as used herein means a protein molecule that plays the role of a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. For the purposes of the present invention, the antibody may be an antibody that specifically binds to CTHRC1 (Collagen triple helix repeat containing-1) protein. The antibody also includes chimeric antibodies, humanized antibodies, human antibodies, bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies. The antibody further includes single-chain antibodies, scabs (single-chain antibodies), derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that have a binding function to FcRn. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG is a subtype that includes IgG1, IgG2, IgG3, and IgG4. The antibody fragment means a fragment having an antigen-binding function, and includes Fd, Fab, Fab', F(ab')2, Fv, and the like. The Fd means a heavy chain portion contained in a Fab fragment. The Fab has a structure having a light chain and a heavy chain variable region, a light chain constant region, and a first heavy chain constant region (CH1 domain), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated by forming a disulfide bond between the cysteine ​​residues in the hinge region of Fab'. The Fv (variable fragment) means a minimum antibody fragment having only a heavy chain variable region and a light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable domain and the light chain variable domain are linked by a disulfide bond, while in single chain Fv (scFv), the heavy chain variable domain and the light chain variable domain are generally linked by a covalent bond via a peptide linker.Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab fragments can be obtained by specifically cleaving a whole antibody with papain, and F(ab')2 fragments can be obtained by cleaving the whole antibody with pepsin), and specifically, can be produced by genetic recombination techniques.

[0027] By "monoclonal antibody" in this invention is meant an antibody molecule of single molecular composition obtained from a population of substantially identical antibodies and such a monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0028] Typically, immunoglobulins have heavy and light chains, each of which contains a constant region and a variable region (these regions are also called domains). The variable regions of the light and heavy chains contain three variable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs mainly play a role in binding to an epitope of an antigen. The CDRs of each chain are typically called CDR1, CDR2, and CDR3 in order from the N-terminus, and are also identified by the chain in which a particular CDR is located.

[0029] In the present invention, the term "human antibody" refers to a molecule derived from human immunoglobulin, in which the entire amino acid sequence constituting the antibody, including the complementarity determining region and framework region, is composed of the amino acid sequence of human immunoglobulin. Human antibodies are usually used to treat human diseases, and they have at least three potential advantages. First, they interact better with the human immune system and can destroy target cells more efficiently, for example by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, they have the advantage that the human immune system does not recognize them as foreign. Third, they have the advantage that their half-life in the human circulation is comparable to that of natural antibodies, even when smaller and less frequent doses of the drug are administered.

[0030] Furthermore, when such an antibody of the present invention contains a constant region, it may contain a constant region derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid of these.

[0031] In the present invention, the term "combination" refers to the binding of a polypeptide encoding a single-chain immunoglobulin constant region of the same origin to a single-chain polypeptide of a different origin when forming a dimer or multimer. For example, a dimer or multimer can be formed from at least two constant regions selected from the group consisting of IgG, IgA, IgD, IgE, and IgM constant regions.

[0032] In the present invention, the term "hybrid" means that a single-chain immunoglobulin heavy chain constant region contains sequences corresponding to immunoglobulin heavy chain constant regions of at least two different origins, and may be, for example, a hybrid of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM.

[0033] In the present invention, an "antibody that specifically binds to CTHRC1 (Collagen triple helix repeat containing-1)" means an antibody that binds to the CTHRC1 protein and inhibits the activity of the CTHRC1 protein.

[0034] The antibodies of the present invention that specifically bind to CTHRC1 have the property of binding to human CTHRC1 protein with high affinity.

[0035] The antibody may comprise (a) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1 or 11, a heavy chain CDR2 of SEQ ID NO: 2 or 12, and a heavy chain CDR3 of SEQ ID NO: 3 or 13, and (b) a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4 or 14, a light chain CDR2 of SEQ ID NO: 5 or 15, and a light chain CDR3 of SEQ ID NO: 6 or 16.

[0036] In addition, the antibody of the present invention may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7, 17, 21 or 25, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 9, 19, 23 or 27, but is not limited to these.

[0037] As an example of the present invention, the antibody of the present invention with improved affinity and specificity for CTHRC1 protein may specifically comprise a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6, or a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16, but is not limited to these.

[0038] In one embodiment of the present invention, an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 9 was named "4H5" or "cCMAb45," and an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 19 was named "9E6" or "cCMAb96."

[0039] In addition, an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 21 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 23 was named "4H5" or "hCMAb45," and an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 was named "9E6" or "hCMAb96."

[0040] In one embodiment of the present invention, B lymphocytes that bind to CTHRC1 protein were screened to select four novel antibodies with the highest antigen binding activity: 4H5, 9E6, 6C1, and 1D1.The epitopes of 4H5 and 9E6 were confirmed to specifically bind to THRC1 protein with high affinity, and therefore the two novel antibodies mentioned above are provided.

[0041] This suggests that antibodies that bind to the CTHRC1 protein of the present invention will be useful in fields where recognition of the CTHRC1 protein is required, such as the diagnosis and treatment of diseases in which the CTHRC1 protein is overexpressed.

[0042] Another aspect of the invention provides a method for producing the antibody.

[0043] The antibody of the present invention can be easily produced by known antibody production techniques. For example, a method for producing a monoclonal antibody can be performed by preparing a hybridoma using B lymphocytes obtained from an immunized animal (Non-Patent Document 2), or by using a phage display technique, but is not limited thereto. A polyclonal antibody can be easily produced by known antibody production techniques.

[0044] The antibody library using the phage display technology is a method in which antibody genes are obtained directly from B lymphocytes without producing hybridomas, and the antibody is expressed on the surface of a phage. The phage display technology overcomes many of the conventional difficulties in generating monoclonal antibodies by B cell immortalization. In general, the phage display technology consists of the steps of: 1) inserting an oligonucleotide with a random sequence into a gene site corresponding to the N-terminus of the phage coat protein pIII (or pIV); 2) expressing a fusion protein of a part of the natural coat protein and a polypeptide encoded by the oligonucleotide with the random sequence; 3) treating a substance that binds to the polypeptide encoded by the oligonucleotide; 4) eluting the antibody-phage particles bound to the substance using low pH or a molecule that is competitive with the binding; 5) amplifying the phage eluted by panning in a host cell; 6) repeating the above process to obtain a desired amount; and 7) determining the sequence of an active antibody from the DNA sequence of the phage clone selected by panning.

[0045] The method for producing the monoclonal antibody of the present invention can be carried out by using phage display technology. Those skilled in the art can easily carry out each step of the above-mentioned production method of the present invention by referring to known phage display technology, for example, the methods disclosed in the papers of Barbas et al. (Non-Patent Documents 3 and 4) and Winter et al. (Non-Patent Document 5). Phages used to construct an antibody library include, for example, filamentous phages, such as fd, M13, f1, If1, Ike, Zj / Z, Ff, Xf, Pf1 or Pf3 phages, but are not limited thereto. In addition, vectors used to express a heterologous gene on the surface of the filamentous phages include, for example, phage vectors such as fUSE5, fAFF1, fd-CAT1, fdtetDOG, and phagemid vectors such as pHEN1, pComb3, pComb8, and pSEX, but are not limited thereto. Additionally, helper phages that can be used to provide the wild-type coat protein necessary to reinfect the recombinant phage for amplification include, but are not limited to, M13K07, VSCM13, and the like.

[0046] Polynucleotides encoding the monoclonal antibody clones of the present invention can be easily isolated using routine procedures and sequenced. For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from a phage template DNA can be used. Once the polynucleotides are isolated, they can be incorporated into an expression vector, which can then be introduced into a suitable host cell to produce the desired monoclonal antibody from the transformed host cell (i.e., the transformant). Thus, the method for producing the human monoclonal antibody of the present invention can be, but is not limited to, a method for producing a human monoclonal antibody, which includes the step of amplifying a polynucleotide encoding the human monoclonal antibody from an expression vector comprising the polynucleotide encoding the human monoclonal antibody.

[0047] Yet other aspects of the invention provide polynucleotides encoding the antibodies, expression vectors comprising the polynucleotides, and host cells into which the vectors have been introduced.

[0048] The antibody is as described above.

[0049] The expression vector provided in the present invention containing the polynucleotide encoding the antibody may be a vector that replicates and / or expresses the polynucleotide in eukaryotic or prokaryotic cells, including, but not limited to, 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 specifically, a vector that is operably linked to an appropriate promoter so that the nucleotide is expressed in a host cell and contains at least one selection marker. For example, the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus, retrovirus vector, etc.

[0050] The expression vector comprising a polynucleotide encoding the antibody may be an expression vector comprising either a polynucleotide encoding the heavy chain or the light chain of the antibody, or may be an expression vector comprising all of the polynucleotides encoding the heavy chain or the light chain.

[0051] The host cell provided in the present invention into which the expression vector is introduced may be, but is not limited to, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc., yeast cells, fungal cells such as Pichia pastoris, etc., insect cells such as Drosophila and Spodoptera Sf9 cells, etc., animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS (monkey kidney fibroblast cells), NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc., transformed with the expression vector, or plant cells.

[0052] In the present invention, "introduction" refers to a method of delivering a vector containing a polynucleotide encoding the antibody to a host cell. The introduction can be performed by various methods known in the art, such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electron bombardment, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Transduction refers to the delivery of a target substance into a cell by infection using virus particles. Furthermore, a vector can be introduced into a host cell by gene bombardment or the like. In the present invention, introduction may be used interchangeably with transformation.

[0053] Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above-mentioned antibody.

[0054] The antibody binds with high affinity to the CTHRC1 protein, which is known to be overexpressed in cancer and to play an important role in the growth and metastasis of cancer cells, and thus can suppress, neutralize, or / and induce a cytotoxic reaction in the activity of the CTHRC1 protein, thereby preventing or treating diseases in which the CTHRC1 protein is overexpressed. The antibody is as described above.

[0055] In the present invention, the "cancer" may be any type of cancer that can be prevented or treated by the antibody of the present invention, such as pancreatic cancer, ovarian cancer, breast cancer, melanoma, liver cancer, cervical cancer, gastric cancer, lung cancer, colon cancer, oral cancer, and bladder cancer. In the present invention, "prevention" refers to any action that suppresses or delays the onset of cancer by administering the composition, and the "treatment" refers to any action that improves or favorably changes the symptoms of cancer by administering the composition.

[0056] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0057] In the present invention, the term "pharmaceutical acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not impair the biological activity and properties of the compound administered. As pharmaceutical carriers acceptable for compositions formulated as liquid solutions, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerin, ethanol, and at least one of these components, which are excellent in sterility and biocompatibility, can be used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be further added to formulate the compositions into injection forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0058] The pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant. Oral solid preparations include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to the usual excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, oral liquids, emulsions, syrups, etc., and in addition to the usual diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavorings, and preservatives are included. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, lyophilized agents, and suppositories. Examples of non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Examples of suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, etc.

[0059] The pharmaceutical composition has any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0060] The compositions of the present invention are administered in a pharma- ceutically effective amount.

[0061] In the present invention, the term "pharmacologically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level is determined by factors including the type and severity of the individual, age, sex, type of cancer, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, co-administered drugs, and other factors known in the medical field. The composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered singly or multiple times. It is important to administer an amount that provides maximum effect with minimal amount without side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0062] Yet another aspect of the invention provides a method of treating cancer using the antibody.

[0063] The antibody and the cancer are as described above.

[0064] The method for treating cancer may be a method for treating cancer comprising a step of administering a pharmaceutical composition further comprising an antibody and a pharma- ceutically acceptable carrier to an individual who has developed cancer or is suspected of developing cancer, and the pharma- ceutically acceptable carrier is as described above. Specifically, the method for treating cancer may be a method for treating cancer comprising a step of administering a composition comprising an antibody to an individual who has developed cancer.

[0065] The individual includes mammals such as cows, pigs, sheep, chickens, dogs, and humans, as well as birds, and may be any individual whose cancer can be treated by administration of the composition of the present invention.

[0066] Here, the composition may be administered in a single or multiple doses in a pharma- ceutical effective amount. Here, the composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric coated tablet or capsule, or suppository. Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration. However, oral compositions must be formulated to coat the active agent or protect it from digestion in the stomach, since the peptide is digested when administered orally. Pharmaceutical compositions may also be administered by any device capable of delivering the active agent to target cells.

[0067] Yet another aspect of the present invention provides a method for providing information for diagnosing cancer, comprising the step of detecting CTHRC1 protein in a biological sample isolated from an individual suspected of having cancer by antigen-antibody reaction using the antibody. The method may also be a method for diagnosing cancer.

[0068] Yet another aspect of the present invention provides a method for providing information for predicting the prognosis of cancer, comprising a step of detecting CTHRC1 protein in a biological sample isolated from an individual suffering from cancer by an antigen-antibody reaction using the antibody. The method may also be a method for predicting the prognosis of cancer. The antibody, cancer, individual, and CTHRC1 protein are as described above. Since CTHRC1 protein is known to be overexpressed in various cancers, the antibody of the present invention is useful for diagnosing cancers in which CTHRC1 protein is known to be overexpressed. It has also been reported that overexpression of CTHRC1 protein induces EMT (epithelial-mesenchymal transition) associated with metastasis and is associated with poor prognosis of cancer, so the antibody according to the present invention is useful for predicting the prognosis of cancer.

[0069] The method for providing information for diagnosing cancer or for predicting the prognosis of cancer can detect CTHRC1 protein by reacting a human monoclonal antibody specific to CTHRC1 of the present invention with an isolated biological sample from an individual suspected of having cancer or an individual who has developed cancer and detecting the formation of an antigen-antibody complex, thereby providing information for diagnosing or predicting the prognosis of cancer.

[0070] Specifically, the method may be a method for providing information for diagnosing cancer or a method for diagnosing cancer, comprising the steps of (a) treating a biological sample isolated from an individual suspected of having cancer with the antibody and detecting CTHRC1 protein by antigen-antibody reaction, and (b) comparing the level of CTHRC1 protein detected in (a) with that of a control group, and determining that the individual is a cancer patient if the CTHRC1 protein level is higher than that of the control group.

[0071] Specifically, the method may be a method for providing information for predicting the prognosis of cancer or a method for predicting the prognosis of cancer, comprising the steps of (a) treating a biological sample isolated from an individual suffering from cancer with the antibody and detecting CTHRC1 protein by antigen-antibody reaction, and (b) comparing the level of CTHRC1 protein detected in (a) with that of a control group.

[0072] In the present invention, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, bacterial expression systems, etc. By reacting these biological samples with or without manipulation with the antibodies of the present invention, the presence of CTHRC1 protein or the presence or absence of cancer can be confirmed.

[0073] In the present invention, the term "antigen-antibody complex" refers to a conjugate of the CTHRC1 protein antigen in a sample and the monoclonal antibody according to the present invention that recognizes the antigen-antibody complex, and the formation of the antigen-antibody complex can be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment, and a scintillation counting method. However, the method is not necessarily limited to these, and various applications and applications are possible.

[0074] In the present invention, various labels can be used to detect antigen-antibody complexes, including, but not limited to, those selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioisotopes.

[0075] Enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-lactamase. Fluorescent substances include fluorescein, Eu 3+ ,EU 3+ Examples of the ligand include biotin derivatives. Examples of the luminescent substance include acridinium esters and isoluminol derivatives. Examples of the fine particles include gold colloids and colored latex. Examples of the radioisotopes include 57 Co, 3 H, 125 I, 125 I-Bolton Hunter Reagent and the like.

[0076] Specifically, the antigen-antibody complex can be detected by enzyme linked immunosorbent assay (ELISA). ELISA includes various ELISA methods such as direct ELISA using a labeled antibody that recognizes the antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes the capture antibody from the complex of the antibody that recognizes the antigen attached to the solid support, direct sandwich ELISA using another labeled antibody that recognizes the antigen from the complex of the antibody and antigen attached to the solid support, indirect sandwich ELISA using a labeled secondary antibody that reacts with another antibody that recognizes the antigen from the complex of the antibody and antigen attached to the solid support and then recognizes that antibody, and competitive ELISA in which antigens having the same antibody binding site compete with each other.

[0077] The monoclonal antibodies may carry a detectable label, or if they do not, they may be captured and identified by treatment with another antibody that carries a detectable label.

[0078] Yet another aspect of the present invention provides a composition for diagnosing or predicting the prognosis of cancer, comprising the antibody.

[0079] The antibody and cancer are as described above. Using a diagnostic or prognostic composition comprising an antibody specific to the CTHRC1 protein of the present invention, a disease associated with the presence or absence of expression or the level of expression of the CTHRC1 protein, such as cancer, can be diagnosed or predicted.

[0080] Yet another aspect of the present invention provides a kit for diagnosing or predicting prognosis of cancer, comprising the above-mentioned composition for diagnosing or predicting prognosis of cancer.

[0081] The composition and cancer are as described above. The cancer diagnostic kit may further include a composition, solution, or device having one or more other components suitable for an analytical method.

[0082] Yet another aspect of the present invention provides a composition and a kit for detecting Collagen triple helix repeat containing-1 (CTHRC1) protein, comprising an antibody that specifically binds to the CTHRC1 protein.

[0083] Yet another aspect of the present invention provides a method for detecting CTHRC1 protein in a sample, comprising detecting a CTHRC1 antigen-antibody complex using an antibody that specifically binds to the CTHRC1 protein, which method can be used to quantify a conjugate vaccine.

[0084] In the present invention, the term "antigen-antibody complex" refers to a binding product of the protein antigen in a sample and an antibody that recognizes it. The antigen-antibody complex may be detected by a method known in the art, for example, a spectroscopic, photochemical, biochemical, immunochemical, electrical, absorptive, chemical, or other method. Specifically, the antigen-antibody complex may be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment, and a scintillation counting method. The antigen-antibody complex may be detected by any method selected from the group consisting of Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (flow cytometry), protein chip, or the like, but is not limited thereto.

[0085] In the present invention, various labels can be used to detect antigen-antibody complexes, including, but not limited to, those selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioisotopes.

[0086] Enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-lactamase. Fluorescent substances include fluorescein, Eu 3+ ,EU 3+ Examples of the ligand include biotin derivatives. Examples of the luminescent substance include acridinium esters and isoluminol derivatives. Examples of the fine particles include gold colloids and colored latex. Examples of the radioisotopes include 57 Co, 3 H, 125 I, 125 I-Bolton Hunter reagent and the like, but are not limited to these.

[0087] The kit of the present invention may be in the form of an ELISA (Enzyme-linked immunosorbent assay) kit. Specifically, the kit contains the above-mentioned antibody as a capture antibody, and the ELISA may contain (i) a polyclonal antibody that binds to CTHRC1, (ii) a capture antibody in the form of an antibody that specifically binds to the CTHRC1 (Collagen triple helix repeat containing-1) protein of the present invention, and (iii) an antibody bound to IgG Fc to which a detection label is bound, but is not limited thereto.

[0088] In the present invention, "ELISA (Enzyme-linked immunosorbent assay)" is also called enzyme immunoassay, and refers to a method in which an enzyme is bound to an antibody to form an antigen-antibody complex, and the enzyme reacts with a substrate to quantify the amount of the antibody using absorbance. The ELISA includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody from an antibody complex that recognizes an antigen attached to a solid support, direct sandwich ELISA using another labeled antibody that recognizes an antigen from an antibody-antigen complex attached to a solid support, indirect sandwich ELISA using a labeled secondary antibody that reacts with another antibody that recognizes the antigen from an antibody-antigen complex attached to a solid support and then recognizes the antibody, and competitive ELISA in which antigens having the same antibody binding site compete with each other.

[0089] The antibody that specifically binds to the CTHRC1 protein of the present invention, a detection composition containing the same, a kit, and a method for detecting CTHRC1 protein using the same can detect extremely small amounts of CTHRC1 protein, and the amount of protein that can be detected can be specifically less than 1 mg or less than 100 ug, and more specifically less than 10 ug, less than 1 ug, less than 100 ng, less than 10 ng, or less than 1 ng.

[0090] In one embodiment of the present invention, it was confirmed that CTHRC1 protein can be detected at the level of several ng by performing indirect ELISA using 4H5 and 9E6 antibodies that specifically bind to CTHRC1 as capture antibodies, and that conjugate vaccines can also be detected by performing the above-mentioned ELISA.

[0091] Thus, by using the antibody of the present invention, much smaller amounts of CTHRC1 protein can be detected compared to conventional CTHRC1 protein detection kits, and therefore the antibody of the present invention is useful for quantifying CTHRC1 protein and vaccines using the same. EXAMPLES

[0092] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples. EXAMPLES

[0093] <Mouse antibodies cCMAb45 and cCMAb96> Example 1-1: Selection of 4H5 and 9E6 To develop an antibody targeting the CTHRC1 protein, we selected a mouse monoclonal antibody that binds to the CTHRC1 protein. Specifically, we injected the CTHRC1 protein into mice, and once antibodies against CTHRC1 were produced, we isolated B lymphocytes from the mouse spleen. The isolated B lymphocytes were diluted so that only one lymphocyte was placed in a 96-well plate coated with the CTHRC1 protein. We then screened for B lymphocytes that bind to the CTHRC1 protein using an anti-mouse antibody labeled with HRP (Horseradish peroxidase).

[0094] As a result, four anti-CTHRC1 mouse monoclonal antibodies, 4H5, 9E6, 6C1, and 1D1, were selected.

[0095] Example 1-2: Confirmation of affinity to CTHRC1 In order to select the antibody having the highest affinity for the CTHRC1 protein from the four anti-CTHRC1 mouse monoclonal antibodies selected in Example 1-1, indirect ELISA was performed.

[0096] Immuno plates coated with CTHRC1-Fc and CTHRC1-His proteins were treated with four antibodies, 4H5, 9E6, 6C1, and 1D1, at various concentrations for 2 hours, and then the absorbance was measured using an anti-mouse antibody labeled with HRP as the secondary antibody. Here, the higher the absorbance (optical density, OD), the stronger the binding to CTHRC1.

[0097] As a result, the OD values ​​of all four antibodies increased depending on the concentration of CTHRC1 protein, but it was confirmed that two antibodies, 4H5 and 9E6, bound to CTHRC1 with higher affinity than the other antibodies (Figure 1).

[0098] Example 1-3: Identification of epitopes In order to confirm the epitopes of 4H5 and 9E6, which have high affinity among the four anti-CTHRC1 mouse antibodies, competitive ELISA was performed. Here, epitope means the position of the antigen recognized by the antibody.

[0099] CTHRC1 protein was coated on an immunoplate, and then biotin-labeled 4H5 and 9E6 antibodies and unlabeled 4H5 and 9E6 antibodies were added at ratios of 1:0, 1:3, 1:6, 1:12.5, 1:25, 1:50, and 1:100. The OD value of each antibody was measured using streptavidin-HRP as the secondary antibody. Here, if 4H5 and 9E6 have similar epitopes, the OD value will decrease as the concentration of unlabeled antibodies increases, but if the epitopes are different, the OD value will not change even if the concentration of unlabeled antibodies increases.

[0100] Therefore, it was confirmed that 4H5 and 9E6 have different epitopes (Figure 2).

[0101] Example 1-4: Construction of chimeric antibodies cCMAb45 and cCMAb96 To minimize the immunogenicity of the 4H5 and 9E6 mouse antibodies, which are specific to CTHRC1 and have high affinity, we constructed chimeric antibodies in which the variable regions of the mouse antibodies were retained and only the constant regions were replaced with human proteins. These were named cCMAb45 and cCMAb96.

[0102] Examples 1-5: Affinity Analysis (Affinity: Indirect ELISA) The affinity of the anti-CTHRC1 chimeric antibodies cCMAb45 and cCMAb96 prepared in Examples 1-3 for the CTHRC1 protein was examined. Indirect ELISA was performed to test the affinity between the antigen and the antibody.

[0103] CTHRC1 was dispensed at 100 ul per well at a concentration of 5 μg / ml onto an immunoplate and coated overnight at 4°C. The next day, the coated CTHRC1 solution was discarded and blocking was performed for 2 hours. After that, the plate was treated with cCMAb45 and cCMAb96 antibodies at various concentrations, and then reacted at room temperature for 2 hours. In order to remove unbound antibodies, the antibodies were discarded and the plate was washed. The OD value was measured using anti-human Fc-HRP, which recognizes the human constant region, as the secondary antibody. Here, the higher the OD value, the higher the affinity between CTHRC1 and the antibody.

[0104] As a result, the K D The value is 5.2 x 10 -10 M, K of cCMAb96 D The value is 4.3 x 10 -10 It was confirmed that both antibodies have very high affinity for the CTHRC1 protein (Figure 3).

[0105] Example 1-6: Specificity Analysis (Indirect ELISA) Indirect ELISA was performed to confirm whether cCMAb45 and cCMAb96 specifically bind to CTHRC1. Similar to the affinity analysis, CTHRC1 protein and multiple non-specific antigen proteins were coated on an immunoplate. The next day, the coating solution was discarded and the plate was blocked, and then cCMAb45 and cCMAb96 were added at concentrations of 0, 1, and 5 μg / ml and reacted for 2 hours. The solution containing the antibodies was then discarded, and a washing process was performed to remove unbound antibodies. The OD value was measured using anti-human kappa light chain-HRP as the secondary antibody.

[0106] As a result, it was confirmed that cCMAb45 and cCMAb96 have high specificity for CTHRC1 (FIG. 3).

[0107] Example 1-7: Confirmation of the effect of inhibiting cancer cell migration To verify the therapeutic efficacy of cCMAb45 and cCMAb96, the migration inhibitory effects were confirmed using pancreatic cancer, breast cancer, ovarian cancer, and bladder cancer cell lines.

[0108] Specifically, cCMAb45 and cCMAb96 were prepared at 1, 5 μg / ml or 1, 10 μg / ml, diluted with serum-free medium together with the cell lines, and dispensed into the upper chamber of a transwell. A serum-containing medium was placed in the lower chamber to create an environment in which the cells could migrate. Each cell line was cultured for a specified time, and the number of migrated cells was measured.

[0109] As a result, it was confirmed that the degree of cancer cell migration was significantly reduced in the groups treated with cCMAb45 and cCMAb96, compared to the control group treated with IgG (FIGS. 4 to 7).

[0110] Example 1-8: Confirmation of the effect of suppressing cancer cell invasion As in Examples 1-7, the efficacy of cCMAb45 and cCMAb96 was verified by examining their inhibitory effects on cancer cell invasion in pancreatic cancer, patient-derived pancreatic cancer cell lines, breast cancer, ovarian cancer, and bladder cancer.

[0111] Matrigel, an artificial imitation of extracellular matrix, was coated on the upper chamber of the transwell to confirm the invasive ability of cancer cells. 1, 5μg / ml or 1, 10μg / ml cCMAb45 and cCMAb96 were prepared and diluted with serum-free medium together with the cell line, and dispensed into the upper chamber of the transwell, and serum-containing medium was placed in the lower chamber. After culturing for a specified time, the number of cells that invaded and migrated through the Matrigel was measured.

[0112] As a result, it was confirmed that the invasive ability of cancer cells was reduced in the groups treated with cCMAb45 and cCMAb96, compared to the IgG control group (FIGS. 8 to 12). EXAMPLES

[0113] <Humanized antibodies hCMAb45 and hCMAb96> Example 2-1: Construction of a humanized antibody library In order to reduce the immunogenicity of the anti-CTHRC1 chimeric antibodies cCMAb45 and cCMAb96, we produced humanized antibodies in which the framework region (hereinafter referred to as "FR") supporting the CDR region was replaced with a human region, excluding the CDR region, which is the most important part for antigen binding.

[0114] Using the CDR grafting method, a humanized antibody library having various FR regions including the CDR regions of cCMAb45 and cCMAb96 was constructed.

[0115] Example 2-2: Selection of humanized antibodies and IgG conversion From the constructed humanized antibody library, clones specific to CTHRC1 and with high affinity were selected. IgG transient expression tests were performed in HEK293F cells, and eight antibodies against 4H5 and 9E6 were purified and their purity was confirmed by SDS-PAGE.

[0116] As a result, the productivity of the 4H5 humanized antibody was 15 to 67.3 mg / L, and the productivity of the 9E6 humanized antibody was 3.1 to 33.3 mg / L (FIG. 13).

[0117] Example 2-3: Analysis of humanized antibody affinity Indirect ELISA was performed to analyze the affinity of the eight candidate clones of 4H5 produced in Example 2-2. Immunoplates were coated with CTHRC1-His, and the next day, the eight humanized candidate antibodies and cCMAb45 were added at concentrations of 1 / 3 serial dilutions starting from 500ng / ml. Anti-human Fc-HRP was used as the secondary antibody to measure the OD value. The affinity was evaluated using K D The values ​​are shown as K D A lower value means a higher affinity.

[0118] As a result, the K D The values ​​were comparable to or lower than cCMAb45.

[0119] Similarly to the above method, the affinity of eight candidate clones of the 9E6 humanized antibody was analyzed. The K D The values ​​were equivalent to or slightly higher than those of cCMAb96.

[0120] Specifically, the affinity of the 4H5 humanized antibody is 0.82 × 10 -10 M~2.5×10 -10 M, and the affinity of the 9E6 humanized antibody is 1.61 × 10 -10 M~4.8×10 -10 It was confirmed that the strain was M (Figure 14).

[0121] Example 2-4: Efficacy testing of humanized antibodies To test the efficacy of the 4H5 and 9E6 humanized antibody candidate clones, migration assays were performed using the pancreatic cancer cell line Panc-1. 4 The cells were diluted with serum-free medium and dispensed into the upper chamber of the transwell. Next, serum-containing medium was placed in the lower chamber to create an environment in which the cells could migrate. After 24 hours of culture, the number of migrated cells was counted to evaluate the efficacy of the candidate antibodies.

[0122] As a result, it was confirmed that the 4H5 humanized antibody candidate clone reduced cell migration ability by 30-50% compared to the control IgG, and the 9E6 humanized antibody candidate reduced cell migration ability by 30-40%. In conclusion, both the 4H5 and 9E6 humanized antibody candidates were confirmed to have efficacy equivalent to or superior to cCMAb45 and cCMAb96 (Figure 15).

[0123] Examples 2-5: Selection of humanized antibodies and re-evaluation of affinity and potency The 4H5 and 9E6 humanized antibody candidate clones were evaluated based on three items: affinity, potency, and yield, and antibodies were selected. For the 4H5 humanized antibody, SA2759, SA2761, and SA2762 were selected. , SA2765 were selected, and for the 9E6 humanized antibody, SA2768, SA2769, and SA2770 were selected.

[0124] As described above, the affinity and efficacy of the selected antibodies were re-evaluated using indirect ELISA for affinity and migration assay for efficacy evaluation, and the experimental methods were the same as those in Examples 2-3 and 2-4.

[0125] As a result, the affinity was similar to that of cCMAb45 and cCMAb96, with K values ​​lower than those of cCMAb45 and cCMAb96, as in the first measurement. D It was confirmed that the selected humanized antibody 4H5 showed an affinity of several pM and reduced the cell migration ability by 40%, and the humanized antibody 9E6 showed an affinity of several tens of pM and reduced the cell migration ability by 50% (FIGS. 16 and 17).

[0126] Finally, SA2765 was designated hCMAb45 and SA2770 was designated hCMAb96.

[0127] Example 2-6: Specificity analysis of hCMAb45 and hCMAb96 To confirm whether hCMAb45 and hCMAb96 specifically bind to CTHRC1, indirect ELISA was performed. CTHRC1-His and multiple unspecific antigens were coated onto an immunoplate. The next day, hCMAb45 and hCMAb96 were reacted with the antigens at concentrations of 0, 1, and 5 μg / ml, and then anti-human kappa light chain-HRP was used as the secondary antibody to measure the OD value.

[0128] As a result, it was confirmed that hCMAb45 and hCMAb96 specifically bind to CTHRC1 (FIG. 18).

[0129] Example 2-7: Confirmation of the effect of inhibiting cancer cell migration To verify the therapeutic efficacy of hCMAb45 and hCMAb96, the inhibitory effect on cancer cell migration was confirmed. Pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, and malignant melanoma were used. The experimental method was the same as in Examples 1-7.

[0130] As a result, it was confirmed that the migration ability of cancer cells was significantly reduced in the groups treated with hCMAb45 and hCMAb96, compared to the control IgG group (FIGS. 19 to 22).

[0131] Example 2-8: Confirmation of the effect of suppressing cancer cell invasion The inhibitory effects of hCMAb45 and hCMAb96 on cancer cell invasion were confirmed using the same experimental method as in Examples 1-8.

[0132] As a result, the invasive ability of cancer cells was significantly reduced in the groups treated with hCMAb45 and hCMAb96, compared to the control IgG group (FIGS. 23 to 26).

[0133] Example 2-9: Confirmation of in vivo anticancer effects in pancreatic cancer animal models In Examples 2-7 and 2-8, the cancer cell migration and invasion inhibitory ability of the humanized antibodies hCMAb45 and hCMAb96, which specifically bind to the CTHRC1 protein, was confirmed in vitro. Therefore, it was confirmed whether they also exerted a similar anticancer effect in vivo. Specifically, a subcutaneous xenograft mouse model was prepared using the pancreatic cancer cell line BxPC-3, which overexpresses CTHRC1. 1×10 6 The cells were subcutaneously transplanted into the right leg of a mouse, and the size of the tumor formed after 6 days was 73 to 77 mm. 3 Mice were selected and divided into groups. Drugs were injected into the tail vein. The control IgG group and the experimental groups hCMAb45 and hCMAb96 were administered at a dose of 10 mg / kg twice a week for a total of 4 weeks, and tumor size was measured twice a week using calipers. The experiment was terminated on the 28th day after the start of antibody treatment (8 treatments), and the tumor size was compared with that of the control IgG group.

[0134] As a result, it was confirmed that the tumor size was reduced in the groups administered with hCMAb45 and hCMAb96 compared to the group administered with control IgG (Figure 27). In particular, it was confirmed that the anti-cancer effect was superior in the group treated with hCMAb96 compared to the group treated with hCMAb45.

[0135] The above examples confirmed the in vivo anti-cancer effects of the humanized antibodies hCMAb45 and hCMAb96 that specifically bind to CTHRC1, and verified their potential as substances that delay or suppress tumor growth.

[0136] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. (a) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 13; (b) a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; An antibody that specifically binds to CTHRC1 (Collagen triple helix repeat containing-1) protein.

2. The antibody of claim 1 , wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 17 or 25, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 19 or 27.

3. A polynucleotide encoding the antibody of claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. A pharmaceutical composition for preventing or treating cancer, comprising the antibody according to claim 1 or 2.

7. 7. The composition of claim 6, wherein the cancer is selected from the group consisting of pancreatic cancer, ovarian cancer, breast cancer, melanoma, liver cancer, gastric cancer, lung cancer, colon cancer, oral cancer, cervical cancer and bladder cancer.

8. A method for providing information for diagnosing cancer, comprising the step of detecting CTHRC1 protein in a biological sample isolated from an individual suspected of having cancer through an antigen-antibody reaction using the antibody described in claim 1 or 2.

9. A method for providing information for predicting the prognosis of cancer, comprising a step of detecting CTHRC1 protein in a biological sample isolated from an individual suffering from cancer by antigen-antibody reaction using the antibody described in claim 1 or 2.

10. A composition for diagnosing or predicting the prognosis of cancer, comprising the antibody of claim 1 or 2.

11. A kit for diagnosing or predicting the prognosis of cancer, comprising the composition of claim 10.

12. A kit for detecting CTHRC1, comprising the antibody according to claim 1 or 2.

13. The kit of claim 12, wherein the kit is in the form of an ELISA.

14. The kit of claim 13 , wherein the antibody is a capture antibody.

15. The ELISA comprises: (i) a polyclonal antibody that binds to CTHRC1; (ii) a capture antibody in the form of an antibody according to claim 1 or 2; (iii) an antibody bound to IgG Fc having a detection label attached thereto; The kit of claim 13, which is an indirect ELISA.

16. A method for detecting CTHRC1 protein in a sample, comprising the step of detecting a CTHRC1 antigen-antibody complex using the antibody described in claim 1 or 2.

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