An epitope and monoclonal antibody of EPB41L5

Abstract

The present disclosure identifies the mechanism of action of TGF-β / Smad3 / EPB41L5 molecules on cancer cells and finds that overexpression of EPB41L5 is associated with poor overall survival in cancer patients, suggesting that EPB41L5 is a potential cancer prognostic marker. Therefore, the present disclosure specifies an epitope of EPB41L5 that can recognize EPB41L5 as an antigen and relates to antibodies or fragments thereof that specifically bind to the epitope. The antibodies disclosed herein can be used as effective therapeutic agents for EPB41L5-associated cancers.

Description

Technical Field

[0001] The present disclosure relates to an epitope of EPB41L5 (EPB41L5 erythrocyte membrane band 4.1 protein analog 5) protein and a monoclonal antibody specifically binding thereto. Background Art

[0002] Cancer is a highly lethal disease that causes abnormal and indefinite proliferation of tissue cells, forming tumors that prevent organs from functioning normally and threaten a person's life. In 2017, malignant tumors (cancer) were the leading cause of death in South Korea, accounting for 27.6% of all deaths.

[0003] Gastric cancer is one of the leading causes of death among cancers. Metastatic gastric cancer, in particular, is a malignant disease with a 5-year survival rate of less than 30% worldwide. While research has been conducted to identify new biomarkers for treating this disease, the specific pathogenesis of metastatic gastric cancer remains unclear.

[0004] Targeted therapies developed to date include HER2-targeted therapies and VEGFR2-targeted therapies. In particular, Genetec / Roche discovered that HER2 is associated with a poor prognosis in breast, ovarian, or gastric cancers. Consequently, Genetec / Roche developed a humanized monoclonal antibody (Herceptin or Trastuzumab) that binds to the extracellular domain of HER2 / neu. Furthermore, Herceptin / Trastuzumab is currently a best-selling drug sold worldwide, generating $950 million in sales in the United States in the first half of 2011 alone. However, it is well known that Herceptin / Trastuzumab has the following drawbacks: Besides breast cancer, it has no clinical effect against other cancers that significantly overexpress HER2 / neu, and patients who take Herceptin / Trastuzumab for a long time can develop resistance to the drug.

[0005] Therefore, there is a need to identify biomolecules associated with cancer growth and metastasis and to discover new biomarkers for cancer targeted therapy that control cancer growth and metastasis by targeting these biomolecules.

[0006] The information disclosed in the above “Background” section is only for enhancement of understanding of the background of the disclosure and should not be regarded as an admission that the information forms the general technology already known to those skilled in the art. Summary of the Invention

[0007] Technical issues

[0008] One object of the present disclosure is to provide a 1-6-mer epitope selected from the 619th to 624th amino acid residues of the EPB41L5 (EPB41L5 erythrocyte membrane band 4.1 protein analog 5) protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Another object of the present disclosure is to provide a monoclonal antibody or a fragment thereof, wherein the monoclonal antibody recognizes the EPB41L5 protein as an antigen and specifically binds thereto, and the amino acid sequence of the EPB41L5 protein is shown in SEQ ID NO: 1.

[0010] Another object of the present disclosure is to provide a nucleic acid molecule encoding a monoclonal antibody or a fragment thereof and a vector comprising the nucleic acid molecule.

[0011] Another object of the present disclosure is to provide a vaccine composition for preventing or treating cancer, which comprises the following active ingredients: a 1-6-mer epitope selected from the 619th to 624th amino acid residues of the EPB41L5 (EPB41L5 erythrocyte membrane band 4.1 protein analog 5) protein represented by the amino acid sequence of SEQ ID NO: 1; a nucleic acid molecule encoding the epitope; or a vector comprising the nucleic acid molecule.

[0012] Another object of the present disclosure is to provide a pharmaceutical composition for preventing, alleviating or treating cancer, wherein the pharmaceutical composition comprises as an active ingredient the above-mentioned monoclonal antibody or a fragment thereof; a nucleic acid molecule encoding the monoclonal antibody or a fragment thereof; or a vector comprising the nucleic acid molecule.

[0013] Another object of the present disclosure is to provide a method for providing information for diagnosing a disease caused by overexpression of EPB41L5.

[0014] Other objects and advantages of the present disclosure will be more clearly understood from the following detailed description of the present disclosure, the appended claims and the accompanying drawings.

[0015] Technical Solution

[0016] The present inventors have discovered that overexpression of the EPB41L5 (EPB41L5 erythrocyte membrane band 4.1 protein analog 5) protein is associated with cancer growth and metastasis compared to a normal control group. They have conducted extensive research to identify new biomarkers for targeted cancer therapy, targeting the EPB41L5 protein to control cancer growth and metastasis. As a result, the present inventors have identified an epitope of the EPB41L5 protein that inhibits cancer progression and metastasis through transforming growth factor β (TGF-β)-EPB41L5 signaling, thereby completing the present disclosure.

[0017] According to one aspect of the present disclosure, the present disclosure provides a 1-6-mer epitope selected from the 619th to 624th amino acid residues of the EPB41L5 (erythrocyte membrane band 4.1 protein analog 5) protein, and the amino acid sequence of the EPB41L5 protein is shown in SEQ ID NO: 1.

[0018] In the present disclosure, the EPB41L5 protein can be represented by SEQ ID NO: 1, and its sequence information can be found in GenBank with accession number NM_020909.

[0019] In the present disclosure, the EPB41L5 (erythrocyte membrane band 4.1 protein analog 5) protein belongs to the NBL4 subfamily of the erythrocyte membrane band 4.1 protein superfamily, has a FERM domain at its N-terminus and a non-homologous sequence at its C-terminus.

[0020] In this specification, the term "epitope" refers to a local region of an antigen that can specifically bind to an antibody or its fragment. Epitopes are usually composed of surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that, in the presence of a denaturing solvent, the conformational epitope is bound rather than the non-conformational epitope is lost. Epitopes may include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by the specific antigen-binding peptide (i.e., the amino acid residue is within the binding region of the specific antigen-binding peptide).

[0021] In the present disclosure, the epitope may comprise any one or more of amino acid residues 619, 620, 621, 622, 623 and 624 of the EPB41L5 protein.

[0022] In the present disclosure, the epitope may consist of amino acids 619 to 624 of the EPB41L5 protein, preferably consisting of the amino acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

[0023] In the present disclosure, when an epitope containing amino acids at the above positions is used in a vaccine or composition, it may be used in combination with a carrier to maintain its structure. Although the carrier in the present disclosure is not particularly limited as long as it is biocompatible and can achieve the intended effect of the present disclosure, it is preferably selected from peptides, serum albumin, immunoglobulin, hemocyanin, and polysaccharides.

[0024] According to another aspect of the present disclosure, the present disclosure provides a monoclonal antibody or a fragment thereof, which recognizes the EPB41L5 protein represented by SEQ ID NO: 1 as an antigen and specifically binds thereto.

[0025] According to a preferred embodiment of the present disclosure, the antibody or fragment thereof of the present disclosure can bind to a 1-6-mer epitope selected from amino acids 619 to 624 of the EPB41L5 protein.

[0026] Epithelial-mesenchymal transition (EMT) is the primary cause of gastric cancer metastasis. Numerous studies have reported that the transforming growth factor β (TGF-β) signaling pathway regulates EMT. TGF-β ligands bind to TGF-β receptor I via the serine / threonine kinase TGF-β receptor II. TGF-β receptor dimers are activated by phosphorylation of Smad2 / 3. Phosphorylated Smad2 / 3 then bind to a shared Smad (Smad4). This protein complex enters the cell nucleus and regulates EMT-related genes, such as PAI-1, ZEB1, and Slug. Studies have shown that TGF-β1 expression is enhanced in the mucosa, serum, and tissues of gastric cancer patients, and overexpression of TGF-β1 is associated with poor survival in gastric cancer patients.

[0027] In the present disclosure, oligonucleotide microarray analysis and Kaplan-Meier survival analysis confirmed that when the expression level of the EPB41L5 gene and its encoded protein is high, the survival rate of cancer patients is lower. In addition, it was confirmed that TGF-β1 increases the mRNA and protein expression of EPB41L5 in four gastric cancer cell lines (KATOIII, MKN28, SNU1, and SNU719), and that the TGF-β1-induced increase in EPB41L5 expression is regulated by Smad-dependent transforming growth factor β (TGF-β) signaling. The increase in EPB41L5 protein expression induced by transforming growth factor β (TGF-β) signaling may affect tumor metastasis by interacting with the cell adhesion molecule p120-catenin. Therefore, the monoclonal antibody or fragment thereof disclosed herein specifically binds to an epitope, which is an amino acid sequence bound when the EPB41L5 / p120-catenin complex is formed, and thus the monoclonal antibody or fragment thereof can very effectively inhibit the occurrence, metastasis or growth of cancer by effectively inhibiting the formation of the EPB41L5 / p120-catenin complex.

[0028] According to a preferred embodiment of the present disclosure, the monoclonal antibody and its fragments specifically bind to EPB41L5 as set forth in SEQ ID NO:1 and block the interaction between EPB41L5 and p120-catenin. While the monoclonal antibody and its fragments are not particularly limited, they can be produced using the EPB41L5 protein set forth in SEQ ID NO:1 as an antigen. Most preferably, but not limited to, the monoclonal antibody and its fragments are produced using the amino acid sequence set forth in SEQ ID NO:2 as an antigen.

[0029] In this specification, the term "epitope" refers to a local region of an antigen that can specifically bind to an antibody or its fragment. For example, continuous amino acids of an antigen polypeptide can serve as an epitope, or two or more non-continuous regions of the tertiary folding of the polypeptide can serve together as an epitope. An epitope can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 or at least 15 continuous or non-continuous amino acids in the unique three-dimensional structure of the antigen. The antibodies or fragments thereof disclosed herein recognize EPB41L5 as an antigen and specifically bind to it. Specifically, the antibody or its fragment can specifically bind to a 1 to 6-mer epitope selected from the 619th to 624th amino acid residues of EPB41L5, and the epitope can comprise one or more amino acids.

[0030] Methods for determining the epitope to which a given antibody binds (e.g., epitope mapping) include, for example, various methods based on immunoblotting and immunoprecipitation assays for antibody reactivity testing. Various methods for determining the three-dimensional structure of an epitope include x-ray crystallography, two-dimensional nuclear magnetic resonance (NMR) and HDX-MS (Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0031] According to a preferred embodiment of the present disclosure, the epitope to which the antibodies or fragments thereof of the present disclosure can be bound can be determined by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, enzyme-linked immunosorbent assay (ELISA), hydrogen / deuterium exchange mass spectrometry (HDX-MS), array-based oligopeptide scanning analysis and / or mutation mapping (Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303).

[0032] As used herein, the term "antibody" may be any type of antibody (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) among immunoglobulin molecules, or may be any isotype of antibody (e.g., human IgG1, IgG2, IgG3, and IgG4; mouse IgG1, IgG2a, IgG2b, and IgG3). Immunoglobulins (e.g., IgG1) can exist in several isotypes, and the term "antibody" used herein includes well-known isotypes and allotypes. In addition, the term "antibody" used herein may be IgG1, IgG2, IgG3, IgG4, or a hybrid thereof (e.g., a hybrid of IgG2 and IgG4).

[0033] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a particular epitope.

[0034] The monoclonal antibodies disclosed herein can be prepared by, for example, the hybridoma method first described by Kohler et al. in Nature 256, 495 (1975) or by recombinant DNA methods. In addition, monoclonal antibodies can be isolated from phage antibody libraries using techniques such as those described by Clackson et al. in Nature 352, 624-628 (1991) and Marks et al. in Journal of Mol. Cell Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. The monoclonal antibodies disclosed herein can be obtained from hybridomas produced by cells expressing the EPB41L5 antigen, or from cells obtained by immunizing mice with the target antigen in the form of a nucleic acid encoding the EPB41L5 antigen. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals (e.g., rats, dogs, primates, etc.).

[0035] In this specification, the use of the monoclonal antibody includes the use of its fragments, and the fragment is preferably an antigen-binding fragment. Various methods known in the art can be used to produce fragments. For example, Fab fragments and F(ab')2 fragments can be produced by hydrolyzing and cleaving immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0036] As used herein, the term "fragment" may be Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv) or sdAb fragment containing a monomer structure (VH or VL domain), and such fragments are well known in the art.

[0037] In the present disclosure, the antibody may be, but is not limited to, a chimeric antibody, a humanized antibody, a bivalent bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a trimer, a tetramer, or a fragment thereof.

[0038] In the present disclosure, the "chimeric antibody" is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and has significantly improved immunogenicity compared to mouse antibodies.

[0039] In addition, in the present disclosure, the term "humanized antibody" refers to an antibody obtained by modifying the protein sequence of an antibody derived from a non-human species so that the protein sequence is similar to that of naturally occurring human antibody variants. For example, a humanized antibody can be produced by recombining murine CDRs with human antibody FRs to produce a humanized variable region, which is then recombined with the desired human antibody constant region.

[0040] The monoclonal antibodies or fragments thereof disclosed herein can inhibit the physiological effects associated with cancer growth and metastasis mediated by the EPB41L5 gene or its encoded protein. Specifically, increased TGF-β1 expression in tumors was identified, and TGF-β1 increased the expression level of the EPB41L5 protein, but the expression level of the EPB41L5 protein was regulated by Smad-dependent transforming growth factor β (TGF-β) signaling. In other words, TGF-β1-induced increases in EPB41L5 protein expression levels affect tumor metastasis and invasion through epithelial-mesenchymal transition. Furthermore, treatment with monoclonal antibodies or fragments thereof containing the above-mentioned antigen-binding proteins can effectively inhibit cell signaling mediated by the EPB41L5 protein.

[0041] As used herein, the expression "inhibits growth" is intended to include any measurable reduction in the growth of cells contacted with the monoclonal antibodies or fragments thereof of the present disclosure, compared to the growth of the same cells not contacted with the monoclonal antibodies or fragments thereof of the present disclosure, for example, an inhibition of growth of the cell culture by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The reduction in cell growth may be caused by a variety of mechanisms.

[0042] As described herein, the monoclonal antibody or its fragment comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). The monoclonal antibody or its fragment that binds to the EPB41L5 protein as an antigen is a polypeptide comprising one or more complementary determining regions (CDRs). In the antigen binding protein, the CDRs are oriented to obtain the appropriate antigen binding properties of the CDRs. In general, the antigen binding proteins provided herein can interfere with, block, reduce, or modulate the interaction between EPB41L5 and p120-catenin. That is, the antigen binding proteins can inhibit TGF-β1-mediated cancer metastasis and growth by inhibiting the formation of the EPB41L5 / p120-catenin complex in a subject.

[0043] In the present disclosure, monoclonal antibodies or fragments thereof may include, but are not limited to:

[0044] A heavy chain variable region comprising a heavy chain CDR1 represented by SEQ ID NO:6, a heavy chain CDR2 represented by SEQ ID NO:7, and a heavy chain CDR3 represented by SEQ ID NO:8; and a light chain variable region comprising a light chain CDR1 represented by SEQ ID NO:9, a light chain CDR2 represented by SEQ ID NO:10, and a light chain CDR3 represented by SEQ ID NO:11.

[0045] In the present disclosure, monoclonal antibodies or fragments thereof may include, but are not limited to:

[0046] The heavy chain variable region is represented by SEQ ID NO: 12 and the light chain variable region is represented by SEQ ID NO: 13.

[0047] The heavy chain variable region (VH) or one or more CDRs thereof can be linked to the constant region to form a heavy chain. Similarly, the light chain variable region (VL) or one or more CDRs thereof can be linked to the constant region to form a light chain. The full-length heavy chain and full-length light chain combine to form a full-length antibody.

[0048] In the present disclosure, as described above, a monoclonal antibody or a fragment thereof that binds to a 1- to 6-mer epitope selected from amino acid residues 619 to 624 of the EPB41L5 protein represented by the amino acid sequence of SEQ ID NO: 1 specifically binds to EPB41L5 represented by SEQ ID NO: 1, thereby exhibiting the effect of blocking the interaction between EPB41L5 and p120-catenin.

[0049] In the present disclosure, the monoclonal antibodies or fragments thereof can be used to treat diseases. Specifically, they can inhibit, control, or regulate one or more biological activities of the EPB41L5 gene or the protein it encodes, and can specifically bind to the EPB41L5 protein, thereby substantially inhibiting EPB41L5 protein-induced cell signaling through competitive binding with proteins that bind to the EPB41L5 protein (e.g., TGF-β1). Therefore, the monoclonal antibodies or fragments thereof can be very effectively used for treatment.

[0050] The variable regions of immunoglobulin chains typically exhibit the same overall structure, consisting of relatively conserved framework regions (FRs) connected by three hypervariable regions (often referred to as "complementarity determining regions" or CDRs). The CDRs from the two chains of each heavy chain / light chain pair described above are typically ordered by the framework regions to form a structure that specifically binds to a specific epitope on a target protein (e.g., PCSK9). From N-terminus to C-terminus, naturally occurring light and heavy chain variable regions typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In order to assign numbers to the amino acids occupying each position in these domains, a numbering system was devised and is described in the Kabat Immunological Protein Sequence Database (1987 and 1991, NIH, Bethesda, MD) or in Chothia and Lesk, 1987, J. Mol. Cell Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.

[0051] Various heavy and light chain variable regions can be provided herein, and as described above, each of these variable regions can be connected to the above-mentioned heavy and light chain constant regions to form complete antibody heavy and light chains, respectively. In addition, each of the formed heavy and light chain sequences can be combined to form a complete antibody structure.

[0052] The monoclonal antibodies or fragments thereof disclosed herein may include amino acid sequence variants, as long as they can specifically bind to EPB41L5. For example, the amino acid sequence of the antibody may be modified to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of one or more residues in the amino acid sequence of the antibody.

[0053] Such amino acid variants are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side-chain substituents indicates that arginine, lysine, and histidine residues are all positively charged; alanine, glycine, and serine are all similar in size; and phenylalanine, tryptophan, and tyrosine are all similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be defined as biologically functional equivalents.

[0054] To introduce variants, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge properties, as follows: 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); Arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is generally understood in the art. It is well known that certain amino acids can be substituted with amino acids having similar hydropathic indexes and still retain similar biological activity. In order to introduce variants with reference to the hydropathic index, the range of the hydropathic index difference between the replaced amino acids is preferably ±2, more preferably ±1, and more preferably ±0.5.

[0055] It is also well known that substitutions between amino acids having similar hydrophilicity values ​​produce proteins with equivalent biological activity. For example, the hydropathic index of the following amino acid residues is disclosed in U.S. Patent No. 4,554,101: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); 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); and tryptophan (-3.4). For the purpose of introducing variants with reference to the hydropathic index, the range of difference in the hydropathic index between substituted amino acids is preferably ±2, more preferably ±1, and even more preferably ±0.5.

[0056] It is well known in the art that amino acid exchanges in proteins generally do not change the activity of the molecule (H. Neurath, RL Hill, the proteins, Academic Press, New York (1979)). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val and Gln / Glu.

[0057] Taking into account the above variants having bioequivalent activities, it can be interpreted that the binding molecules of the present disclosure also include sequences showing substantial identity to the sequences described in the sequence listing.

[0058] As used herein, the term "substantially identical" refers to sequences that exhibit at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology, as determined by aligning the disclosed sequence with any other sequence so as to correspond to each other as closely as possible and analyzing the alignment using algorithms commonly used in the art. Methods for sequence alignment are known in the art. Smith and Waterman disclose various methods and algorithms for alignment. Adv.Appl.Math.2:482 (1981); Needleman and Wunsch, J.Mol.Bio.48:443 (1970); Pearson and Lipman, Methods in Mol.Biol.24:307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc.Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is available from NCBI (National Center for Biotechnology Information) and can be used in conjunction with sequencing programs available on the Internet, such as blastp, blasm, blastx, tblastn, and tblastx. BLAST can be accessed at http: / / www.ncbi.nlm.nih.gov / BLAST. Sequence homology comparison methods can be performed online using this program (http: / / www.ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0059] In the present disclosure, although binding molecules (preferably antibodies) can be produced by conventional methods for producing antibodies, they can also be produced by affinity maturation.

[0060] As used herein, the term "affinity maturation" refers to the process by which activated B cells produce antibodies with enhanced affinity for antigens during an immune response. For the purposes of this disclosure, affinity maturation can be based on the principles of mutation and selection, with antibodies or antibody fragments produced by affinity maturation in the same process that occurs in nature.

[0061] According to another aspect of the present disclosure, the present disclosure provides: a nucleic acid molecule encoding a monoclonal antibody or a fragment thereof; a vector comprising the nucleic acid molecule; and a host cell comprising the vector.

[0062] The nucleic acid molecules disclosed herein can be isolated or recombinant nucleic acid molecules. Such nucleic acid molecules include single-stranded and double-stranded DNA and RNA and their corresponding complementary sequences. "Isolated nucleic acid" can be isolated from a natural source. In this case, the isolated nucleic acid is separated from the surrounding gene sequences present in the genome of the subject from which the nucleic acid is isolated. Isolated nucleic acid can be understood as nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide. In this case, the nucleic acid produced from this process can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule represents a nucleic acid molecule that exists as a separate fragment or as a component of a larger nucleic acid construct. When a nucleic acid is arranged in a functional relationship with another nucleic acid sequence, it is "operably linked." For example, when expressed as a preprotein, a presequence or secretory leader DNA (i.e., a pre-secretory polypeptide) is "operably linked" to the DNA of the polypeptide. When they are arranged to promote translation, a promoter or enhancer that affects the transcription of the polypeptide sequence is operably linked to the coding sequence, or a ribosome binding site is operably linked to the coding sequence. In general, the term "operably linked" means that the DNA sequences to be connected are adjacent to each other. In the context of secretory leaders, the term "operably linked" means that the secretory leaders are adjacent in the same leader frame. However, enhancers are not located adjacent to each other. Linkage is achieved by ligation at convenient restriction endonuclease sites. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0063] As used herein, the term "vector" refers to a vehicle into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The nucleic acid sequence may be foreign or heterologous.

[0064] As used herein, the term "expression vector" refers to a vector that contains at least a portion of a nucleic acid sequence encoding a gene product that can be transcribed. In some cases, the RNA molecule is translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of control sequences. In addition to control sequences that regulate transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that provide other functions.

[0065] In a specific embodiment of the present disclosure, the expression vector can be selected from the commercially widely used pCDNA vector, F, R1, RP1, Col, pBR322, ToL, Ti vector; cosmid; phage, such as lambda, lambdoid, M13, Mu, p1, P22, Qμ, T-even, T2, T3, T7, etc.; plant virus, but not limited thereto. Any expression vector known to those skilled in the art can be used in the present disclosure, and the selection of the expression vector depends on the properties of the selected host cell. The vector can be introduced into the host cell by, but not limited to, calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, liposome transfection or electroporation, and those skilled in the art can select and use an introduction method suitable for the expression vector and host cell used. Preferably, the vector includes, but is not limited to, one or more selective tags, and a vector that does not include a selective tag can also be used to determine whether a product has been produced. The selection of the selective tag can depend on, but is not limited to, the selected host cell, as the selection is performed using methods known to those skilled in the art.

[0066] To facilitate purification of the disclosed nucleic acid molecules, a tag sequence can be inserted and fused into the expression vector. Examples of tags include, but are not limited to, a hexahistidine tag, a hemagglutinin tag, a myc tag, or a marker tag. Any tag known to those skilled in the art that facilitates purification can be used in the present disclosure.

[0067] As used herein, the term "host cell" includes eukaryotic and prokaryotic organisms and refers to any transformable organism capable of replicating a vector or expressing a gene encoded by a vector. A host cell can be transfected or transformed by a vector. Transfection or transformation refers to the process of transferring or introducing an exogenous nucleic acid molecule into a host cell.

[0068] Preferred examples of the host cells disclosed herein include, but are not limited to, bacterial cells, CHO cells, HeLa cells, HEK293 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, NIH3T3 cells, and the like.

[0069] According to another aspect of the present disclosure, the present disclosure provides a vaccine composition for preventing or treating cancer, which contains the following active ingredients: a 1-6-mer epitope of the 619th to 624th amino acid residues of the EPB41L5 (EPB41L5 erythrocyte membrane band 4.1 protein analog 5) protein represented by the amino acid sequence of SEQ ID NO: 1; a nucleic acid molecule encoding the epitope; or a vector containing the nucleic acid molecule.

[0070] In the vaccine composition of the present disclosure, the contents related to EPB41L5, epitope, nucleic acid molecule and vector are the same as those described above, and thus their description is omitted to avoid over-complication of this specification.

[0071] The vaccine can be a live vaccine, an attenuated vaccine or an inactivated vaccine, and the vaccine can be used directly or as an adjuvant.

[0072] The vaccine composition disclosed herein can prevent or treat cancer by inducing an immune response to EPB41L5 and a systemic immune response through active immunization. Active immunization refers to the immunity of an organism by producing antibodies in its own body when a pathogen invades.

[0073] Furthermore, the vaccine composition of the present disclosure can be used to prevent or treat cancer by administering it to a subject.

[0074] The cancer in the present disclosure may be a cancer determined to overexpress the EPB41L5 gene or a protein encoded by it.

[0075] As used herein, the term "overexpression" means that the expression level of the EPB41L5 gene or the protein encoded by it is 1.1 to 2 times the expression level in control cells (eg, normal cells of relevant organs) when the expression level of EPB41L5 is determined by an appropriate expression assay. The types of cancers disclosed herein are not limited, and the vaccine compositions disclosed herein can be used to treat a variety of cancers, including lymphomas, such as leukemia, acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and multiple myeloma; solid tumors in children, such as brain tumors, glioblastomas, neuroblastomas, rhabdomyosarcomas, retinoblastomas, Wilms' tumors, bone tumors, and soft tissue sarcomas; and common solid tumors in adults, such as lung cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, oral cancer, pancreatic cancer, melanoma, skin cancer, gastric cancer, ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer, and testicular cancer.

[0076] The cancer in the present disclosure may be cancer stem cells.

[0077] As used herein, the term "cancer stem cell" refers in a broad sense to cancer cells that have the ability to self-renew or differentiate, which is a unique ability of stem cells. Tumor stem cells are a type of stem cell known to exist in tumors and are believed to occur due to the abnormal transfer of genetic information from normal stem cells. It is well known that the survival and proliferation of tumor stem cells are due to the presence of the microenvironment (niches) in which they survive, and the normal cells, immune-related cells, or differentiated cancer cells present around cancer stem cells affect the maintenance and proliferation of these cancer stem cell characteristics. Unlike ordinary cancer cells, under normal tumor growth conditions for cancer stem cells (the "normal tumor growth conditions" refer to a state in which the nutrients (glucose) required for cell growth are sufficient, the tumor microenvironment growth conditions are rich, and there is no cell stress), cancer stem cells can slowly proliferate, or may remain dormant, and thus may be resistant to anticancer drugs. For example, unlike the expression in normal tumor cells, the expression of transcriptional regulators may be controlled, so the function of the main metabolic regulatory substances may be different from that in ordinary cancer cells. Therefore, the term "cancer stem cell" generally refers to cells that acquire resistance to apoptosis under nutrient deprivation conditions and possess invasive and / or metastatic potential through this differential metabolic regulation and the regulation of cell signaling systems associated with this mechanism. However, cancer stem cells can include, but are not limited to, any cells that can differentiate into common cancer cells.

[0078] According to another aspect of the present disclosure, the present disclosure provides a composition comprising: the above-mentioned monoclonal antibody or a fragment thereof; a nucleic acid molecule encoding the monoclonal antibody or a fragment thereof; a vector comprising the nucleic acid molecule; or an inhibitor that inhibits the expression of the EPB41L5 gene.

[0079] According to a preferred embodiment of the present disclosure, the composition of the present disclosure can be used as a pharmaceutical composition for preventing or treating cancer or inhibiting cancer metastasis.

[0080] In the compositions disclosed herein, the contents related to antibodies or fragments thereof, nucleic acid molecules, cancer, cancer stem cells, etc. are the same as the contents of the above-mentioned epitopes, monoclonal antibodies or fragments thereof, etc. Therefore, their description is omitted to avoid excessive complexity of this specification.

[0081] The pharmaceutical composition of the present disclosure may comprise: (a) an antibody or a fragment thereof; a nucleic acid molecule encoding the antibody or a fragment thereof; a vector comprising the nucleic acid molecule; or an inhibitor that inhibits EPB41L5 gene expression; and (b) a pharmaceutically acceptable carrier.

[0082] The greatest feature of the pharmaceutical composition disclosed herein is that the pharmaceutical composition treats cancer or inhibits cancer metastasis by targeting the EPB41L5 gene or the protein encoded by it and inhibiting the expression level of the EPB41L5 gene or the expression or activity of the EPB41L5 protein.

[0083] The inhibitor disclosed herein can be one or more selected from siRNA (small interfering RNA), shRNA (short hairpin RNA), microribonucleic acid (miRNA), ribozyme, DNA enzyme, PNA (peptide nucleic acid) and antisense oligonucleotide. Preferably, the inhibitor is siRNA (small interfering RNA) that specifically binds to the mRNA of the gene. The siRNA, as an inhibitor of EPB41L5 gene expression, can have a nucleotide sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 5.

[0084] According to another aspect of the present disclosure, the present disclosure provides a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition.

[0085] In the method for preventing or treating cancer disclosed herein, the contents related to the pharmaceutical composition are the same as those described above regarding the pharmaceutical composition, and thus description thereof is omitted to avoid excessive complexity of the specification.

[0086] As used herein, the term "prevention" may include, but is not limited to, any effect in which symptoms caused by cancer are blocked, inhibited, or delayed by the application of the composition of the present disclosure.

[0087] As used herein, the term "treatment" may include, but is not limited to, any effect of the symptoms caused by the application of the composition of the present disclosure to alleviate or improve cancer. The pharmaceutically acceptable carriers included in the vaccine composition and pharmaceutical composition of the present disclosure are commonly used formulation carriers, examples of which include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl dihydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above-mentioned components, the pharmaceutical composition of the present disclosure may also include lubricants, wetting agents, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0088] The vaccine composition and pharmaceutical composition of the present disclosure can be administered orally or parenterally, preferably parenterally. For example, the composition can be administered by intravenous injection, local injection or intraperitoneal injection.

[0089] The applicable dosage of each vaccine composition and pharmaceutical composition disclosed herein depends on factors such as the formulation method, mode of administration, patient age, weight, sex, and pathological condition, diet, duration of administration, route of administration, excretion rate, and reaction sensitivity. A skilled physician can readily determine and prescribe an effective dosage for treatment or prevention. According to a preferred embodiment of the present disclosure, the daily dosage of each vaccine composition and pharmaceutical composition disclosed herein is 0.0001 to 100 mg / kg.

[0090] The vaccine composition and pharmaceutical composition of the present disclosure can be formulated into a unit dosage form or a multi-dose packaged formulation using pharmaceutically acceptable carriers and / or excipients according to methods readily administered by those skilled in the art. In this case, the formulation can be a solution, suspension, or emulsion in an oil or aqueous medium, or an extract, powder, granule, tablet, or capsule, and may further contain a dispersant or stabilizer.

[0091] Each vaccine composition and pharmaceutical composition disclosed herein can be used as a monotherapy, but can also be used in combination with other conventional chemotherapy or radiotherapy. When this combination therapy is performed, the treatment of cancer may be more effective. Chemotherapeutic drugs that can be used with the compositions disclosed herein include cisplatin, carboplatin, procarbazine, dichloromethyldiethylamine, cyclophosphamide, ifosfamide, melphalan, chloramphenicol, busulfan, nitrosoureas, dactinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide, tamoxifen, paclitaxel, new platinums, 5-fluorouracil, vincristine, vinblastine and methotrexate. Radiotherapy that can be used with the compositions disclosed herein includes X-ray radiation and gamma-ray radiation.

[0092] According to another aspect of the present disclosure, the present disclosure provides a method for providing information for diagnosing a disease caused by overexpression of EPB41L5.

[0093] In the present disclosure, the method includes the following steps: (a) obtaining a sample isolated from a subject in vitro; (b) treating the sample with a monoclonal antibody or a fragment thereof; and (c) determining whether the expression level of EPB41L5 contained in the sample from the subject is higher than the expression level of EPB41L5 contained in a normal sample.

[0094] In the method of providing information disclosed herein, the contents related to diseases caused by overexpression of EPB41L5, cancer, cancer stem cells, monoclonal antibodies, etc. are the same as those described above, and thus their description is omitted to avoid excessive complexity of the specification.

[0095] According to another aspect of the present disclosure, the present disclosure provides a method for quantifying EPB41L5 protein contained in a sample, the method comprising the step of treating the sample with a monoclonal antibody or a fragment thereof.

[0096] Since the monoclonal antibody or fragment thereof disclosed herein specifically binds to EPB41L5, the method disclosed herein can accurately measure the amount of EPB41L5 contained in a sample.

[0097] In the quantitative method according to the present disclosure, the contents related to diseases caused by overexpression of EPB41L5, cancer, cancer stem cells, monoclonal antibodies, etc. are the same as those described above, and thus their description is omitted to avoid excessive complexity of the specification.

[0098] According to another aspect of the present disclosure, the present disclosure provides a kit for quantifying EPB41L5 protein, wherein the kit comprises the monoclonal antibody or a fragment thereof.

[0099] The kit for quantification disclosed herein can be used to quantify EPB41L5 by analyzing an antigen against an antibody through an antigen-antibody binding reaction. The antigen-antibody binding reaction is preferably selected from, but not limited to, one of the following: conventional ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), immunosandwich method, Western blotting on polyacrylamide gel, immunoblotting, and immunohistochemical staining.

[0100] The carrier used for the antigen-antibody binding reaction of the present disclosure can be selected from, but not limited to, nitrocellulose membranes, PVDF membranes, well plates synthesized using polyethylene resins or polystyrene resins, and glass slides.

[0101] Secondary antibody is preferably labeled with conventional coloring agents for color development reaction. Specifically, any labeling agent selected from, but not limited to, such as fluorescein (such as HRP (horseradish peroxidase), alkaline phosphatase, colloidal gold, FITC (polylysine-fluorescein isothiocyanate) or RITC (rhodamine B isothiocyanate)) and dye composition can be used. Preferably, the substrate for inducing color development is selected with a labeling agent according to color development reaction. Specifically, substrate is preferably selected from, but not limited to, any one of TMB (3,3', 5,5'-tetramethylbenzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid] and OPD (o-phenylenediamine).

[0102] Beneficial effects

[0103] The features and advantages of the present disclosure are summarized as follows:

[0104] (1) The present disclosure provides a monoclonal antibody and a fragment thereof that recognizes EPB41L5 as an antigen and specifically binds thereto.

[0105] (2) The present disclosure can be used to identify antibodies that specifically bind to the EPB41L5 epitope, and the antibodies identified by the method inhibit EPB41L5 signaling, which enables the antibodies to be effectively used as vaccines or therapeutic drugs to combat EPB41L5-associated cancers, particularly gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1a The results of microarray analysis of gastric cancer patient specimens were shown, indicating that when the expression level of EPB41L5 was high, the prognosis of gastric cancer patients was poor. Figure 1a Figure 4 shows the results of oligonucleotide microarray analysis in gastric cancer patients without lymph node metastasis. Gastric cancer patients were divided into two groups based on the median expression level of the EPB41L5 gene. Figure 1b This is the analysis result of the 10-year survival rate of two groups of gastric cancer patients. Figure 1c Shown are the results of Kaplan-Meier plot analysis of two groups of gastric cancer patients.

[0107] Figure 2 shows data from various experiments performed to analyze whether EPB41L5 and mesenchymal gene expression are associated with TGF-β signaling. Figure 2a Shown are the results of Western blot analysis of various gastric cancer cells.

[0108] Figure 2b A bar graph showing the results of real-time qPCR analysis of EPB41L5 gene expression levels in gastric cancer cells treated with TGF-β1 (10 ng / ml) for 24 hours and in gastric cancer cells not treated with TGF-β1. The control group represents the EPB41L5 mRNA expression level in gastric cancer cells not treated with TGF-β1, while the TGF-β1 group represents the EPB41L5 mRNA expression level in gastric cancer cells treated with TGF-β1.

[0109] Figure 2c The results of analyzing the expression levels of EMT-related genes (EPB41L5, p-Smad2, Smad2, p-Smad3, Smad3, PAI-1, and β-actin) by Western blotting of gastric cancer cells treated with TGF-β1 (10 ng / ml) for 24 hours and gastric cancer cells not treated with TGF-β1 are shown.

[0110] Figure 2dShown are the results of analyzing the expression levels of endogenous EPB41L5 and E-cadherin in NCI-N87 and KATO III cells treated with TGF-β1 (10 ng / ml) for 24 hours, as well as in NCI-N87 and KATO III cells not treated with TGF-β1 (control). Here, green represents EPB41L5, red represents E-cadherin, and blue represents Hochest33258. The scale bar represents 20 μm.

[0111] Figure 2e Images showing the cell morphology of KATOIII and SNU719 cells treated with or without TGF-β1. Figure 2e Cell morphology was observed in the presence and absence of TGF-β1. White arrows indicate cell transformation into mesenchymal cells. KATOIII and SNU719 cells are shown. Scale bar represents 20 μm.

[0112] Figure 3 shows the results of experiments performed to demonstrate that Smad-dependent TGF-β signaling regulates EPB41L5 expression. Figure 3a Shown are the results of Western blot analysis of Smad4-deficient MKN45 and NCI-N87 cells treated with or without TGF-β1.

[0113] Figure 3b Shown are the results of immunofluorescence staining analysis of the expression levels of endogenous EPB41L5 and E-cadherin in NCI-N87 cells treated with or without TGF-β1. Green represents EPB41L5, red represents E-cadherin, and the scale bar represents 20 μm.

[0114] Figure 3c The graph shows the mRNA expression (fold induction) of Smad4, EPB41L5, Slug, and PAI-1 in KATO III cells treated with or without TGF-β1 or siSMAD4#1 or #2. The results are presented as mean ± sem, and the scale bar represents 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

[0115] Figure 3d Shown are the results of analyzing the expression levels of EMT-related genes (EPB41L5, p-Smad4, αPAI-1, αSlug, and α-tubulin) by Western blotting of KATOIII cells treated with TGF-β1 (10 ng / ml) or siSmad4#1 or #2 and untreated KATOIII cells.

[0116] Figure 4 Shown are the results of immunofluorescence staining for analysis of endogenous EPB41L5 and E-cadherin expression in untreated KATO III cells, KATO III cells treated with TGF-β1 alone, and KATO III cells treated with TGF-β1 followed by a TGF-β inhibitor (LY2157299). Green indicates EPB41L5, red indicates E-cadherin, and blue indicates Hochest 33258. Scale bar represents 20 μm.

[0117] Figure 5a Shown are the results of real-time qPCR of KATOIII cells transfected with NC siRNA and KATOIII cells transfected with EPB41L5 siRNA#1 or EPB41L5 siRNA#2 in the presence and absence of TGF-β1 to confirm that the metastasis of gastric cancer cells induced by EPB41L5 is regulated by TGF-β1 treatment.

[0118] Figure 5b Shown are the results of Western blot analysis of KATOIII cells transfected with NC siRNA and KATOIII cells transfected with EPB41L5 siRNA#1 or EPB41L5 siRNA#2 in the presence and absence of TGF-β1.

[0119] Figure 5c The results show the results of measuring morphological changes in NC siRNA-transfected KATO III cells, EPB41L5 siRNA #1- or EPB41L5 siRNA #2-transfected KATO III cells, and SNU719 cells in the presence and absence of TGF-β1. White arrows indicate cell transformation into mesenchymal cells, and the scale bar represents 20 μm.

[0120] Figure 5d Shown are the results of in vitro migration and invasion analysis of KATO III cells transfected with NC siRNA and EPB41L5 siRNA #1 or EPB41L5 siRNA #2 in the presence and absence of TGF-β1. Scale bar represents 20 μm.

[0121] Figure 5e Figure 2 shows in vitro migration and invasion analysis of KATO III cells transfected with NC (negative control) siRNA and EPB41L5 siRNA#1 or EPB41L5 siRNA#2 in the presence and absence of TGF-β1. Results are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

[0122] FIG6 shows the specificity of anti-EPB41L5 monoclonal antibodies. Figure 6a Shown are the results of Western blot analysis of MKN28 cells transfected with the Flag-EPB41L5 construct. Figure 6b Shown are the results of Western blot analysis of MKN28 cells transfected with EPB41L5 siRNA (#1 or #2).

[0123] Figure 6c The results of immunofluorescence staining of MKN28 cells transfected with EPB41L5 siRNA (EPB41L5 siRNA) are shown, where green represents EPB41L5.

[0124] Figure 6d The structure of the EPB41L5 domain is shown.

[0125] Figure 6e Shown are the results of Western blot analysis of 293T cells transfected with FL (full length) of EPB41L5, FERM of EPB41L5, and the C-terminus of EPB41L5, respectively.

[0126] Figure 6f Shown are the results of Western blot analysis of total lysates from 293T cells transfected with plasmids (1-633, 1-628, 1-623, 1-618, and 1-613) in which 5 amino acid residues at the C-terminus of EPB41L5 were deleted.

[0127] Figure 6g Shown are the results of Western blot analysis of total lysates from 293T cells transfected with plasmids consisting of FL (full length) of EPB41L5 and Δ619-624 of EPB41L5, respectively.

[0128] Figure 6h Shown are the results of immunofluorescence staining of MKN28 cells co-transfected with EPB41L5 siRNA and EPB41L5 FL (full-length), EPB41L5 FERM, EPB41L5 C-terminus, and EPB41L5 Δ619-624. Immunofluorescence staining was performed using anti-EPB41L5 monoclonal antibodies and anti-Flag antibodies. Green represents EPB41L5, red represents the Flag tag, and blue represents the host. Scale bar represents 20 μm.

[0129] Figure 7 shows images of cell migration ( Figure 7a ) and result analysis diagram ( Figure 7bKATO III cells were treated with various concentrations of anti-EPB41L5 monoclonal antibody in the presence and absence of TGF-β1 to confirm that TGF-β1-induced increases in gastric cancer cell invasion and metastasis were inhibited by anti-EPB41L5 monoclonal antibody. Data are presented as mean ± SEM. Scale bar represents 20 μm. **P < 0.01 and ***P < 0.001.

[0130] Figure 8 The results show a comparison of the extent to which the invasion and metastasis of gastric cancer cells increased by TGF-β1 are inhibited by the anti-EPB41L5 monoclonal antibody between the monoclonal antibody of the present disclosure and antibodies recognizing other epitopes.

[0131] FIG9 shows the results of experiments conducted to confirm the correlation between EPB41L5 and lung metastasis of gastric cancer cells. Figure 9a The results of in vitro migration and invasion assays of KATOIII cells overexpressing EPB41L5 and control cells (vector) are shown. Here, 1% FBS was used as a chemoattractant.

[0132] Figure 9b Shown are quantifications of in vitro metastasis and invasion assays of KATOIII cells overexpressing EPB41L5 and control cells (vector). Fifteen cells were randomly selected from each group and counted using Fusion-Capt-Advanced image acquisition and analysis software. Data are presented as mean ± sem.

[0133] Figure 9c Shown are the results obtained by injecting EPB41L5-overexpressing KATOIII cells (GFP-labeled) (EPB41L5(GFP)) into nude mice via the tail vein (10 mice per group) and analyzing the lung metastasis of the cells using an IVIS optical imaging system. Figure 9d Shows the use of the ROI tool to Figure 9c The image intensity was measured to quantify the results. Experimental data are expressed as mean radiant efficiency ± SEM. *P < 0.05 and **P < 0.01.

[0134] Figure 10a Schematic diagram showing that EPB41L5-overexpressing KATOIII cells were injected into nude mice via the tail vein, and 5 mg / kg of anti-EPB41L5 monoclonal antibody was injected into nude mice to prepare the (EPB41L5+mAb) group, which was injected once every 2 days for 2 weeks.

[0135] Figure 10bThe figures show the analysis results of the control group (vector group), the (EPB41L5) group (EPB41L5-overexpressing KATOIII cells were injected into nude mice via the tail vein), and the (EPB41L5+mAb) group (EPB41L5-overexpressing KATOIII cells were injected into nude mice via the tail vein, and 5 mg / kg of anti-EPB41L5 monoclonal antibody was injected into nude mice, once every 2 days for 2 weeks) by the IVIS optical imaging system.

[0136] Figure 10c Shows the ROI tool from Figure 9b The image intensity quantification results are shown in Figure 2. Experimental data are mean radiant efficiency ± SEM. **P < 0.01.

[0137] Figure 11 The results of Western blotting analysis using an EBP41L5 antibody to analyze EBP41L5 protein expression in gastric cancer, lung cancer, and breast cancer cells and the function of the antibody are shown.

[0138] Figure 12 Graphs showing the results of cell viability of gastric cancer, lung cancer, and breast cancer cells treated with different concentrations of EBP41L5 antibodies. DETAILED DESCRIPTION

[0139] According to one embodiment of the present disclosure, the present disclosure provides an epitope selected from the 619th to 624th amino acid residues of the EPB41L5 protein represented by the amino acid sequence of SEQ ID NO: 1; and a vaccine composition for preventing or treating cancer, comprising the epitope.

[0140] According to another embodiment of the present disclosure, the present disclosure provides a monoclonal antibody or a fragment thereof, comprising: a heavy chain variable region comprising a heavy chain CDR1 represented by SEQ ID NO: 6, a heavy chain CDR2 represented by SEQ ID NO: 7, and a heavy chain CDR3 represented by SEQ ID NO: 8; and a light chain variable region comprising a light chain CDR1 represented by SEQ ID NO: 9, a light chain CDR2 represented by SEQ ID NO: 10, and a light chain CDR3 represented by SEQ ID NO: 11.

[0141] According to another embodiment of the present disclosure, the present disclosure provides a composition for preventing or treating cancer, comprising a monoclonal antibody or a fragment thereof as an active ingredient.

[0142] Invention Solution

[0143] Hereinafter, the present disclosure will be described in more detail with reference to Examples. Obviously, for those skilled in the art, these Examples are only used to describe the present disclosure in more detail, and the scope of the present disclosure is not limited by these Examples.

[0144] Experimental methods

[0145] 1. Patient specimens

[0146] Gastric cancer patient specimens for oligonucleotide microarray and survival analysis were obtained from the Severance Hospital Clinical Trials Center (IRB number: 4-2016-0013).

[0147] 2. Cell Culture and Reagents

[0148] Human gastric cancer cell lines: AGS, NCI-N87, KATO 2 , SK-GT-4, MKN1, MKN28, MKN45, SNU1, SNU5, SNU16, SNU216, SNU484, SNU638, SNU668, and SNU719 were obtained from the laboratory of Professor Cheong Jae Ho at Yonsei University (Seoul, South Korea).

[0149] All gastric cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic / antimycotic solution (Corning, Manassas, VA, USA) at 37°C under 5% CO2. 293FT cells were cultured in Dulbecco's modified Eagle's medium (DMEM). TGF-β1 was purchased from Prospec (East Brunswick, NJ, USA), and LY2157299 was purchased from Selleckchem (Houston, TX, USA).

[0150] 3. Plasmid

[0151] DNA constructs encoding the full-length EPB41L5, the FERM domain, the C-terminus, Δ619–624, and the C-terminal five amino acids were generated by PCR and cloned into pSG5-KF2M1-Flag (Sigma-Aldrich, St. Louis, MI, USA). All plasmid constructs were verified by DNA sequencing.

[0152] 4. siRNA Transfection

[0153] Cells were transfected with siRNA using Lipofectamine RNAiMAX (ThermoFisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. The siRNAs used were synthesized by Genolution (Seoul, South Korea) and had the following siRNA sequences: EPB41L5-1 (EPB41L5 siRNA#1; siEPB41L5#1) (5′-GC AAUUGGCAGCUUAUAAUUU-3′), EPB41L5-2 (EPB41L5 siRNA#2; siEPB41L5#2) (5′-UUCAGAUUC GUGCCUAUUCAGUU-3′), Smad4-1 (5′-GCUACUUACCAUCAUAACAUU-3′), and Smad4-2 (5′-GUUCCAUUGCUUACUUUUUUUUU-3′).

[0154] 5. Monoclonal Antibody - Anti-EPB41L5 Monoclonal Antibody

[0155] Monoclonal antibodies were prepared by immunizing mice with amino acid residues 386 to 637 of the human EPB41L5 antigen (SEQ ID NO: 3). The production process was completed by ATgen (Seongnam, South Korea).

[0156] 1) Mouse immunization and hybridoma cell production

[0157] Monoclonal antibodies were prepared using amino acid residues 386 to 637 of the human EPB41L5 antigen (SEQ ID NO: 3). To efficiently prepare antibodies, SEQ ID NO: 3 was selected as the target peptide for antibody preparation.

[0158] The target peptide (SEQ ID NO: 3) for antibody production is injected into mice, and cells producing antibodies against the antigen are generated. Antibody-producing cells (B lymphocytes) obtained from the mouse spleen are fused with myeloma cells to produce hybridoma cells. The hybridoma cells are cultured in HAT medium, a medium in which only hybridoma cells can survive, and the antibody activity is measured using ELISA.

[0159] 2) Selection and identification

[0160] Among the hybridoma cells, monoclonal hybridoma cells that specifically recognize EPB41L5 are selected, the selected monoclonal hybridoma cells are injected into the peritoneal cavity of mice, and then the monoclonal antibodies are recovered from the ascites. The monoclonal antibodies of the present disclosure recovered from the ascites are purified using protein A and protein G columns.

[0161] Thereafter, monoclonal antibodies that specifically recognize EPB41L5 (EPB41L5 monoclonal antibodies) were screened and identified by Western blot analysis and immunofluorescence staining using the EPB41L5 construct and siRNA.

[0162] 6. Establishment of EPB41L5 Overexpression Stable Cell Line

[0163] EPB41L5 DNA was cloned into the pCDH-CMV-MCS-EF1-puro vector (Addgene, Cambridge, MA, USA). In 293FT cells, pCDH-EPB41L5 or pLECE3-GFP was transfected with the packaging plasmids pRSV-Rev and pMD2.G to prepare lentiviral particles. After 48 hours of culture, the supernatant was collected and filtered with a 0.45 μm filter. KATO2 cells were infected with the lentiviral particles and then screened with 1 μg / mL puromycin (Sigma-Aldrich). Selected stable EPB41L5 overexpressing cells were sorted using GFP using an Aria II flow cytometer (BD Biosciences, Sparks, MD, USA).

[0164] 7. Western Blot Analysis

[0165] The cells were lysed in lysis buffer (20 mmol / LTris-Cl, 150 mmol / LNaCl, 1% Triton X-100, 1.5% MgCl2, 1 mM EDTA, 1 mM Na2VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (pH 7.5)). The lysate was vortexed briefly and centrifuged at 13000 rpm for 20 minutes at 4°C. The supernatant was collected and transferred to a new test tube. Protein concentration was measured at 660 nm using a protein assay reagent (Thermo Fisher Scientific). Protein samples of equal concentration were prepared, and each sample was electrophoresed on an SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). The supernatant was lysed in a 4% (v / v) PBS containing 0.1% (v / v) Tween 20 (Sigma-Aldrich) and 5% (w / v) Difco TMThe membrane was blocked in Tris buffer (pH 7.4) with skim milk (BD Biosciences) and probed with primary antibodies. The following antibodies were used: polyclonal EPB41L5 (Thermo Fisher Scientific, Niton); EPB41L5 (ATGen, Seongnam, South Korea); Flag tag antibodies, β-actin (Sigma-Aldrich); α-tubulin (Abcam, Cambridge, GB), TβRI, TβRII, Smad2, Smad3, Smad4, p-Smad2, p-Smad3, Slug, ZEB1 (Cell Signaling, Danvers, MA, USA), and PAI-1 (Santa Cruz, Dallas, TX, USA). Signals were developed using substrates (Thermo Fisher Scientific, Niton) according to the manufacturer's instructions.

[0166] 8. RNA Isolation and Real-time Quantitative PCR

[0167] Total RNA was extracted using TRIzol reagent (Takara Bio, Otsu, Shiga, Japan), and cDNA was synthesized using PrimeScript reverse transcriptase (Takara Bio, Otsu, Shiga, Japan) and oligo(dT) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed using SYBR Green Master (Roche, Basel, Switzerland) and an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). Transcripts were detected using the following primers: α-tubulin (5′-TTCTCCATTTACCCGGCACC-3′) and (5′-GTTAGTGTAGGTTGGGCGCT-3′); EPB41L5 (5′-GAAAGAAGGCCCAGCAAACG-3′) and (5′-AGATCTCATCCCCCAAGCCT-3′); PAI-1 (5′-CCCCACTTCTTCAGGCTGTT-3′) and (5′-GCCGTTGAA The expression levels of the cDNAs were 5'-GTAGAGGGCAT-3'); Slug (5'-TCATCTTTGGGGCGAGTGAG-3') and (5'-TGCAGCTGCTTATGTTTGGC-3'); ZEB1 (5'-TATGAATGCCCAAACTGCAA-3') and (5'-TGGTGATGCTGAAAGAGACG-3'); and Smad4 (5'-TGCATGACTTTGAGGGACAG-3') and (5'-GTGGAAGCCACAGGAATGTT-3'). The amount of cDNA was compared with α-tubulin. All experiments were performed in triplicate, and relative expression levels and standard deviations were calculated using the comparative method.

[0168] 9. Immunofluorescence Analysis

[0169] Cells were grown on glass slides (SPL Life Sciences, Pocheon, South Korea) and fixed in 4% paraformaldehyde for 30 minutes at room temperature. Fixed cells were washed with PBS and incubated with 3% BSA for 1 hour to block nonspecific antibodies. Anti-EPB41L5, anti-E-cadherin, and anti-Flag tags were incubated overnight at 4°C and then stained with Alexa-Fluor 488- or Alexa-Fluor 549-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific). Cell nuclei were stained with Hoechst 33258. Samples were imaged using an LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).

[0170] 10. In vitro metastasis and invasion assays

[0171] Cell migration was measured by Transwell using 8.0 μm pore polycarbonate membrane inserts (Corning, Manassas, VA, USA). For invasion assays, the inserts in the Transwell were coated with Matrigel (BD Biosciences). 1 × 10 5 Cells were plated in the lower chamber and filled with 600 μL of serum-free medium with or without the addition of TGF-β1, LY2157299 (TGF-β inhibitor), or anti-EPB41L5 monoclonal antibody (mAb) as a chemoattractant. After 24 hours of culture, non-metastatic or non-invasive cells were carefully removed from the upper chamber using a cotton swab. Migrating or invasive cells were stained with 0.2% crystal violet (Sigma-Aldrich) in 20% methanol and counted under a microscope at ×200 magnification. Migration and invasion assays were performed in triplicate.

[0172] 11. In vivo metastasis assay

[0173] Five-week-old athymic BALB / c nu / nu mice were obtained from Orient (Seoul, South Korea). KATO2 cells (containing 2 × 10 7 The cells were then injected into the lateral tail vein (200 μL of PBS solution). Anti-EPB41L5 monoclonal antibody was administered at 5 mg / kg daily for 2 weeks. Fluorescence images were acquired and analyzed using an IVIS imaging system (Caliper Life Sciences, Hopkinton, MA, USA).

[0174] 12. Statistical Analysis

[0175] Overall survival was analyzed using the Kaplan-Meier plotter (http: / / kmplot.com / analysis) using Affymetrix ID 220977_x_at. Kaplan-Meier survival plots were analyzed using the log-rank test. Statistical analysis was performed using the two-sided t-test to compare two independent groups. Data are presented as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

[0176] Example

[0177] Example 1 Analysis of EPB41L5 gene expression levels in gastric cancer patient specimens

[0178] To identify potential target molecules in GC, oligonucleotide microarray analysis was performed on 78 gastric cancer patients without lymph node metastasis. The patients were divided into two groups (Group 1 and Group 2) based on the median expression level of the EPB41L5 gene. In terms of the median expression level of the EPB41L5 gene, Group 1 was located on the left and Group 2 was located on the right.

[0179] It was confirmed that the recurrence rate or mortality rate of the first group with overexpression of APEG1, SMPX, GPR177 and EPB41L5 genes was higher than that of the second group with low gene expression levels ( Figure 1a Among APEG1, SMPX, GPR177 and EPB41L5 genes, EPB41L5 was selected as a target protein for antibody-based tumor therapy. According to the median expression level of EPB41L5 gene ( Figure 1b )Analysis of the 10-year survival rates of the two groups showed that the survival rate of patients with high expression levels of the EPB41L5 gene was lower than that of patients with low expression levels of the EPB41L5 gene.

[0180] In addition, by performing Kaplan-Meier curve analysis on two groups of cancer patients (876 gastric cancer patients), it was confirmed in the Kaplan-Meier curve using databases such as GEO, EGA or TCGA that gastric cancer patients with high EPB41L5 expression had a lower survival rate (HR 1.73, P2.8E-10) ( Figure 1c These clinical results indicate that high expression levels of EPB41L5 are associated with poor prognosis in gastric cancer patients. In other words, gastric cancer patients with high expression of the EPB41L5 gene have a lower survival rate.

[0181] Example 2 Analysis of TGF-β1 and EPB41L5 protein expression patterns in gastric cancer cells

[0182] The present inventors examined which gastric cancer cells responded to TGF-β signaling. Protein levels associated with the TGF-β signaling cascade were analyzed by Western blot analysis of several gastric cancer cell lines ( Figure 2a The results confirmed that Smad3 or Smad4 were not detected in NCI-N87, MKN45, SNU216, and SNU484 cells.

[0183] Next, gastric cancer cells were treated with TGF-β1 to examine whether EPB41L5 gene expression is regulated by TGF-β signaling. The results confirmed that TGF-β1 treatment significantly increased the expression level of EPB41L5 gene in KATOIII, MKN28, SNU1, and SNU719 cells, but not in Smad3 / Smad4-deficient GC cells ( Figure 2bFurthermore, the expression of mesenchymal genes such as PAI-1, Slug, and phosphorylated Smad2 and Smad3 was significantly increased by TGF-β1 treatment ( Figure 2c ).

[0184] According to the results of immunofluorescence staining analysis ( Figure 2d ) confirmed that the expression of the epithelial marker E-cadherin in the transmembrane was reduced, and TGF-β1 in the cell membrane of KATOIII increased the expression level of EPB41L5 protein. In addition, the morphological characteristics of gastric cancer cells KATOIII and SNU719 changed to mesenchymal characteristics after TGF-β1 treatment ( Figure 2e ).

[0185] These results indicate that TGF-β signaling regulates the expression levels of the EPB41L5 gene and its encoded protein and epithelial-mesenchymal transition in cancer cells, such as gastric cancer cells.

[0186] Example 3 Regulation of EPB41L5 protein expression levels by Smad-dependent TGF-β signaling

[0187] To examine whether the TGF-β1-induced increase in EPB41L5 protein expression levels is Smad-dependent, we performed Western blot analysis and immunofluorescence staining in Smad4-deficient MKN45 (Smad4 knockout MKN45) and NCI-N87 cells (gastric cancer). It was confirmed that the levels of these genes and EPB41L5 protein were not altered in each cell line treated with TGF-β1 ( Figure 3a and 3b ). In addition, it was confirmed that EPB41L5, PAI-1, and Slug were upregulated in each type of cells treated with TGF-β1, and that the expression of EPB41L5, PAI-1, and Slug was inhibited in MKN28 cells in which Smad4 was knocked down using siRNA ( Figure 3c and 3d ).

[0188] Taken together these results, it can be seen that TGF-β signaling is dependent on Smad, indicating that TGF-β signaling is involved in the regulation of EPB41L5 expression in gastric cancer cells.

[0189] Example 4 Correlation Analysis between TGF-β1 and EPB41L5 Protein

[0190] The present disclosure analyzes changes in EPB41L5 protein expression levels after treatment with LY2157299 (Galunisertib), a potent TGF-β receptor I inhibitor.

[0191] Figure 4Shown are the results of immunofluorescence staining for analyzing the expression of endogenous EPB41L5 and E-cadherin in untreated KATO III cells, KATO III cells treated only with TGF-β1, and KATO III cells treated with TGF-β1 followed by a TGF-β inhibitor (LY2157299). Green indicates EPB41L5, red indicates E-cadherin, and blue indicates Hochest 33258. Scale bar represents 20 μm.

[0192] like Figure 4 As shown, immunofluorescence staining of KATOIII cells treated with TGF-β1 alone and sequentially treated with TGF-β1 and LY2157299 showed that TGF-β1 increased the expression level of EPB41L5 protein, but this was inhibited by LY2157299.

[0193] Example 5 Analysis of the effect of EPB41L5 on gastric cancer cell metastasis

[0194] The present disclosure verifies whether EPB41L5 affects the response of gastric cancer cells to TGF-β signaling. To this end, KATO III cells or SNU719 cells were transfected with EPB41L5 siRNA. Specifically, two types of siRNA (EPB41L5 siRNA #1 (SEQ ID NO 4: 5'-GCAAUUGGCAGCUUAUAAUUU-3') and EPB41L5 siRNA #2 (SEQ ID NO 5: 5'-UUCAGAUUC GUGCCUAUUCAGUU-3')) were used to knock down EPB41L5. It was confirmed that the gene and protein levels of EPB41L5 and PAI-1 were significantly reduced in KATO III cells transfected with EPB41L5 siRNA #1 or EPB41L5 siRNA #2 compared to KATO III cells transfected with NC siRNA ( Figure 5a and 5b However, no changes in phosphorylated Smad3 (αp-Smad3) and Smad3 (αSmad3) were observed in KATOIII cells transfected with EPB41L5siRNA#1 or EPB41L5siRNA#2 ( Figure 5b ).

[0195] In addition, by analyzing the results of epithelial-mesenchymal transition and gastric cancer cell metastasis in KATOIII cells transfected with NC siRNA and KATOIII cells transfected with EPB41L5 siRNA#1 or EPB41L5 siRNA#2 after treatment with TGF-β1, it was confirmed that knockout of EPB41L5 could block the effect of TGF-β1 on epithelial-mesenchymal transition and gastric cancer cell metastasis ( Figure 5c 、 5d and 5e). In other words, EPB41L5 is an important factor in TGF-β-induced metastasis and migration of gastric cancer cells.

[0196] Example 6 Anti-EPB41L5 mAb (monoclonal antibody) recognizes the C-terminal 619-624 amino acid sequence

[0197] The present disclosure considers that since cell adhesion molecules play an important role in metastasis, new therapeutic drugs can be developed using monoclonal antibodies and peptides. Therefore, a monoclonal antibody (mAb) against EPB41L5 was developed.

[0198] To verify the specificity of the developed antibody, gastric cancer cells transfected with Flag-EPB41L5 or EPB41L5 siRNA were analyzed by Western blot analysis and immunofluorescence analysis. The results confirmed that overexpression of EPB41L5 was detected in gastric cancer cells transfected with Flag-EPB41L5 ( Figure 6a ), and EPB41L5 was silenced in gastric cancer cells transfected with EPB41L5 siRNA (NC, siRNA#1, or siRNA#2). Figure 6b Furthermore, the results of immunofluorescence staining analysis of gastric cancer cells transfected with EPB41L5 siRNA showed that EPB41L5 was clearly detected in gastric cancer cells by anti-EPB41L5 monoclonal antibody ( Figure 6c ). The anti-EPB41L5 monoclonal antibody of the present disclosure was analyzed by Western blotting, and the results confirmed that the anti-EPB41L5 monoclonal antibody recognized the C-terminus of EPB41L5, rather than the N-terminal FERM domain of EPB41L5 ( Figure 6d and 6e ).

[0199] Furthermore, by analyzing the antibody with the five amino acids at the C-terminus of EPB41L5 sequentially removed, it was confirmed that the antibody recognized the 619th to 624th amino acids at the C-terminus of EPB41L5 ( Figure 6f and 6gConsidering the characteristics of non-permeability immunofluorescence staining, in order to inhibit the expression of endogenous EPB41L5, any plasmid selected from EPB41L5 FL (full length), EPB41L5 FERM, EPB41L5 C-terminus, and EPB41L5 △619~624 was co-transfected with EPB41L5 siRNA into cells. The expression of exogenous EPB41L5 was analyzed using anti-Flag antibody. EPB41L5 expression was detected in FK cells transfected with FL and C-terminus constructs and in MKN28 cells not transfected with △619~624 ( Figure 6h ). Based on these results, it can be seen that the anti-EPB41L5 monoclonal antibody specifically recognizes the 619-624 amino acid region of EPB41L5.

[0200] Example 7 Determination of variable region sequence of anti-EPB41L5 monoclonal antibody

[0201] The sequences of the variable regions of the monoclonal antibodies produced in Example 6 were analyzed by ATgen (Korea). As a result, the sequences of the heavy chain variable regions and light chain variable regions of the monoclonal antibodies of the present disclosure were as follows:

[0202] Heavy chain variable region

[0203] (SEQ ID NO: 12)

[0204] QVQLKESGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTSYNQKFKDKAKLTAVTSTSTAYMELSSLTDEASAVYYCTRGGKLPFAMDYWGQGTSVTVSS

[0205] Light chain variable region

[0206] (SEQ ID NO: 13)

[0207] DVLMTQTPLSLPVSLGDQASMSCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0208] In addition, the sequences of the heavy chain variable region CDR1 to CDR3 and the light chain variable region CDR1 to CDR3 of the monoclonal antibody of the present disclosure are as follows:

[0209] Heavy chain variable region CDR1: Gly Tyr Thr Phe Thr Ser Tyr Trp (SEQ ID NO: 6)

[0210] Heavy chain variable region CDR2: Ile TyrPro GlyAsn SerAsp (SEQ ID NO: 7)

[0211] Heavy chain variable region CDR3: ThrArg Gly Gly Lys LeuPro PheAlaMetAsp Tyr (SEQ ID NO: 8)

[0212] Light chain variable region CDR1: Gln Ser LeuVal His Ser Asn GlyAsn Thr Tyr (SEQ ID NO: 9)

[0213] Light chain variable region CDR2: Lys Val Ser (SEQ ID NO: 10)

[0214] Light chain variable region CDR3: Ser Gln SerThr His Val Pro Trp Thr (SEQ ID NO: 11)

[0215] Example 8 Therapeutic Effect of Anti-EPB41L5 Monoclonal Antibody

[0216] The present disclosure validates the specificity of anti-EPB41L5 monoclonal antibodies. In this experiment, by evaluating the effect of anti-EPB41L5 monoclonal antibodies on the metastasis of gastric cancer (GC) cells with or without TGF-β1 treatment, it was confirmed that TGF-β1 can effectively promote the metastasis of KATOIII. In addition, it was confirmed that anti-EPB41L5 monoclonal antibodies inhibited the metastasis of KATOIII cells increased by TGF-β1 ( Figure 7a and 7b ).

[0217] Example 9 Comparison of Metastasis Inhibitory Effects of Anti-EPB41L5 Monoclonal Antibodies

[0218] In the same manner as in Example 8, the extent to which anti-EPB41L5 monoclonal antibodies inhibited cell metastasis was compared between different epitope sequences recognized by the monoclonal antibodies. Figure 8 As shown, it was confirmed that the monoclonal antibody of the present disclosure (AtGen) significantly (100%) inhibited cell metastasis increased by TGF-β1, while the antibody (Novus antibody) that recognizes the 638-708 amino acid sequence of the EPB41L5 protein was almost unable to inhibit cell metastasis increased by TGF-β1. Therefore, it can be seen that the antibody that recognizes the 619-624 amino acid sequence of the C-terminal region of EPB41L5 can very effectively inhibit cell metastasis increased by TGF-β1 compared to antibodies that specifically bind to epitopes composed of other amino acid sequences.

[0219] Example 10 Correlation Analysis between EPB41L5 and Gastric Cancer Cell Metastasis

[0220] The above experiments confirmed that the increased expression of EPB41L5 due to TGF-β1 is involved in the metastasis and invasion of gastric cancer cells in vivo. Therefore, in the present disclosure, the effect of EPB41L5 on the metastasis of gastric cancer cells was analyzed by in vitro metastasis and invasion assays.

[0221] Specifically, the present disclosure prepared KATOIII cells overexpressing EPB41L5 as a vector and untreated KATOIII cells.

[0222] By analyzing the in vitro migration and invasion assays of EPB41L5-overexpressing KATOIII cells, it was confirmed that the migration and invasion of EPB41L5-overexpressing KATOIII cells were significantly increased ( Figure 9a and 9b To observe the expression of EPB41L5 in gastric cancer cells, KATOIII cells overexpressing EPB41L5 were injected into nude mice via the tail vein. The results showed that the lung metastasis of gastric cancer cells in the EPB41L5 (GFP) group was significantly increased compared with the vector (GFP) group ( Figure 9c and 9d ). In summary, EPB41L5 can promote the metastasis of gastric cancer cells in vivo.

[0223] Example 11 Detection of the ability of anti-EPB41L5 monoclonal antibody to inhibit gastric cancer cell metastasis

[0224] The in vivo efficacy of the anti-EPB41L5 monoclonal antibody was tested. To this end, the following groups were prepared: a vector group; an (EPB41L5) group, in which EPB41L5-overexpressing KATOIII cells were injected into nude mice via the tail vein; and an (EPB41L5+mAb) group, in which EPB41L5-overexpressing KATOIII cells were injected into nude mice via the tail vein and 5 mg / kg of the anti-EPB41L5 monoclonal antibody was injected into the nude mice every 2 days for 2 weeks. Figure 10a ).

[0225] It was confirmed that in nude mice injected with EPB41L5-overexpressing cells, lung metastasis of cancer increased, but in the group injected with anti-EPB41L5 monoclonal antibody, lung metastasis of gastric cancer cells was reduced compared with the vehicle group ( Figure 10b and 10c ). Therefore, it can be seen that the anti-EPB41L5 monoclonal antibody disclosed in the present invention has a therapeutic or preventive effect on advanced gastric cancer.

[0226] Example 12 Confirmation of the Function of Anti-EPB41L5 Monoclonal Antibodies That Specifically Recognize EBP41L5 Protein

[0227] To confirm the specificity of the anti-EPB41L5 monoclonal antibody for EBP41L5, we generated KATOIII and MKN28 cells as gastric cancer cell lines. Lung cancer cell lines A549 and H226, and breast cancer cell lines MCF7 and MDA-MB-231 were also generated. These cells were obtained from Professor Cheong Jae Ho's laboratory at Yonsei University and the Korean Cell Bank. Each cell line was cultured at 37°C in 5% CO2 in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic / antimycotic solution (Corning, Manassas, VA, USA).

[0228] The expression of EBP41L5 protein in each cell group was analyzed by Western blotting using anti-EPB41L5 monoclonal antibody ( Figure 11 As a result, EBP41L5 protein expression was detected not only in gastric cancer cell lines but also in lung cancer cell lines and breast cancer cell lines by anti-EPB41L5 monoclonal antibody ( Figure 11 This indicates that the anti-EPB41L5 monoclonal antibody according to the present disclosure can be effectively used to treat or prevent not only gastric cancer, but also lung cancer and breast cancer.

[0229] Example 13 Cell-specific toxicity detection of anti-EPB41L5 monoclonal antibody

[0230] To examine the effects of the disclosed anti-EPB41L5 monoclonal antibodies on the cellular activity of various cells, we prepared KATO III and MKN28 cells as gastric cancer cell lines. Lung cancer cell lines A549 and H226, and breast cancer cell lines MCF7 and MDA-MB-231 were also prepared. These cells were obtained from Professor Cheong Jae Ho's laboratory at Yonsei University and the Korea Cell Bank.

[0231] Each cell line was cultured at 37° C. and 5% CO 2 in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic / antimycotic solution (Corning, Manassas, VA, USA).

[0232] 0, 2, 4 or 6 μg of anti-EPB41L5 monoclonal antibody was injected into each group of cells, and after 24 hours, the cell viability (%) of each group of cells was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method ( Figure 12 The results confirmed that when the anti-EPB41L5 monoclonal antibody was injected, the cell viability decreased in a concentration-dependent manner, indicating that the anti-EPB41L5 monoclonal antibody disclosed herein exhibits a therapeutic or preventive effect on advanced cancer or gastric cancer ( Figure 12 ).

[0233] in conclusion

[0234] Cell adhesion proteins play a key role in tumor metastasis. Therefore, the possibility of developing anti-tumor metastasis drugs using cell adhesion protein antibodies has been discussed. The present inventors have worked hard to develop a new therapy based on a new adhesion molecule or complex related to metastasis, thereby completing the present disclosure. In EMT (epithelial-mesenchymal transition), epithelial cells lose intercellular connections and cell polarity, and the actin cytoskeleton is reorganized to achieve a mesenchymal phenotype. It is reported that EPB41L5 interacts with p120-catenin (which destroys E-cadherin) and paxillin (paxillin focal adhesion kinase). In addition, EPB41L5 is known to bind to MPP5, a Crumbs complex component that negatively regulates cell polarity. Another connexin, β-catenin, co-localizes with EPB41L5 on the basolateral membrane of renal epithelial cells, but its binding has not yet been confirmed. FAK has a FERM domain at the N-terminus, which binds to ASAP1 / AMAP1 and paxillin (another binding partner of C-terminal EPB41L5).

[0235] The present disclosure has found that EPB41L5 is associated with poor prognosis in gastric cancer patients. EPB41L5 has been shown to be highly expressed in cancer cells. Furthermore, since TGF-β promotes gastric cancer metastasis, it is hypothesized that TGF-β expression is associated with lymph node metastasis and prognosis in gastric cancer. Analysis of the correlation between TGF-β and EPB41L5 revealed a large number of Smad binding motifs within the EPB41L5 gene promoter.

[0236] Therefore, the present disclosure confirms that knockout of EPB41L5 abolishes TGF-β1-induced mesenchymal transition and increased GC cell metastasis, indicating that EPB41L5 is a major factor in TGF-β signaling. In summary, EPB41L5 gene knockout can regulate the metastasis and invasion of gastric cancer cells caused by epithelial-mesenchymal transition through the TGF-β / Smad3 / EPB41L5 pathway.

[0237] The present disclosure demonstrates that EPB41L5 is located on the cell surface and promotes the metastasis of gastric cancer cells in vitro and in vivo, suggesting that antibodies that specifically bind to EPB41L5 can be used as therapeutic monoclonal antibodies. Therefore, the present invention developed an EPB41L5 monoclonal antibody. The EPB41L5 monoclonal antibody was shown to effectively block TGF-β1-induced metastasis and invasion of gastric cancer cells and significantly inhibit lung metastasis in EPB41L5-overexpressing cells.

[0238] Furthermore, it was demonstrated that the anti-EPB41L5 monoclonal antibodies of the present disclosure significantly inhibited EPB41L5 protein expression in gastric cancer, lung cancer, and breast cancer cell lines (especially gastric cancer cell lines), and reduced the viability of cancer cells.

[0239] Therefore, it can be seen that the EPB41L5 monoclonal antibody disclosed in the present invention is a very effective method for preventing, treating or inhibiting cancer metastasis, especially gastric cancer metastasis.

[0240] Although the present disclosure has been described in detail with reference to specific features, it is obvious to those skilled in the art that the description of the present disclosure is only its preferred embodiment and does not limit the scope of the present disclosure. Therefore, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.

[0241] Industrial Applicability

[0242] The features and advantages of the present disclosure are summarized as follows:

[0243] (1) The present disclosure provides a monoclonal antibody and a fragment thereof that recognizes EPB41L5 as an antigen and specifically binds thereto.

[0244] (2) The present disclosure can be used to identify antibodies that specifically bind to the EPB41L5 epitope, and the antibodies identified by the method have the function of inhibiting EPB41L5 signaling, and the function enables the antibodies of the present disclosure to be effectively used as vaccines or therapeutic drugs against EPB41L5-related cancers (particularly gastric cancer). <110> Sellast Ltd. <120> An epitope and monoclonal antibody of EPB41L5 <130> POPB187366PCT <150> KR 10-2018-0093045 <151> 2018-08-09 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 733 <212> PRT <213> Artificial sequence <220> <223> EPB41L5 protein <400> 1 Met Leu Ser Phe Phe Arg Arg Thr Leu Gly Arg Arg Ser Met Arg Lys 1 5 10 15 His Ala Glu Lys Glu Arg Leu Arg Glu Ala Gln Arg Ala Ala Thr His 20 25 30 Ile Pro Ala Ala Gly Asp Ser Lys Ser Ile Ile Thr Cys Arg Val Ser 35 40 45 Leu Leu Asp Gly Thr Asp Val Ser Val Asp Leu Pro Lys Lys Ala Lys 50 55 60 Gly Gln Glu Leu Phe Asp Gln Ile Met Tyr His Leu Asp Leu Ile Glu 65 70 75 80 Ser Asp Tyr Phe Gly Leu Arg Phe Met Asp Ser Ala Gln Val Ala His 85 90 95 Trp Leu Asp Gly Thr Lys Ser Ile Lys Lys Gln Val Lys Ile Gly Ser 100 105 110 Pro Tyr Cys Leu His Leu Arg Val Lys Phe Tyr Ser Ser Glu Pro Asn 115 120 125 Asn Leu Arg Glu Glu Leu Thr Arg Tyr Leu Phe Val Leu Gln Leu Lys 130 135 140 Gln Asp Ile Leu Ser Gly Lys Leu Asp Cys Pro Phe Asp Thr Ala Val 145 150 155 160 Gln Leu Ala Ala Tyr Asn Leu Gln Ala Glu Leu Gly Asp Tyr Asp Leu 165 170 175 Ala Glu His Ser Pro Glu Leu Val Ser Glu Phe Arg Phe Val Pro Ile 180 185 190 Gln Thr Glu Glu Met Glu Leu Ala Ile Phe Glu Lys Trp Lys Glu Tyr 195 200 205 Arg Gly Gln Thr Pro Ala Gln Ala Glu Thr Asn Tyr Leu Asn Lys Ala 210 215 220 Lys Trp Leu Glu Met Tyr Gly Val Asp Met His Val Val Lys Ala Arg 225 230 235 240 Asp Gly Asn Asp Tyr Ser Leu Gly Leu Thr Pro Thr Gly Val Leu Val 245 250 255 Phe Glu Gly Asp Thr Lys Ile Gly Leu Phe Phe Trp Pro Lys Ile Thr 260 265 270 Arg Leu Asp Phe Lys Lys Asn Lys Leu Thr Leu Val Val Val Glu Asp 275 280 285 Asp Asp Gln Gly Lys Glu Gln Glu His Thr Phe Val Phe Arg Leu Asp 290 295 300 His Pro Lys Ala Cys Lys His Leu Trp Lys Cys Ala Val Glu His His 305 310 315 320 Ala Phe Phe Arg Leu Arg Gly Pro Val Gln Lys Ser Ser His Arg Ser 325 330 335 Gly Phe Ile Arg Leu Gly Ser Arg Phe Arg Tyr Ser Gly Lys Thr Glu 340 345 350 Tyr Gln Thr Thr Lys Thr Asn Lys Ala Arg Arg Ser Thr Ser Phe Glu 355 360 365 Arg Arg Pro Ser Lys Arg Tyr Ser Arg Arg Thr Leu Gln Met Lys Ala 370 375 380 Cys Ala Thr Lys Pro Glu Glu Leu Ser Val His Asn Asn Val Ser Thr 385 390 395 400 Gln Ser Asn Gly Ser Gln Gln Ala Trp Gly Met Arg Ser Ala Leu Pro 405 410 415 Val Ser Pro Ser Ile Ser Ser Ala Pro Val Pro Val Glu Ile Glu Asn 420 425 430 Leu Pro Gln Ser Pro Gly Thr Asp Gln His Asp Arg Lys Cys Ile Pro 435 440 445 Leu Asn Ile Asp Leu Leu Asn Ser Pro Asp Leu Leu Glu Ala Thr Ile 450 455 460 Gly Asp Val Ile Gly Ala Ser Asp Thr Met Glu Thr Ser Gln Ala Leu 465 470 475 480 Asn Asp Val Asn Val Ala Thr Arg Leu Pro Gly Leu Gly Glu Pro Glu 485 490 495 Val Glu Tyr Glu Thr Leu Lys Asp Thr Ser Glu Lys Leu Lys Gln Leu 500 505 510 Glu Met Glu Asn Ser Pro Leu Leu Ser Pro Arg Ser Asn Ile Asp Val 515 520 525 Asn Ile Asn Ser Gln Glu Glu Val Val Lys Leu Thr Glu Lys Cys Leu 530 535 540 Asn Asn Val Ile Glu Ser Pro Gly Leu Asn Val Met Arg Val Pro Pro 545 550 555 560 Asp Phe Lys Ser Asn Ile Leu Lys Ala Gln Val Glu Ala Val His Lys 565 570 575 Val Thr Lys Glu Asp Ser Leu Leu Ser His Lys Asn Ala Asn Val Gln 580 585 590 Asp Ala Ala Thr Asn Ser Ala Val Leu Asn Glu Asn Asn Val Pro Leu 595 600 605 Pro Lys Glu Ser Leu Glu Thr Leu Met Leu Ile Thr Pro Ala Asp Ser 610 615 620 Gly Ser Val Leu Lys Glu Ala Thr Asp Glu Leu Asp Ala Leu Leu Ala 625 630 635 640 Ser Leu Thr Glu Asn Leu Ile Asp His Thr Val Ala Pro Gln Val Ser 645 650 655 Ser Thr Ser Met Ile Thr Pro Arg Trp Ile Val Pro Gln Ser Gly Ala 660 665 670 Met Ser Asn Gly Leu Ala Gly Cys Glu Met Leu Leu Thr Gly Lys Glu 675 680 685 Gly His Gly Asn Lys Asp Gly Ile Ser Leu Ile Ser Pro Pro Ala Pro 690 695 700 Phe Leu Val Asp Ala Val Thr Ser Ser Gly Pro Ile Leu Ala Glu Glu 705 710 715 720 Ala Val Leu Lys Gln Lys Cys Leu Leu Thr Thr Glu Leu 725 730 <210> 2 <211> 6 <212> PRT <213> Artificial sequence <220> <223> EPB41L5 epitope <400> 2 Ile Thr Pro Ala Asp Ser 1 5 <210> 3 <211> 270 <212> PRT <213> Artificial sequence <220> <223> Human EPB41L5 antibody amino acid residues 386-637 <400> 3 Glu Arg Arg Pro Ser Lys Arg Tyr Ser Arg Arg Thr Leu Gln Met Lys 1 5 10 15 Ala Cys Ala Thr Lys Pro Glu Glu Leu Ser Val His Asn Asn Val Ser 20 25 30 Thr Gln Ser Asn Gly Ser Gln Gln Ala Trp Gly Met Arg Ser Ala Leu 35 40 45 Pro Val Ser Pro Ser Ile Ser Ser Ala Pro Val Pro Val Glu Ile Glu 50 55 60 Asn Leu Pro Gln Ser Pro Gly Thr Asp Gln His Asp Arg Lys Cys Ile 65 70 75 80 Pro Leu Asn Ile Asp Leu Leu Asn Ser Pro Asp Leu Leu Glu Ala Thr 85 90 95 Ile Gly Asp Val Ile Gly Ala Ser Asp Thr Met Glu Thr Ser Gln Ala 100 105 110 Leu Asn Asp Val Asn Val Ala Thr Arg Leu Pro Gly Leu Gly Glu Pro 115 120 125 Glu Val Glu Tyr Glu Thr Leu Lys Asp Thr Ser Glu Lys Leu Lys Gln 130 135 140 Leu Glu Met Glu Asn Ser Pro Leu Leu Ser Pro Arg Ser Asn Ile Asp 145 150 155 160 Val Asn Ile Asn Ser Gln Glu Glu Val Val Lys Leu Thr Glu Lys Cys 165 170 175 Leu Asn Asn Val Ile Glu Ser Pro Gly Leu Asn Val Met Arg Val Pro 180 185 190 Pro Asp Phe Lys Ser Asn Ile Leu Lys Ala Gln Val Glu Ala Val His 195 200 205 Lys Val Thr Lys Glu Asp Ser Leu Leu Ser His Lys Asn Ala Asn Val 210 215 220 Gln Asp Ala Ala Thr Asn Ser Ala Val Leu Asn Glu Asn Asn Val Pro 225 230 235 240 Leu Pro Lys Glu Ser Leu Glu Thr Leu Met Leu Ile Thr Pro Ala Asp 245 250 255 Ser Gly Ser Val Leu Lys Glu Ala Thr Asp Glu Leu Asp Ala 260 265 270 <210> 4 <211> 21 <212> RNA <213> artificial sequence <220> <223> EPB41L5 siRNA (EPB41L5 siRNA#1) <400> 4 gcaauuggca gcuuauaau u 21 <210> 5 <211> 23 <212> RNA <213> artificial sequence <220> <223> EPB41L5 siRNA (EPB41L5 siRNA#2) <400> 5 uucagauucg ugccuauuca guu 23 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 <400> 6 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 <400> 7 Ile Tyr Pro Gly Asn Ser Asp 1 5 <210> 8 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 <400> 8 Thr Arg Gly Gly Lys Leu Pro Phe Ala Met Asp Tyr 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 <400> 9 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 10 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 <400> 10 Lys Val Ser 1 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 <400> 11 Ser Gln Ser Thr His Val Pro Trp Thr 1 5 <210> 12 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 12 Gln Val Gln Leu Lys Glu Ser Gly Thr Val Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Asp Glu Ala Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Gly Lys Leu Pro Phe Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 13 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 13 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Met Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody recognizes the EPB41L5 protein as an antigen and specifically binds thereto, wherein the amino acid sequence of the EPB41L5 protein is as shown in SEQ ID NO: 1, and the monoclonal antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region comprising a heavy chain CDR1 represented by SEQ ID NO: 6, a heavy chain CDR2 represented by SEQ ID NO: 7, and a heavy chain CDR3 represented by SEQ ID NO: 8; and A light chain variable region comprising a light chain CDR1 represented by SEQ ID NO:9, a light chain CDR2 represented by SEQ ID NO:10, and a light chain CDR3 represented by SEQ ID NO:

11. 2 . The monoclonal antibody or antigen-binding fragment thereof according to claim 1 , which blocks the interaction between EPB41L5 and TGF-β1.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a humanized antibody. A nucleic acid molecule encoding the monoclonal antibody or a fragment thereof according to claim 1 . A vector comprising the nucleic acid molecule of claim 4 . A host cell comprising the vector according to claim 5 .

7. A pharmaceutical composition for preventing or treating cancer, comprising at least one of the monoclonal antibody or antigen-binding fragment thereof according to claim 1; a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof; and a vector containing the nucleic acid molecule as an active ingredient.

8. A pharmaceutical composition for inhibiting cancer metastasis, comprising at least one of the monoclonal antibody or antigen-binding fragment thereof according to claim 1; a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof; and a vector comprising the nucleic acid molecule as an active ingredient. 9 . A kit for quantifying EPB41L5 protein, comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1 .

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