Anti-epithelial cell adhesion molecule monoclonal antibody and use thereof

Abstract

The present invention relates to an anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody and use thereof. In the anti-EpCAM antibody of the present invention, a biopanning technique using an OPAL library having a diversity of 1×1010 was used, and a group of anti-EpCAM therapeutic antibody candidates was selected through an enzyme-linked immunosorbent assay. FACS verification was performed to confirm the binding of anti-EpCAM candidate antibodies, and the binding affinity of the candidate antibodies in tumor tissues was confirmed through IHC. In addition, the affinity of the candidate antibodies was verified through surface plasmon response analysis. For the two types of candidate antibodies verified above, the potential thereof as ADCs was evaluated, and the spheroid formation inhibitory activity of the candidate antibodies was verified to confirm the degree of inhibition of cancer cell proliferation induction. As a result of evaluating the anticancer efficacy of the candidate antibodies in an orthotropic colon cancer mice model, the candidate antibodies exhibited an excellent anticancer effect. Therefore, the antibodies produced in the present invention are likely to be effectively used as antibody therapeutic agents having an anticancer effect.

Description

Anti-epithelial cell adhesion molecule monoclonal antibody and its uses

[0001] The present invention relates to an anti-epithelial cell adhesion molecule (Anti-EpCAM) monoclonal antibody and its uses.

[0002] EpCAM consists of a protein domain protruding outside the cell (EpEX), a domain penetrating the cell membrane (TM), and a domain located inside the cell (EpICD). Claudin-7 is bound to the EpEX portion of EpCAM (Fig. 1). EpCAM interacts with various tetraspanin proteins (e.g., CD9, CD44v6, CO-029), which regulate cell adhesion and migration. Furthermore, ADAM10 and ADAM17 promote cell division by cleaving the outer domain of EpCAM, allowing the EGF-like domain to bind to EGFR. Additionally, EpCAM enhances tumorigenic ability by binding to E-Chdherin and regulating intercellular adhesion. Therefore, there is potential to develop antibody therapies and antibody-drug conjugates (ADCs) with potent anticancer efficacy by discovering antibodies that bind to EpCAM and simultaneously conjugating them with natural products and compounds.

[0003] The object of the present invention is to provide an anti-EpCAM antibody or an antigen-binding fragment thereof.

[0004] Another object of the present invention is to provide a nucleic acid molecule encoding the anti-EpCAM antibody or its antigen-binding fragment, a recombinant expression vector comprising the nucleic acid molecule, and a cell isolated by transforming it with the recombinant expression vector.

[0005] Another objective of the present invention is to provide a composition for detecting EpCAM antigens comprising the above-mentioned anti-EpCAM antibody or its antigen-binding fragment as an active ingredient.

[0006] Another objective of the present invention is to provide a cancer diagnostic composition in which EpCAM is overexpressed, comprising the above-mentioned Anti-EpCAM antibody or its antigen-binding fragment as an active ingredient.

[0007] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the above-mentioned Anti-EpCAM antibody or its antigen-binding fragment as an active ingredient.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the above-mentioned Anti-EpCAM antibody or its antigen-binding fragment; and an antibody-drug conjugate (ADC) bound to a drug, and said ADC as an active ingredient.

[0009] To achieve the above objective, the present invention comprises a heavy chain variable region comprising: a heavy chain FR1 having an amino acid sequence represented by SEQ ID NO. 1; any one heavy chain CDR1 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 2, SEQ ID NO. 10, and SEQ ID NO. 13; a heavy chain FR2 having an amino acid sequence represented by SEQ ID NO. 3; any one heavy chain CDR2 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, and SEQ ID NO. 24; a heavy chain FR3 having an amino acid sequence represented by SEQ ID NO. 5; any one heavy chain CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, and SEQ ID NO. 25; and a heavy chain FR4 having an amino acid sequence represented by SEQ ID NO. 7;and light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, any one light chain CDR1 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 27, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 45, SEQ ID NO. 48, and SEQ ID NO. 52, light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, any one light chain CDR2 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 29, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 40, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 50, and SEQ ID NO. 53, light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, and any one light chain selected from the group consisting of amino acid sequences represented by SEQ ID NO. 31, SEQ ID NO. 35, SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, and SEQ ID NO. 54 Provides an anti-epithelial cell adhesion molecule (Anti-EpCAM) antibody or an antigen-binding fragment thereof, comprising a light chain variable region including a light chain FR4 consisting of an amino acid sequence represented by CDR3 and SEQ ID NO. 32.

[0010] In addition, the present invention provides a nucleic acid molecule encoding the anti-epCAM antibody or its antigen-binding fragment.

[0011] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0012] In addition, the present invention provides a cell isolated by transforming with the recombinant expression vector.

[0013] In addition, the present invention provides a composition for detecting EpCAM antigens comprising the above-mentioned anti-EpCAM antibody or an antigen-binding fragment thereof as an active ingredient.

[0014] In addition, the present invention provides a cancer diagnostic composition in which EpCAM is overexpressed, comprising the above-mentioned Anti-EpCAM antibody or an antigen-binding fragment thereof as an active ingredient.

[0015] In addition, the present invention provides an antibody-drug conjugate (ADC) in which the anti-epCAM antibody or its antigen-binding fragment; and a drug are bound.

[0016] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the antibody-drug conjugate as an active ingredient.

[0017] The present invention relates to an anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody and its uses, wherein the anti-EpCAM antibody of the present invention is 1×10 10 Biopanning technology was utilized using an OPAL library with diverse characteristics, and candidate EpCAM therapeutic antibodies were selected via enzyme-linked immunosorbent assay. FACS was performed to confirm the binding of anti-EpCAM candidate antibodies, and the binding affinity of the candidate antibodies in tumor tissue was confirmed via IHC. Additionally, the affinity of the candidate antibodies was verified through surface plasmon response analysis. The potential of the two candidate antibodies verified above as ADCs was evaluated, and the spheroid formation inhibitory ability of the candidate antibodies was verified to determine the degree of inhibition of cancer cell proliferation. As a result of evaluating the anticancer efficacy of the candidate antibodies in an orthotropic colon cancer mouse model, the candidate antibodies exhibited excellent anticancer effects. Therefore, the antibodies produced in this invention have the potential to be usefully utilized as antibody therapeutics with anticancer effects.

[0018] Figure 1 shows the mechanism of EpCAM proliferation.

[0019] Figure 2 shows the process of selecting EpCAM therapeutic antibody candidates through enzyme-linked immunosorbent assay.

[0020] Figure 3 shows the results of confirming the binding affinity of the EpCAM therapeutic antibody through the enzyme-linked immunosorbent assay.

[0021] Figure 4 shows the FACS verification results for confirming the binding of anti-EpCAM candidate antibodies.

[0022] Figure 5 shows the results of confirming the binding affinity of candidate antibodies in tumor tissue through IHC.

[0023] Figure 6 shows the results of verifying the affinity of candidate antibodies through surface plasmon response analysis.

[0024] Figures 7 and 8 show the results of verifying the spheroid formation inhibitory ability of the SKAI-Ep126 antibody.

[0025] Figure 9 shows the results of the anticancer efficacy evaluation of SKAI-Ep66 and SKAI-Ep126 in an orthotropic colon cancer mouse model.

[0026] Figure 10 shows the survival rate, tumor weight, and hEpCAM mRNA level results for SKAI-Ep66 and SKAI-Ep126 treated mouse groups.

[0027] The present invention comprises a heavy chain variable region comprising a heavy chain FR1 having an amino acid sequence represented by SEQ ID NO. 1, any one heavy chain CDR1 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 2, SEQ ID NO. 10, and SEQ ID NO. 13, a heavy chain FR2 having an amino acid sequence represented by SEQ ID NO. 3, any one heavy chain CDR2 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, and SEQ ID NO. 24, a heavy chain FR3 having an amino acid sequence represented by SEQ ID NO. 5, any one heavy chain CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, and SEQ ID NO. 25, and a heavy chain FR4 having an amino acid sequence represented by SEQ ID NO. 7;and light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, any one light chain CDR1 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 27, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 45, SEQ ID NO. 48, and SEQ ID NO. 52, light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, any one light chain CDR2 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 29, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 40, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 50, and SEQ ID NO. 53, light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, and any one light chain selected from the group consisting of amino acid sequences represented by SEQ ID NO. 31, SEQ ID NO. 35, SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, and SEQ ID NO. 54 Provides an anti-epithelial cell adhesion molecule (Anti-EpCAM) antibody or an antigen-binding fragment thereof, comprising a light chain variable region including a light chain FR4 consisting of an amino acid sequence represented by CDR3 and SEQ ID NO. 32.

[0028] Preferably, the anti-epCAM antibody or the antigen-binding fragment thereof comprises: 1) a heavy chain variable region comprising a heavy chain FR1 having an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 having an amino acid sequence represented by SEQ ID NO. 2, a heavy chain FR2 having an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO. 4, a heavy chain FR3 having an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 having an amino acid sequence represented by SEQ ID NO. 6, and a heavy chain FR4 having an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 27, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 29, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 31, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0029] 2) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 8, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 9, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 33, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 34, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 35, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0030] 3) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 10, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 11, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 12, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 36, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 37, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 38, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0031] 4) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 14, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 15, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 39, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 40, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 41, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0032] 5) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 16, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 17, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 43, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 44, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0033] 6) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 10, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 18, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 19, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 45, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 46, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 47, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0034] 7) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 20, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 21, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 48, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 29, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 49, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0035] 8) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 23, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 33, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 50, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 51, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or

[0036] 9) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 10, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 24, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 25, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; It may include a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 52, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 53, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 54, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, but is not limited thereto.

[0037] In the present invention, the term “antibody” refers to a protein molecule that acts as a receptor for specifically recognizing an antigen, comprising an immunoglobulin molecule that is immunologically reactive with a specific antigen, and may include, for example, monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antibody fragments. Additionally, the term “antibody” may include bivalent or bispecific molecules (e.g., bispecific antibodies), diabadies, triabadies, or tetraabadies.

[0038] In the present invention, the term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same group of antibodies, and unlike polyclonal antibodies which can bind to multiple epitopes, such monoclonal antibodies exhibit single binding ability and affinity for a specific epitope. In the present invention, the term “full-length antibody” refers to a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3, and IgG4.

[0039] In the present invention, the term “heavy chain” may include both a full-length heavy chain and fragments thereof, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, a variable region VH, and three constant regions CH1, CH2, and CH3. Additionally, in the present invention, the term “light chain” may include both a full-length light chain and fragments thereof, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, a variable region VL, and a constant region CL.

[0040] In the present invention, the terms “fragment,” “antibody fragment,” and “antigen-binding fragment” are used interchangeably to refer to any fragment of the antibody of the present invention having the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

[0041] The anti-EpCAM antibody of the present invention or its antigen-binding fragment may include not only the sequence of the antibody described herein but also biological equivalents thereof, to the extent that it can exhibit the ability to specifically bind to EpCAM. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such changes include, for example, the deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. By analysis of the size, shape, and type of amino acid side chain substituents, it can be seen that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Accordingly, based on this, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0042]

[0043] In addition, the present invention provides a nucleic acid molecule encoding the anti-epCAM or its antigen-binding fragment.

[0044] As used herein, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified. The sequence of a nucleic acid molecule encoding the heavy chain and light chain variable regions of the present invention may be modified, and said modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0045]

[0046] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0047] In the present invention, "vector" means a self-replicating DNA molecule used to carry a clonal gene (or another piece of clonal DNA).

[0048] In the present invention, “expression vector” refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a specific host organism. The expression vector may preferably include one or more selectivity markers. The marker is a nucleic acid sequence having characteristics that can typically be selected by chemical methods, and includes any gene capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin, and chloramphenicol, and can be appropriately selected by a person skilled in the art.

[0049] To express the DNA sequence of the present invention, any of the very diverse expression regulatory sequences may be used in the vector. Examples of useful expression regulatory sequences may include, for example, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTRs of retroviruses, the lac system, the trp system, the TAC or TRC system, T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of fd code proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of said phosphatase, for example Pho5, promoters of the yeast alpha-mating system, and other sequences of configuration and induction known to regulate the expression of genes of prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof.

[0050] The vector expressing the antibody of the present invention may be either a vector system in which the light chain and heavy chain are simultaneously expressed in a single vector, or a system in which the light chain and heavy chain are expressed in separate vectors. In the latter case, the two vectors are introduced into host cells through co-transformation and targeted transformation. Co-transformation is a method of simultaneously introducing the respective vector DNA encoding the light chain and heavy chain into host cells and then selecting cells that express both the light chain and the heavy chain. Targeted transformation is a method of selecting cells transformed with a vector containing the light chain (or heavy chain), and then transforming the selected cells expressing the light chain back into a vector containing the heavy chain (or light chain) to finally select cells that express both the light chain and the heavy chain.

[0051]

[0052] In addition, the present invention provides a cell isolated by transforming it with a recombinant expression vector.

[0053] Cells capable of stably and continuously cloning and expressing the vector of the present invention may be any host cell known in the relevant art, and include, but are not limited to, prokaryotic host cells such as, for example, Escherichia coli, Bacillus subtilis and Bacillus churingensis strains, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0054] In the above method for preparing the anti-epCAM antibody or its antigen-binding fragment, the culture of the transformed cells may be carried out according to suitable media and culture conditions known in the relevant art. A person skilled in the art can easily adjust and use this culture process depending on the selected strain. Cell culture is classified into suspension culture and attachment culture according to the cell growth method, and into batch, fed-batch, and continuous culture methods according to the culture method. The media used for culture must adequately satisfy the requirements of the specific strain.

[0055]

[0056] In addition, the present invention provides a composition for detecting EpCAM antigens comprising the above-mentioned anti-EpCAM antibody or an antigen-binding fragment thereof as an active ingredient.

[0057]

[0058] In addition, the present invention provides a cancer diagnostic composition in which EpCAM is overexpressed, comprising the above-mentioned Anti-EpCAM antibody or an antigen-binding fragment thereof as an active ingredient.

[0059] Preferably, the cancer may be colorectal cancer or ovarian cancer, but is not limited thereto.

[0060]

[0061] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the above-mentioned Anti-EpCAM antibody or an antigen-binding fragment thereof as an active ingredient.

[0062] Preferably, the cancer may be colorectal cancer or ovarian cancer, but is not limited thereto.

[0063]

[0064] In addition, the present invention provides an antibody-drug conjugate (ADC) in which the anti-epCAM antibody or its antigen-binding fragment; and a drug are bound.

[0065] Preferably, the drug may be an anticancer agent, and more preferably, the anticancer agent is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, Cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine,Clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E It may be one or more selected from the group consisting of auristatin E (MMAE) and monomethyl auristatin F, but is not limited thereto.

[0066] In the present invention, the term “antibody drug conjugate (ADC)” implies that the anticancer drug must remain stably bound to the antibody until it is delivered to target cancer cells. Once delivered to the target, the drug must be released from the antibody to induce apoptosis in the target cells. To achieve this, the drug must be stably bound to the antibody while simultaneously possessing sufficient cytotoxicity to induce apoptosis in the target cells upon release.

[0067] Meanwhile, the above antibody may be bound to a drug through a linker. The linker is a region connecting the antibody and the drug of the present invention, allowing the drug to be released from the antibody in an intracellular environment, and reflecting the long half-life of the antibody, the antibody must be stable during systemic circulation, and the binding of the linker to the drug must not affect the stability and pharmacokinetics of the antibody. The linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, it may be cleaved by intracellular peptidases or protease enzymes, such as lysosomes or endosomes, such as peptide linkers, and in the case of a non-cleavable linker, for example, a thioether linker, the drug may be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0068]

[0069] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the antibody-drug conjugate as an active ingredient.

[0070] Preferably, the cancer may be colorectal cancer or ovarian cancer, but is not limited thereto.

[0071] The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizing agents such as excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, or flavoring agents. The pharmaceutical composition of the present invention may preferably be formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, which are sterile and biocompatible, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components may be used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, by additionally adding diluents, dispersants, surfactants, binders, and lubricants, it can be formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0072] The pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable liquids, as well as sustained-release formulations of the active compound. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, nasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes. The effective amount of the active ingredient in the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Accordingly, it may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health status, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and concurrently used drugs.

[0073] The present invention is described in detail below according to embodiments that do not limit the invention. It should be understood that the following embodiments of the present invention are merely for the purpose of embodying the invention and do not limit or restrict the scope of the rights of the present invention. Accordingly, anything that can be easily inferred by a person skilled in the art to which the present invention pertains from the detailed description and embodiments of the present invention is interpreted as falling within the scope of the rights of the present invention.

[0074]

[0075] <Example>

[0076] The development of the anti-EpCAM antibody of the present invention was carried out using 1×10 provided by Ewha Womans University 10 Biopanning technology was used with an OPAL library having dog diversity (refer to Korean Registered Patent No. 10-0961392).

[0077] As shown in Figure 2, it was confirmed that antibody diversity was restored as poly scFv bound to the EpCAM protein during the third biopanning. 384 colonies recovered in the third round were acquired, and each was induced to express scFv to analyze individual binding affinities via enzyme-linked immunosorbent assay (ELISA). There were 229 clones with a binding affinity ratio of 5 or higher relative to BSA, and among these, 178 clones with a ratio of 15 or higher were selected for sequence analysis. As a result, 142 different diversitys were analyzed, and among them, 10 clones with a binding affinity ratio of 20 or higher relative to BSA were selected. Tables 1 and 2 show the analyzed different anti-EpCAM antibody sequences.

[0078] Heavy chain variableSerialFR1:CDR1:FR2:CDR2:FR3:CDR3:FR4:SKAI_Ep52EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYDMS(Sequence No. 2)WVRQAPGKGLEWVS(Sequence No. 3)SISPGSGNT(Sequence No. 8)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)RAGLLCYHQVCYSSDAMDV(Sequence No. 9)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep66EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYSMS(Sequence No. 10)WVRQAPGKGLEWVS(Sequence No. 3)SIYPNGSSK(Sequence No. 11)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)RGVLLCQARICYSAYGMDV(Sequence No. 12)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep89EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYAMS(Sequence No. 13)WVRQAPGKGLEWVS(Sequence No. 3)GISYSGDST(Sequence No. 14)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)KVIIRCVQHVCYSAYAMDV(Sequence No. 15)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep94EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYAMS(Sequence No. 13)WVRQAPGKGLEWVS(Sequence No. 3)AISHGGSSK(Sequence No. 16)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)KAALICIHHVCYSDNAMDV(Sequence No. 17)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep107EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYSMS(Sequence No. 10)WVRQAPGKGLEWVS(Sequence No. 3)SISYSGGSI(Sequence No. 18)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No.5)RGAILCVHKLCYSSNAMDV(Sequence No. 19)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep112EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYDMS(Sequence No. 2)WVRQAPGKGLEWVS(Sequence No. 3)GISHSSGSK(Sequence No. 20)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)RDIVTCFTTLCYSDNAMDV(Sequence No. 21)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep126EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYAMS(Sequence No. 13)WVRQAPGKGLEWVS(Sequence No. 3)GISPGGGSK(Sequence No 22)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)KGILLCRESVCYSAYAMDV(Sequence No. 23)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep161EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYDMS(Sequence No. 2)WVRQAPGKGLEWVS(Sequence No. 3)GISYSGGST(Sequence No. 4)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)RDSIICQKRICYSADAMDV(Sequence No. 6)WGQGTLVTVSS(Sequence No. 7)SKAI_Ep163EVQLLESGGGLVQTGGSLRLSCAASGFTFS(Sequence No. 1)GYSMS(Sequence No 10)WVRQAPGKGLEWVS(Sequence No. 3)GIYPGGGSK(Sequence No. 24)YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(Sequence No. 5)RAAVLCHQRLCYSSNAMDV(Sequence No. 25)WGQGTLVTVSS(Sequence No. 7)

[0079]

[0080] light chain variableSerialFR1:CDR1:FR2:CDR2:FR3:CDR3:FR4:SKAI_Ep52QSVLTQPPSASGTPGQRVTISC(SEQ No. 26)SGSSSNIGNNSVY(SEQ No. 33)WYQQLPGTAPKLLIY(SEQ No. 28)SNSH(SEQ No. 34)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(SEQ No. 30)AAWDDSLSG(SEQ No. 35)YVFGGGTKLTVL(SEQ No. 32)SKAI_Ep66QSVLTQPPSASGTPGQRVTISC(SEQ No. 26)IGSSSNIGSNNVY(SEQ No. 36)WYQQLPGTAPKLLIY(SEQ No. 28)YDSN(SEQ No. 37)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)GTWDASLSA(Sequence No. 38)YVFGGGTKLTVL(Sequence No. 32)SKAI_Ep89QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)SGSSSNIGNNAVN(Sequence No. 39)WYQQLPGTAPKLLIY(Sequence No. 28)ANSH(Sequence No. 40)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)GSWDYSLSA(Sequence No. 41)YVFGGGTKLTVL(Sequence No. 32)SKAI_Ep94QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)TGSSSNIGSNDVS(Sequence No 42)WYQQLPGTAPKLLIY(Sequence No. 28)YDNK(Sequence No. 43)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)ATWDDSLSG(Sequence No. 44)YVFGGGTKLTVL(Sequence No. 32)SKAI_Ep107QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)TGSSSNIGNNDVY(Sequence No. 45)WYQQLPGTAPKLLIY(Sequence No. 28)SNNH(Sequence No. 46)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)GAWDASLSG(Sequence No. 47)YVFGGGTKLTVL(Sequence No32)SKAI_Ep112QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)SGSSSNIGSNTVS(Sequence No. 48)WYQQLPGTAPKLLIY(Sequence No. 28)ADNH(Sequence No. 29)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)AAWDDSLNA(Sequence No. 49)YVFGGGTKLTVL(Sequence No. 32)SKAI_Ep126QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)SGSSSNIGNNSVY(Sequence No. 33)WYQQLPGTAPKLLIY(Sequence No. 28)SDSQ(Sequence No. 50)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No 30)GTWDYSLNG(Sequence No. 51)YVFGGGTKLTVL(Sequence No. 32)SKAI_Ep161QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)SGSSSNIGSNDVN(Sequence No. 27)WYQQLPGTAPKLLIY(Sequence No. 28)ADNH(Sequence No. 29)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)GTWDAGLNG(Sequence No. 31)YVFGGGTKLTVL(Sequence No. 32)SKAI_Ep163QSVLTQPPSASGTPGQRVTISC(Sequence No. 26)SGSSSNIGSNAVN(Sequence No. 52)WYQQLPGTAPKLLIY(Sequence No. 28)ADSN(Sequence No 53)RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC(Sequence No. 30)GAWDSSLSA(Sequence No. 54)YVFGGGTKLTVL(Sequence No. 32)

[0081]

[0082] The anti-EpCAM candidate antibody scFv sequence was inserted into a human IgG two-vector system to be expressed as an IgG antibody. The two cloned heavy and light chain IgG plasmids were co-transfected into Expi-CHOs cells, and the monoclonal antibodies were purified using Protein A resin (Amicogen). The binding affinity to the EpCAM antigen was confirmed using enzyme-linked immunosorbent assay (Fig. 3).

[0083]

[0084] EpCAM consists of an extracellular domain (EpEX, 256 amino acids), a transmembrane domain (TM), and an intracellular domain (EpICD, 26 amino acids). The extracellular domain is divided into EpCL (AA 24-80) and EpRE (AA 81-265). EpRE is composed of Thyroglobulin type-1 and a cysteine-poor region (C-domain). Most commercially available mAbs bind to the EpCL domain, while MT201 (Adecatumumab), NM104, etc., bind to the cysteine-poor region (C-domain). Since the EpCL domain is short, making protein expression and purification difficult, Thyroglobulin type-1 and the cysteine-poor region (C-domain) were expressed and purified, and an enzyme-linked immunosorbent assay was performed. SKAI-Ep66 and SKAI-Ep126 were selected, which have high binding affinity in the entire EpCAM form like PC (323 / A3) as shown in Fig. 3rjl and low binding affinity to Thyroglobulin type-1 and cysteine-poor region (C-domain).

[0085] As shown in Figure 4, flow cytometry was performed to determine whether SKAI-Ep66 and SKAI-Ep126 bind to HCT116 cells expressing EpCAM and Raji and A2780 cells known to be EpCAM negative. The results confirmed that they bind well to HCT116 cells, but have low binding affinity or do not bind to Raji and A2780 cells, which are EpCAM negative cells.

[0086]

[0087] The inventors hypothesized that anti-EpCAM candidate antibodies would bind better in the tumor microenvironment and generated tumor tissue by subcutaneously injecting HCT116 cells into mice. As a result of analyzing the binding affinity of SKAI-Ep66 and SKAI-Ep126 using the generated tumor tissue, it was confirmed that both antibodies bound well to the tumor tissue, and it was confirmed through IHC that SKAI-Ep126 had a slightly higher binding affinity (Fig. 5).

[0088] The antigen-antibody affinity between the candidate antibody and the EpCAM antigen was confirmed using the Octat BLI instrument. The affinity measurement results showed that the affinity value (Kd) between the SKAI-Ep66 antibody and the EpCAM antigen was 21 × 10⁻⁶. -9 The M value was checked, and the affinity value (Kd) between the SKAI-Ep126 antibody and the EpCAM antigen was 14 × 10 -9 The M value was checked. Thus, strong binding affinity was confirmed as both SKAI-Ep66 and SKAI-Ep126 antibodies had Kd values ​​at the nM level. Therefore, it was confirmed that they showed higher binding affinity than the reference antibody adcatumumab (Fig. 6).

[0089] The inventors evaluated the potential of the two candidate antibody groups verified above as ADCs. As a result, it was confirmed that both SKAI-Ep66 and SKAI-Ep126 were internalized, and it was verified that SKAI-Ep126 had a larger influx of antibodies into the cytoplasm. Adecatumumab, used as a positive control, showed lower internalization than the antibody developed by the inventors, and it was confirmed that Fab, used as a negative control, did not undergo internalization (Fig. 7). Accordingly, the inventors conducted a follow-up study on efficacy evaluation focusing on antibody 126.

[0090]

[0091] To confirm the degree of inhibition of cancer cell proliferation induction through increased cell-cell adhesion among the EpCAM mechanisms described in Fig. 1, the inventors constructed a 3D HCT116-based spheroid. After treating spheroid cells on day 1 with the SKAI-Ep126 antibody, the size of the spheroids was measured on days 3, 6, 7, and 11, and it was confirmed that the spheroid size did not increase compared to the control. Consequently, it was determined that the SKAI-Ep126 antibody therapeutic inhibited cell proliferation by inhibiting cell-cell adhesion (Fig. 8).

[0092] The inventors constructed an orthotropic colon cancer mouse model using HCT-116. Although there are many reasons for using an orthotropic colon cancer mouse model for efficacy evaluation, the model was established to conduct the evaluation because, due to the characteristics of the EpCAM antigen, the tumor microenvironment must be more natural and metastasis and proliferation must occur in a more complex manner. The inventors treated SKAI-Ep66 and SKAI-Ep126 at a concentration of 10 mg / kg, respectively, and performed laparotomy 35 days later to measure the tumor size. As a result, it was confirmed that the cancer in the PBS group had grown large enough to completely cover the cecum, while in the SKAI-Ep66 group, a small tumor was observed in a specific area but was smaller than that of the PBS group. In the case of SKAI-Ep126, which was one of the candidate groups of interest to the inventors, no tumor could be observed with the naked eye. In the case of adecatumumab used as a positive control, tumors smaller than those observed with PBS were observed, but tumors much larger than those observed with the two candidate antibodies discovered by the inventors were visible to the naked eye.

[0093]

[0094] As shown in Figure 10, it was confirmed that the survival rate of the PBS group decreased starting from day 24, and all mice in the PBS group died by day 34. However, there were no deaths in any of the treatment groups. After sacrificing the mice, the total weight of the tumor and cecum was measured for both SKAI-Ep66 and SKAI-Ep126, and it was confirmed that this weight was lower than that of PBS. Furthermore, qPCR analysis was performed to check for hidden cancer cells undetectable to the naked eye by examining human epcam mRNA within the tissues. As a result, hEpCAM was not observed in either SKAI-Ep66 or SKAI-Ep126, whereas hEpCAM was observed in the control and adecatumumab groups. Accordingly, it was confirmed that both SKAI-Ep66 and SKAI-Ep126 possess anticancer efficacy.

[0095]

[0096] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, any one heavy chain CDR1 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 2, SEQ ID NO. 10, and SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, any one heavy chain CDR2 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, and SEQ ID NO. 24, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, any one heavy chain CDR3 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, and SEQ ID NO. 25, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and Light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26; any one light chain CDR1 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 27, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 45, SEQ ID NO. 48, and SEQ ID NO. 52; light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28; any one light chain CDR2 selected from the group consisting of amino acid sequences represented by SEQ ID NO. 29, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 40, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 50, and SEQ ID NO. 53; light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30; and any one light chain selected from the group consisting of amino acid sequences represented by SEQ ID NO. 31, SEQ ID NO. 35, SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, and SEQ ID NO.

54. An anti-epithelial cell adhesion molecule (Anti-EpCAM) antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising a light chain FR4 consisting of the amino acid sequence represented by CDR3 and SEQ ID NO.

32.

2. In claim 1, the Anti-EpCAM antibody or its antigen-binding fragment is, 1) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 4, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 27, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 29, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 31, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 2) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 8, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 9, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 33, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 34, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 35, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 3) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 10, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 11, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 12, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 36, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 37, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 38, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 4) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 14, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 15, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 39, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 40, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 41, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 5) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 16, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 17, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 43, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 44, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 6) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 10, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 18, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 19, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 45, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 46, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 47, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 7) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 20, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 21, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 48, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 29, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 49, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 8) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 23, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; and includes a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 33, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 50, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 51, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 32, or 9) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 10, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 24, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 25, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 7; An anti-epCAM antibody or an antigen-binding fragment thereof characterized by comprising a light chain variable region including a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 26, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 52, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 28, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 53, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 54, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

32.

3. A nucleic acid molecule encoding the anti-EpCAM antibody of claim 1 or 2 or an antigen-binding fragment thereof.

4. A recombinant expression vector comprising the nucleic acid molecule of claim 3.

5. Cells isolated after being transformed with the recombinant expression vector of paragraph 4.

6. A composition for detecting EpCAM antigens comprising the anti-EpCAM antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.

7. A composition for diagnosing cancer in which EpCAM is overexpressed, comprising the Anti-EpCAM antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.

8. A cancer diagnostic composition according to claim 7, characterized in that the cancer is colorectal cancer or ovarian cancer.

9. A pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the Anti-EpCAM antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.

10. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in claim 9, the cancer is colorectal cancer or ovarian cancer.

11. The anti-epCAM antibody of claim 1 or 2 or an antigen-binding fragment thereof; and antibody-drug conjugates (ADCs) bound to a drug.

12. An antibody-drug conjugate according to claim 11, characterized in that the above drug is an anticancer agent.

13. A pharmaceutical composition for the prevention or treatment of cancer in which EpCAM is overexpressed, comprising the antibody-drug conjugate of claim 11 as an active ingredient.

14. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in paragraph 13, the cancer is colorectal cancer or ovarian cancer.

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