Antibody or antibody fragment thereof specifically binding to tspan1, and therapeutic, prophylactic, or diagnostic use thereof

Abstract

The present invention relates to an antibody or antibody fragment thereof that specifically binds to tetraspanin 1 (TSPAN1), and use thereof. More specifically, the present invention relates to: an antibody or antibody fragment thereof that specifically binds to a site comprising an amino acid fragment at positions 110 to 211 in the TSPAN1 amino acid sequence of SEQ ID NO: 1; a method for preparing the antibody or a fragment thereof; a composition for inhibiting cancer and / or cancer metastasis, comprising the antibody or a fragment thereof; a method for inhibiting cancer and / or cancer metastasis, the method comprising administering the antibody or the composition; a composition for diagnosing cancer and / or cancer metastasis, comprising the antibody; a kit for diagnosing cancer, comprising the composition; a method for diagnosing cancer by using the composition; and the use of an amino acid fragment at positions 110 to 211 of TSPAN1 as an epitope for an antibody for the inhibition of cancer and / or cancer metastasis. Also, the present invention relates to an antibody-drug conjugate comprising the antibody or an antibody fragment thereof of the present invention, to which a drug is conjugated.

Description

An antibody or antibody fragment thereof that specifically binds to TSPAN1, and the therapeutic, prophylactic, or diagnostic use thereof

[0001] The present invention relates to an antibody or antibody fragment that specifically binds to TSPAN1 (tetraspanin 1) and to the use thereof. More specifically, the present invention relates to an antibody or antibody fragment that specifically binds to a region containing amino acid fragments 110 to 211 of the TSPAN1 amino acid sequence of SEQ ID NO. 1, a method for preparing said antibody or said fragment, a composition for treating or preventing cancer and / or inhibiting cancer metastasis comprising said antibody or said fragment, a method for inhibiting cancer and / or cancer metastasis by administering the antibody or the composition, a composition for diagnosing cancer and / or cancer metastasis comprising the antibody, and a cancer diagnostic kit comprising the composition.

[0002] In addition, the present invention relates to an antibody-drug conjugate comprising the antibody of the present invention or a fragment of the antibody thereof, to which a drug is bound.

[0003] Cancer is a condition in which abnormal cell growth is caused by various changes in gene expression that disrupt normal cell proliferation and death, invading and destroying adjacent tissues and metastasizing to other parts of the body, ultimately leading to death. Although the exact cause of cancer—specifically, the mechanism by which normal cells transform into cancer cells—has not been precisely identified, current knowledge suggests that cancer is caused by a complex interplay of external factors such as environmental factors, chemicals, radiation, and viruses, as well as internal factors such as genetic and immunological factors.

[0004] Cancer is broadly classified into hematological cancers, which involve abnormalities in blood cell counts, and solid tumors, which take the form of masses of cells with a specific hardness and shape within the body. Cancer can occur in the blood, tissues, and almost any part of the body; examples include lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer. The primary methods of cancer treatment involve surgery, radiation therapy, and anticancer drug therapy using chemotherapy agents that inhibit cell proliferation. Pharmaceuticals for cancer treatment are broadly divided into small molecule drugs and large molecule drugs; compared to small molecule drugs, which lack specificity and carry relatively significant side effects, large molecule drugs, which possess specificity, are gaining prominence as therapeutic agents.

[0005] In particular, it is important to discover antibodies with high specificity for the target. Therefore, we aim to identify antibodies that yield effective results through a screening process among various antibodies.

[0006] The TSPAN1 (Tetraspanin 1) protein is a protein encoded by the TSPAN1 gene and is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members correspond to cell surface proteins characterized by the presence of four hydrophobic domains. These proteins are known to mediate signal transduction involved in the regulation of cell development, activation, growth, and motility.

[0007] TSPAN1 acts as an oncogene in gastric cancer (FEBS LETTERS, Volume 589, Issue 15, July 08, 2015, Pages 1988-1994) and is known to upregulate MMP2 associated with pancreatic cancer cell metastasis (Oncology Reports, April-2019, Volume 41 Issue 4 pages 2117-2125).

[0008] In addition, the correlation between the expression level of TSPAN1 and high-risk endometriosis is known (Korean Patent Application Publication No. 10-2022-0022772), and a decrease in the expression of TSPAN1 protein can inhibit the progression of endometriosis to cancer (Korean Registered Patent No. 10-2401005). Furthermore, TSPAN1 protein is known to be associated with the prognosis and treatment of metastasis in rectal cancer, breast cancer, gastric cancer, and colorectal cancer, and it is known that the expression level of TSPAN1 protein is increased in these cancers (Chinese Patent Publication No. 108977547, Chinese Patent Application Publication No. 110055334, Japanese Patent Application Publication No. 19175021).

[0009] It has been reported that inhibition of TSPAN1 expression suppresses cancer cell division in pancreatic cancer cell lines and reduces tumor growth in a mouse pancreatic cancer model. (https: / www.tandfonline.com / doi / full / 10.1080 / 15548627.2020.1826689?utm_source=chatgpt.com#d1e539)

[0010] Accordingly, antibodies that specifically bind to TSPAN1 may be useful for the prevention, treatment, or diagnosis of the aforementioned cancers. Furthermore, antibodies that specifically bind to TSPAN1 can also be used as antibody-drug conjugates (ADCs). Antibody-drug conjugates (ADCs) represent a relatively novel class of therapeutic agents comprising antibodies conjugated to cytotoxic drugs via chemical linkers. The therapeutic concept of an ADC is to combine the antibody's ability to bind to a drug, wherein the antibody is used to deliver the drug to tumor cells by binding to a target surface antigen, including target surface antigens that are overexpressed or amplified in tumor cells.

[0011] Under this technical background, the inventors of the present application intend to develop and provide an antibody that specifically binds to TSPAN1, which may be effective for cancer treatment or prevention, prevention of cancer metastasis, or cancer diagnosis.

[0012] [Prior Art Literature]

[0013] [Patent Literature]

[0014] Republic of Korea Patent Application Publication No. 10-2022-0022772

[0015] Republic of Korea Registered Patent No. 10-2401005

[0016] Chinese Patent Publication No. 108977547

[0017] Chinese Patent Application Publication No. 110055334

[0018] Japanese Patent Application Publication No. 19175021

[0019]

[0020] [Non-patent literature]

[0021] FEBS LETTERS, Volume589, Issue15, July 08, 2015, Pages 1988-1994

[0022] Oncology Reports, April-2019, Volume 41 Issue 4 pages 2117-2125

[0023] The present invention aims to provide an antibody or a fragment thereof having high specificity for TSPAN1, a composition for cancer treatment or prevention or metastasis inhibition using the same, or a composition or diagnostic kit for diagnosing cancer and / or cancer metastasis.

[0024] The present invention aims to provide an antibody-drug conjugate that can effectively deliver a drug to tumor cells by having high specificity for TSPAN1.

[0025] One aspect of the present invention relates to an antibody or antibody fragment that specifically binds to TSPAN1, wherein the antibody or antibody fragment specifically binds to a region comprising amino acid fragments 110 to 211 of the TSPAN1 amino acid sequence represented by SEQ ID NO. 1.

[0026] Another aspect of the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer or the inhibition of cancer metastasis, or a composition or diagnostic kit for the diagnosis of cancer and / or cancer metastasis, comprising the antibody or antibody fragment disclosed herein.

[0027] Another aspect of the present invention relates to a polynucleotide encoding an antibody or an antibody fragment thereof disclosed herein, an expression vector comprising said polynucleotide, or a host cell comprising said expression vector.

[0028] Another aspect of the present invention relates to a method for producing an antibody or an antibody fragment thereof, comprising culturing the host cell to produce the antibody described herein by nucleic acid expression.

[0029] Another aspect of the present invention provides an antibody-drug conjugate comprising an antibody or a fragment of the antibody described herein to which a drug is conjugated, wherein the drug is any one selected from the group consisting of toxins, chemotherapy agents, anticancer agents, antibiotics, ADP-ribosyltransferase, radioisotopes, and nucleases.

[0030] The present invention provides antibody recognition site and sequence information of a TSPAN1 antigen, and further provides an antibody or antibody fragment thereof that specifically binds to the antibody recognition site.

[0031] Thus, an antibody or a fragment thereof useful for the treatment or prevention of cancer or for the diagnosis of cancer and / or cancer metastasis is provided.

[0032] In addition, the antibody or fragment thereof of the present invention can effectively kill cancer cells by specifically binding to cells expressing TSPAN1, thereby providing the cancer cells with an antibody for cancer treatment related to TSPAN1 expression or an antibody-drug conjugate having high specificity for TSPAN1.

[0033] Figure 1 shows the results of Fab-Phage Panning and screening to discover antibodies against TSPAN1, Figure 1a shows the results for the constructed HDB001 Phage Library, and Figure 1b shows the results for the HDB002 Phage Library.

[0034] Figure 2 shows the results of a filter lift assay to find hit substances among the various antibodies discovered in the screening of Figure 1. There were 94 clones detected in the fabricated Fab library, of which 8 (left) from HDB001 and 40 (right) from HDB002 were evaluated as valid clones.

[0035] Figure 3 shows the results of the phage ELISA experiment, and Figures 3a and 3b show (indicated by stars) clones with high binding affinity to TSPAN1 compared to IgG-Fc in batch 1 for HDB001. Among these, six clones are unique clones according to sequence analysis.

[0036] Figures 4a and 4b show (starred) clones with high binding affinity to TSPAN1 relative to IgG-Fc in batch 2 for HDB001. Of these, five clones exhibited high binding affinity.

[0037] Figure 5 shows the results of other phage ELISA experiments, and Figures 5a and 5b show (indicated by stars) clones with high binding affinity to TSPAN1 compared to IgG-Fc in Batch 1 for HDB002. Of these, 24 clones are unique clones according to sequence analysis.

[0038] Figures 6a and 6b show (indicated by stars) clones with high binding affinity to TSPAN1 relative to IgG-Fc in batch 2 for HDB002. Of these, 40 clones exhibited high binding affinity.

[0039] Figure 7 shows a total of 29 hits selected in order of highest binding affinity with TSPAN1 among the effective clones detected in Figures 3 to 6.

[0040] Figure 8 shows the antibody recognition site and sequence information of the TSPAN1 antigen investigated through Uniprot.

[0041] Figure 9 shows the VH domain (Figure 9a) and VK domain amino acid sequences (Figure 9b) for 29 hits obtained through Panning phage display, Filter lift assay, and SPE (Solid Phase Extraction) ELISA.

[0042] Figure 10 shows the ELISA test results for 29 hits. Figure 10a shows the ELISA results of 10 antibodies from Batch 1, and Figure 10b shows the ELISA results of 19 antibodies from Batch 2 and Batch 3.

[0043] Fig. 11 is EC 50 This is a graph showing the SPR analysis results for 10 antibodies with low values.

[0044] Figure 12 is a graph showing the ability of the selected antibodies of the present invention to bind to AsPC-1, U87MG, and SH-SY5Y, respectively, which are cells expressing TSPAN1.

[0045]

[0046] One aspect of the present invention relates to an antibody or an antibody fragment thereof that specifically binds to TSPAN1 (tetraspanin 1), wherein the antibody or the antibody fragment thereof may specifically bind to an amino acid fragment of positions 110 to 211 of the TSPAN1 amino acid sequence represented by SEQ ID NO. 1, or to a region containing such a fragment. The amino acid fragment of positions 110 to 211 of SEQ ID NO. 1 is referred to herein as 'Tsbs'.

[0047] The TSPAN1 (Tetraspanin 1) protein of the present invention is a protein encoded by the TSPAN1 gene and is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members correspond to cell surface proteins characterized by the presence of four hydrophobic domains. The protein is known to mediate signal transduction involved in the regulation of cell development, activation, growth, and motility. TSPAN1 may be represented by the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0048] In one embodiment, TSPAN1 may be human TSPAN1 or mouse TSPAN1, and in a specific embodiment, it may be human TSPAN1.

[0049] The amino acid fragment of positions 110 to 211 of the TSPAN1 amino acid sequence represented by SEQ ID NO. 1, i.e., Tsbs, corresponds to the largest loop in the extracellular region, and its sequence specificity is higher than that of other TSPAN types (e.g., TSPAN 7, TSPAN 9, TSPAN 18, etc.).

[0050] In the present invention, "epitope" refers to a portion of an antibody that determines antigen specificity and can be used interchangeably with an antigen determinant or an antigen determinant site. Accordingly, in the present invention, the epitope may refer to an amino acid fragment of positions 110 to 211 of the amino acid sequence of TSPAN1 or a polypeptide having the same function. In addition to the above amino acid fragment, additional regions may be included. Any polypeptide that performs the same role as the above amino acid fragment, for example, a polypeptide having 80%, 85%, 90%, 95%, 98%, or 99% or more of amino acid sequence homology, may be used as an epitope.

[0051] TSPAN1 is known to be associated with the prognosis and treatment of gastric cancer, rectal cancer, pancreatic cancer, uterine cancer, breast cancer, colorectal cancer, etc., or metastasis thereof, and the expression level of TSPAN1 protein is increased in these cancers.

[0052] Accordingly, an antibody that specifically binds to TSPAN1 can specifically bind to the epitope of TSPAN1 and inhibit the cancer and / or cancer metastasis, and thus may be useful for the prevention or treatment of cancers and for preventing cancer metastasis.

[0053] The term "antibody" in the present invention refers to a protein molecule comprising an immunoglobulin molecule that responds immunologically to a specific antigen and functions as a receptor that specifically recognizes the antigen, and refers to a protein molecule intended to include polyclonal antibodies, monoclonal antibodies, total antibodies, and antibody fragments. Additionally, chimeric antibodies (e.g., humanized murine antibodies), divalent or bispecific molecules (e.g., bispecific antibodies), diabadies, triabadies, and tetraabadies fall within the range of antibodies useful in the present invention.

[0054] Total antibodies consist of disulfide bonds between light and heavy chains, two full-length light chains, and two full-length heavy chains. In mammals, there are five antibody isotypes known as IgA, IgD, IgE, IgM, and IgG, and IgG is further classified into four subtypes: IgG1, IgG2, IgG3, and IgG4.

[0055] In the present invention, "antibody fragment" refers to a fragment possessing at least an antigen-binding function and may include Fab, F(ab'), F(ab')2, scFv, (scFv)2, scFv-Fc, and Fv. Fab is an antigen-binding site and is composed of a variable region of the heavy chain and a light chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, respectively. Fab' differs from Fab in that it includes one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 is composed of two Fab' molecules formed by a disulfide bond between the cysteine ​​residues at the hinge site. FV (variable fragment), composed of a variable region of each of the heavy chain and light chain, is a minimal antibody fragment containing the original specificity of the parent immunoglobulin. A disulfide-stabilized FV (hsFv) is formed by connecting the variable region of the heavy chain to the variable region of the light chain through a disulfide bond. A single-chain FV (scFv) is an FV in which the variable regions of the heavy chain and light chain are covalently connected by a peptide linker.

[0056] These antibody fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be broken down into papain or pepsin to obtain Fab or F(ab'), respectively), and preferably can be produced by genetic recombination technology.

[0057] Antibody discovery is used to identify or develop highly specific antibodies against specific antigens by understanding the interactions between proteins or peptides. In the present invention, phage display was performed to discover antibodies with high specificity for TSPAN1. This method studies the interactions between proteins expressed on the surface of bacteriophages using various molecules such as peptides, DNA, and other proteins. The main process of this technique involves reacting a phage library, on which proteins of various sequences are expressed on the surface, with antigens immobilized on a plate to select phages with high affinity; this process is called biopanning. Subsequently, a filter lift assay can be performed to identify hit substances among the selected antibodies. This method measures the degree of protein binding using a filter and can be applied when screening drugs. Among the candidate antibodies with high binding affinity, unique clones that specifically bind to TSPAN1 can be further selected through SPE ELISA, etc.

[0058] In one embodiment, the antibody of the present invention or the antibody fragment thereof comprises a Complementary Determining Regions (CDR) 1 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs. 2 to 4;

[0059] CDR2 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 6; and

[0060] It may include a VH domain comprising a CDR3 including any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 7 to 35.

[0061] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0062] A VH domain comprising CDR 1 having the amino acid sequence of SEQ ID NO. 2, CDR 2 having the amino acid sequence of SEQ ID NO. 6, and CDR 3 having the amino acid sequence of SEQ ID NO. 7, or

[0063] A VH domain comprising a CDR 1 having the amino acid sequence of SEQ ID NO. 3, a CDR 2 having the amino acid sequence of SEQ ID NO. 5, and a CDR 3 having any one selected from the group consisting of the amino acid sequences of SEQ ID NOs. 8 to 17, 19 to 23, 25, and 27 to 35, or

[0064] It may include a VH domain comprising a CDR 1 containing the amino acid sequence of SEQ ID NO. 4, a CDR 2 containing the amino acid sequence of SEQ ID NO. 5, and a CDR 3 containing any one selected from the group consisting of the amino acid sequences of SEQ ID NO. 18, 24, and 26.

[0065] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0066] CDR1 comprising the amino acid sequence of SEQ ID NO. 36 or SEQ ID NO. 37 (wherein SEQ ID NO. 36 is RAS Xa1 Xa2 Xa3 and Xa1 is Q, R or E, Xa2 is GINN, GIGG, GIRN, GISN, GIGT, GIAN, GISS, GIST, GISR, SISS, SISI, SVSR, SVSS, SVNS, NIRH, TITT, DISR, PINN, or NYNY, and Xa3 is YLG, WLA, NLA, YLN, YVN, HLN, FVN, or YLA);

[0067] A CDR2 comprising an amino acid sequence of any one of SEQ ID NOs 38 to 44 (wherein SEQ ID NO 38 is AAS Aa1 Aa2, where Aa1 is S, T, or N, and Aa2 is LES, LQS, LPD, SQS, or LQR; SEQ ID NO 39 is GAS Ba1 Ba2, where Ba1 is S, N, R, or T, and Ba2 is LHS, LQS, VHS, LQS, LRS, LDS, or RAT; and SEQ ID NO 40 is Ca1 ASS Ca2, where Ca1 is S, K, or D, and Ca2 is LQS or LES); and

[0068] It may include a VK domain comprising a CDR3 having an amino acid sequence of any one of SEQ ID NOs 45 to 49 (wherein SEQ ID NO 45 is QQ Da1 Da2, where Da1 is SYST, SYSP, SYSS, GYSS, GYSL, YWSY, SYGT, SHSS, YNSW, ASSL, ASSF, GYIA, YYRS, YHSY, ANSF, ANSL, YGSS, AHSS, ANDF, or AHSF, and Da2 is PLT, PIT, PFT, PPT, PLS, PWT, or QLS).

[0069] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0070] CDR 1 containing the amino acid sequence of SEQ ID NO. 36 (wherein SEQ ID NO. 36 is RAS Xa1 Xa2 Xa3, where Xa1 is Q, Xa2 is GINN, GIGG, GIRN, GISN, GIGT, GIAN, GISS, GIST, GISR, SISS, SISI, SVSR, SVSS, SVNS, TITT, DISR, PINN, or NYNY, and Xa3 is YLG, WLA, NLA, YLN, YVN, HLN, FVN, or YLA);

[0071] A CDR2 comprising an amino acid sequence of any one of SEQ ID NOs 38 to 40 (wherein SEQ ID NO 38 is AAS Aa1 Aa2, where Aa1 is S or T and Aa2 is LES, LQS, LPD, or LQR; SEQ ID NO 39 is GAS Ba1 Ba2, where Ba1 is S or N and Ba2 is LHS, LQS, or LRS; and SEQ ID NO 40 is Ca1 ASS Ca2, where Ca1 is S, K, or D and Ca2 is LQS or LES); and

[0072] A VK domain may be included, comprising a CDR3 having an amino acid sequence of any one of SEQ ID NOs 45 to 49 (wherein SEQ ID NO 45 is QQ Da1 Da2, where Da1 is SYST, SYSP, SYSS, GYSS, GYSL, SYGT, SHSS, ASSL, ASSF, GYIA, YYRS, YHSY, ANSF, ANSL, YGSS, AHSS, ANDF, or AHSF, and Da2 is PLT, PIT, PFT, PPT, PLS, PWT, or QLS).

[0073] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0074] CDR1 containing sequence number 36 (wherein sequence number 36 is RAS Xa1 Xa2 Xa3, where Xa1 is Q, Xa2 is GINN, GIRN, GISN, GIGT, GISS, or SISI, and Xa3 is WLA or YLG);

[0075] A CDR2 comprising SEQ NO 38, SEQ NO 39, or SEQ NO 40 (wherein SEQ NO 38 is AAS Aa1 Aa2, where Aa1 is S or N and Aa2 is LES or LQS; SEQ NO 39 is GAS Ba1 Ba2, where Ba1 is S and Ba2 is LHS or RAT; and SEQ NO 40 is Ca1 ASS Ca2, where Ca1 is D and Ca2 is LQS or LES); and

[0076] It may include a VK domain including a CDR3 including sequence number 45.

[0077] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0078] Among the VH domains,

[0079] CDR1 may include an amino acid sequence selected from the group consisting of SEQ ID NOs 2 to 4, and CDR2 includes the amino acid sequence of SEQ ID NO. 5 or 6, and

[0080] CDR3 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 7, 9 to 31, 35, 57, 61, and 123, and

[0081] Among VK domains,

[0082] CDR1 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 50, 58, 62, 124, 128, 130, 133, 135, 136, 138, 140, 145, 148, 149, 151, 154, 155, 158, 160, 162, 54, 37, 166, 170, 172, and 175, and

[0083] CDR2 may include an amino acid sequence selected from the group consisting of SEQ ID NOs 41, 42, 43, 51, 55, 59, 63, 125, 129, 131, 141, 143, 146, 152, 156, 163, 165, 167, 168, 173, and 176, and

[0084] CDR3 may include an amino acid sequence selected from the group consisting of sequence numbers 46, 47, 48, 49, 52, 56, 60, 64, 126, 132, 134, 137, 139, 142, 144, 147, 150, 153, 157, 159, 161, 164, 169, 171, 174, and 177.

[0085] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0086] A VH domain comprising CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 6, and CDR3 of SEQ ID NO. 7, and

[0087] A VK domain comprising CDR1 of SEQ ID NO. 50, CDR2 of SEQ ID NO. 51, and CDR3 of SEQ ID NO. 52;

[0088] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 123, and

[0089] A VK domain comprising CDR1 of SEQ ID NO. 124, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 126;

[0090] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 9, and

[0091] A VK domain comprising CDR1 of SEQ ID NO. 128, CDR2 of SEQ ID NO. 129, and CDR3 of SEQ ID NO. 46;

[0092] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 10, and

[0093] A VK domain comprising CDR1 of SEQ ID NO. 130, CDR2 of SEQ ID NO. 131, and CDR3 of SEQ ID NO. 132;

[0094] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 11, and

[0095] A VK domain comprising CDR1 of SEQ ID NO. 133, CDR2 of SEQ ID NO. 129, and CDR3 of SEQ ID NO. 134;

[0096] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 12, and

[0097] A VK domain comprising CDR1 of SEQ ID NO. 135, CDR2 of SEQ ID NO. 42, and CDR3 of SEQ ID NO. 47;

[0098] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 13, and

[0099] A VK domain comprising CDR1 of SEQ ID NO. 136, CDR2 of SEQ ID NO. 129, and CDR3 of SEQ ID NO. 137;

[0100] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 14, and

[0101] A VK domain comprising CDR1 of SEQ ID NO. 138, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 139;

[0102] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 15, and

[0103] A VK domain comprising CDR1 of SEQ ID NO. 140, CDR2 of SEQ ID NO. 141, and CDR3 of SEQ ID NO. 142;

[0104] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 16, and

[0105] A VK domain comprising CDR1 of SEQ ID NO. 136, CDR2 of SEQ ID NO. 143, and CDR3 of SEQ ID NO. 144;

[0106] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 17, and

[0107] A VK domain comprising CDR1 of SEQ ID NO. 145, CDR2 of SEQ ID NO. 146, and CDR3 of SEQ ID NO. 147;

[0108] A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 18, and

[0109] A VK domain comprising CDR1 of SEQ ID NO. 148, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 48;

[0110] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 19, and

[0111] A VK domain comprising CDR1 of SEQ ID NO. 149, CDR2 of SEQ ID NO. 41, and CDR3 of SEQ ID NO. 150;

[0112] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 20, and

[0113] A VK domain comprising CDR1 of SEQ ID NO. 151, CDR2 of SEQ ID NO. 152, and CDR3 of SEQ ID NO. 153;

[0114] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 21, and

[0115] A VK domain comprising CDR1 of SEQ ID NO. 154, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 56;

[0116] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 22, and

[0117] A VK domain comprising CDR1 of SEQ ID NO. 155, CDR2 of SEQ ID NO. 156, and CDR3 of SEQ ID NO. 157;

[0118] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 23, and

[0119] A VK domain comprising CDR1 of SEQ ID NO. 158, CDR2 of SEQ ID NO. 43, and CDR3 of SEQ ID NO. 159;

[0120] A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 24, and

[0121] A VK domain comprising CDR1 of SEQ ID NO. 160, CDR2 of SEQ ID NO. 55, and CDR3 of SEQ ID NO. 161;

[0122] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 25, and

[0123] A VK domain comprising CDR1 of SEQ ID NO. 162, CDR2 of SEQ ID NO. 163, and CDR3 of SEQ ID NO. 164;

[0124] A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 26, and

[0125] A VK domain comprising CDR1 of SEQ ID NO. 54, CDR2 of SEQ ID NO. 55, and CDR3 of SEQ ID NO. 56;

[0126] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 27, and

[0127] A VK domain comprising CDR1 of SEQ ID NO. 37, CDR2 of SEQ ID NO. 165, and CDR3 of SEQ ID NO. 139;

[0128] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 28, and

[0129] A VK domain comprising CDR1 of SEQ ID NO. 166, CDR2 of SEQ ID NO. 167, and CDR3 of SEQ ID NO. 49;

[0130] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 29, and

[0131] A VK domain comprising CDR1 of SEQ ID NO. 158, CDR2 of SEQ ID NO. 168, and CDR3 of SEQ ID NO. 169;

[0132] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 30, and

[0133] A VK domain comprising CDR1 of SEQ ID NO. 170, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 171;

[0134] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 31, and

[0135] A VK domain comprising CDR1 of SEQ ID NO. 172, CDR2 of SEQ ID NO. 173, and CDR3 of SEQ ID NO. 174;

[0136] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 57, and

[0137] A VK domain comprising CDR1 of SEQ ID NO. 58, CDR2 of SEQ ID NO. 59, and CDR3 of SEQ ID NO. 60;

[0138] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 61, and

[0139] A VK domain comprising CDR1 of SEQ ID NO. 62, CDR2 of SEQ ID NO. 63, and CDR3 of SEQ ID NO. 64;

[0140] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 28, and

[0141] A VK domain comprising CDR1 of SEQ ID NO. 166, CDR2 of SEQ ID NO. 167, and CDR3 of SEQ ID NO. 49;

[0142] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 35, and

[0143] It may include a VK domain including CDR1 of SEQ ID NO. 175, CDR2 of SEQ ID NO. 176, and CDR3 of SEQ ID NO. 177.

[0144] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0145] A VH domain comprising CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 6, and CDR3 of SEQ ID NO. 7, and

[0146] A VK domain comprising CDR1 of SEQ ID NO. 50, CDR2 of SEQ ID NO. 51, and CDR3 of SEQ ID NO. 52;

[0147] A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 26, and

[0148] A VK domain comprising CDR1 of SEQ ID NO. 54, CDR2 of SEQ ID NO. 55, and CDR3 of SEQ ID NO. 56;

[0149] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 57, and

[0150] A VK domain comprising CDR1 of SEQ ID NO. 58, CDR2 of SEQ ID NO. 59, and CDR3 of SEQ ID NO. 60;

[0151] A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 61, and

[0152] It may include a VK domain including CDR1 of SEQ ID NO. 62, CDR2 of SEQ ID NO. 63, and CDR3 of SEQ ID NO. 64.

[0153] In one embodiment, the antibody of the present invention or the antibody fragment thereof is,

[0154] The VH domain sequence of sequence number 65 and the VK domain sequence of sequence number 94

[0155] Includes the VH domain sequence of SEQ ID NO. 66 and the VK domain sequence of SEQ ID NO. 95, or

[0156] Includes the VH domain sequence of SEQ ID NO. 67 and the VK domain sequence of SEQ ID NO. 96, or

[0157] Includes the VH domain sequence of SEQ ID NO. 68 and the VK domain sequence of SEQ ID NO. 97,

[0158] Includes the VH domain sequence of SEQ ID NO. 69 and the VK domain sequence of SEQ ID NO. 98, or

[0159] Includes the VH domain sequence of SEQ ID NO. 70 and the VK domain sequence of SEQ ID NO. 99, or

[0160] Includes the VH domain sequence of SEQ ID NO. 71 and the VK domain sequence of SEQ ID NO. 100, or

[0161] Includes the VH domain sequence of SEQ ID NO. 72 and the VK domain sequence of SEQ ID NO. 101, or

[0162] Includes the VH domain sequence of SEQ ID NO. 73 and the VK domain sequence of SEQ ID NO. 102, or

[0163] Includes the VH domain sequence of SEQ ID NO. 74 and the VK domain sequence of SEQ ID NO. 103, or

[0164] Includes the VH domain sequence of SEQ ID NO. 75 and the VK domain sequence of SEQ ID NO. 104, or

[0165] Includes the VH domain sequence of SEQ ID NO. 76 and the VK domain sequence of SEQ ID NO. 105, or

[0166] Includes the VH domain sequence of SEQ ID NO. 77 and the VK domain sequence of SEQ ID NO. 106, or

[0167] Includes the VH domain sequence of SEQ ID NO. 78 and the VK domain sequence of SEQ ID NO. 107, or

[0168] Includes the VH domain sequence of SEQ ID NO. 79 and the VK domain sequence of SEQ ID NO. 108, or

[0169] Includes the VH domain sequence of SEQ ID NO. 80 and the VK domain sequence of SEQ ID NO. 109, or

[0170] Includes the VH domain sequence of SEQ ID NO. 81 and the VK domain sequence of SEQ ID NO. 110, or

[0171] Includes the VH domain sequence of SEQ ID NO. 82 and the VK domain sequence of SEQ ID NO. 111, or

[0172] Includes the VH domain sequence of SEQ ID NO. 83 and the VK domain sequence of SEQ ID NO. 112, or

[0173] Includes the VH domain sequence of SEQ ID NO. 84 and the VK domain sequence of SEQ ID NO. 113, or

[0174] Includes the VH domain sequence of SEQ ID NO. 85 and the VK domain sequence of SEQ ID NO. 114,

[0175] Includes the VH domain sequence of SEQ ID NO. 86 and the VK domain sequence of SEQ ID NO. 115, or

[0176] Includes the VH domain sequence of SEQ ID NO. 87 and the VK domain sequence of SEQ ID NO. 116, or

[0177] Includes the VH domain sequence of SEQ ID NO. 88 and the VK domain sequence of SEQ ID NO. 117, or

[0178] Includes the VH domain sequence of SEQ ID NO. 89 and the VK domain sequence of SEQ ID NO. 118, or

[0179] Includes the VH domain sequence of SEQ ID NO. 90 and the VK domain sequence of SEQ ID NO. 119,

[0180] Includes the VH domain sequence of SEQ ID NO. 91 and the VK domain sequence of SEQ ID NO. 120, or

[0181] Includes the VH domain sequence of SEQ ID NO. 92 and the VK domain sequence of SEQ ID NO. 121, or

[0182] It may include the VH domain sequence of sequence number 93 and the VK domain sequence of sequence number 122.

[0183] The above antibody or antibody fragment thereof may be isolated from a living organism (not present in the living organism) or non-naturally occurring, for example, may be produced synthetically or recombinantly.

[0184] Another aspect of the present invention relates to radiopharmaceutical therapeutics, antibody drug conjugates, T cell engagers, bispecific antibodies, etc., using the antibody or antibody fragment described herein as a targeting substance.

[0185] Another aspect of the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer or for preventing cancer metastasis, comprising an antibody or an antibody fragment thereof described herein. The cancer may be gastric cancer, uterine cancer, breast cancer, colorectal cancer, ovarian cancer, brain tumor, or pancreatic cancer, but is not limited thereto.

[0186] The above pharmaceutical composition may be provided in the form of capsules, tablets, granules, injections, ointments, powders, or beverages. The above pharmaceutical composition may be intended for humans.

[0187] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a flavoring agent, etc. For parenteral administration, in the case of an injectable, a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used, and for topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used.

[0188] The routes of administration of the pharmaceutical composition of the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0189] The term "parenteral" in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques.

[0190] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific antibody used, age, body weight, general health, gender, diet, time of administration, route of administration, release rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, although it depends on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration. It may be administered once a day or two or more times at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg.

[0191] Another aspect of the present invention relates to a polynucleotide encoding an antibody or an antibody fragment thereof. The polynucleotide encoding the VH and VK domains described herein may comprise DNA and RNA, which may be wholly or partially synthetic.

[0192] The above polynucleotide may be any one of SEQ ID NOs 178 to 206.

[0193] Another aspect of the present invention relates to an expression vector comprising the polynucleotide. The expression vector may be, for example, a plasmid, a virus, for example, a phage, or a phagemid or cosmid, and in some embodiments may be a vector in the form of a transcription or expression cassette comprising one or more of the polynucleotides. A suitable vector containing a suitable regulatory sequence, including a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene, and other suitable sequences, may be selected or constructed. For further details, reference is made to, for example, the literature [Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press].

[0194] In addition, the vector of the present invention is a viral vector capable of infecting a host cell, for example, a

[0195] It includes denovirus, retrovirus, or adeno-associated virus vectors. Such vectors may be useful for generating said antibodies by expressing said antibodies in cells of a human or animal subject and delivering them to said subject.

[0196] Another aspect of the present invention provides a host cell comprising the expression vector. The polynucleotide of the present invention is incorporated into the genome (e.g., chromosome) of the host cell. Incorporation may be facilitated by including a sequence that promotes recombination with the genome, according to standard techniques.

[0197] Systems for cloning and expressing polypeptides in host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for polypeptide expression include Chinese hamster ovary cells, HeLa cells, pup hamster kidney cells, NS0 mouse melanoma cells, YB2 / 0 rat myeloma cells, and many other cells. A specific common bacterial host is E. coli.

[0198] The expression of antibodies and antibody fragments in prokaryotic cells, e.g., E. coli, is well established in the art (Ref. Pluckthun, A. Bio / Technology 9: 545-551 (1991)). Expression in cultured eukaryotic cells is also available to those skilled in the art as an option for the production of target antibodies (Ref. ME (1993) Curr. Opinion Biotech. 4: 573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6: 553-560).

[0199] Another aspect of the present invention provides a method for producing an antibody or an antibody fragment thereof, comprising culturing a host cell of the present invention to produce an antibody by nucleic acid expression. The method comprises culturing a host cell under conditions in which an antibody is produced from an encoding polynucleotide, and then isolating and purifying the antibody using any suitable method.

[0200] The present invention also provides an antibody-drug conjugate comprising an antibody of the present invention to which a drug is conjugated. The drug may be any one selected from the group consisting of toxins, chemotherapy agents, anticancer agents, antibiotics, ADP-ribosyltransferase, radioisotopes, and nucleases. The antibody-drug conjugate may include a linker that links the antibody and the drug. The linker may include a cleavable linker or a non-cleavable linker. The cleavable linker may be cleaved by intracellular peptidases or protease enzymes, such as lysosomes or endosomes, such as peptide linkers. For example, the cleavable linker may be any one selected from the group consisting of a protease cleavable linker, an acid-cleavable linker, a disulfide linker, a beta-glucuronide-based (β-based) linker, and a beta-galactoside-based (β-based) linker, but is not limited thereto. The non-cleavable linker allows the drug to be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0201] For example, a TSPAN1-binding antibody can be used as an antibody-drug conjugate for the treatment of cancer, e.g., solid tumors, more specifically HER2-positive breast cancer, HER2-low expression breast cancer, HER2-positive gastric cancer, etc. In this case, the drug may be emtansine, deruxtecan, or govitecan.

[0202] One aspect of the present invention also provides the use of said antibody for the preparation of a composition for the prevention or treatment of cancer and / or cancer metastasis, or for the diagnosis of cancer.

[0203] One aspect of the present invention also provides an antibody of the present invention for use in the prevention or treatment of cancer and / or cancer metastasis, or in the diagnosis of cancer.

[0204] One aspect of the present invention also provides a method for preventing or treating cancer and / or cancer metastasis, comprising administering the antibody to an individual in need.

[0205] One aspect of the present invention also provides a composition for diagnosing cancer and / or cancer metastasis comprising the antibody. The cancer is a cancer in which TSPAN1 is specifically expressed, and may be, for example, gastric cancer, uterine cancer, breast cancer, colorectal cancer, ovarian cancer, brain tumor, or pancreatic cancer, but is not limited thereto. The term "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to determine the occurrence of cancer or cancer metastasis.

[0206] In another aspect, the present invention provides a diagnostic kit for cancer and / or cancer metastasis comprising a diagnostic composition of the present invention. The kit can detect TSPAN1, specifically TSBS of TSPAN1, as a marker for cancer and / or cancer metastasis. The detection kit of the present invention may include one or more compositions, solutions, or devices suitable for an analytical method, as well as an antibody that selectively recognizes the marker.

[0207] Specifically, the diagnostic kit may comprise a matrix, a suitable buffer solution, a chromogenic enzyme or fluorescent substance, a chromogenic substrate, or a secondary antibody labeled for the immunological detection of the antibody. As the matrix, a nitrocellulose membrane, a 96-well plate made of polyvinyl resin, a 96-well plate made of polystyrene resin, and a slide glass may be used. As the chromogenic enzyme, peroxidase and alkaline phosphatase may be used. As the fluorescent substrate, FITC and RITC may be used, and as the chromogenic substrate solution, ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethyl benzidine) may be used.

[0208] In another aspect, the present invention provides a method for diagnosing cancer and / or cancer metastasis, comprising detecting the presence and / or amount of TSPAN1, specifically TSPAN1-Tsbs, through an antigen-antibody reaction in a biological sample separated from a subject. The biological sample includes, but is not limited to, samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.

[0209] Another aspect of the present invention relates to the use of amino acid fragments 110 to 211 of SEQ ID NO. 1 of TSPAN1 as an epitope in the development or manufacture of antibodies for inhibiting cancer and / or cancer metastasis.

[0210]

[0211] Hereinafter, the present invention will be described in detail with reference to the attached drawings and sequence list, etc., through embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to a person skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited by this. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0212] Furthermore, the terms used in this specification are used to appropriately describe embodiments of the present invention, and these may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0213] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0214] Throughout this specification, conventional one- and three-character codes for naturally occurring amino acids may be used. Additionally, amino acids referred to by abbreviations in this invention are described according to the IUPAC-IUB nomenclature as follows:

[0215] Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V.

[0216]

[0217] [Example]

[0218] 1. Explanation of the antibody discovery process - Screening process

[0219] It is important to identify antibodies with high specificity for the desired target. Therefore, we aim to discover antibodies that yield valid results through a screening process among various antibodies.

[0220] Antibody discovery was carried out using phage display. This method studies the interactions between proteins expressed on the surface of bacteriophages and various molecules, such as peptides, DNA, and other proteins. It is used to identify or develop highly specific antibodies against specific antigens by understanding the interactions between proteins or peptides. A filter lift assay was performed to identify hit substances among the various antibodies discovered in this way. This assay measures the degree of protein binding using a filter and can be applied when screening drugs.

[0221]

[0222] 1.1 Panning phage display

[0223] 1.1.1 Experimental Method

[0224] Day 0

[0225] For each phage library, 1 mL of 1xPBS was added to one tube, and the sample antigen and control antigen (Recombinant Rabbit IgG Protein (Sino Biological, Cat#11047-TNAH)) were coated, respectively. O / N coating was performed at 4℃.

[0226] Day 1

[0227] The immune tubes coated with the sample antigens and control antigens were washed three times with 1xPBS. 4 mL of 1% casein solution was added to each tube, and the tubes were blocked by rolling them up and down at room temperature for 1 hour. For the first panning, 5x10 per phage library 12 pfu IN1 phages were blocked with 1 mL of 1% BSA (Bovine Serum Albumin) (dissolved in 1x PBS (phosphate-buffered saline).

[0228] For the second and third panning, the entire library was blocked with 1 mL of 1% BSA. It was incubated at room temperature for 1 hour while being rolled up and down in a rotary rotator. After washing the control antigen tubes three times with 1x PBS, the phages from each blocked library were placed into the washed tubes, sealed with new stoppers, and incubated at room temperature for 2 hours while being rolled up and down for 1.5 hours, followed by 0.5 hours in a fixed state.

[0229] After washing the prepared sample antigen-coated tubes three times with 1x PBS, negative selection phages were placed into each coating tube and sealed with a new stopper. The tubes were incubated at room temperature for 2 hours, with rolling up and down for 1.5 hours, and then fixed for 0.5 hours. The unbound phages in each tube were discarded, and the tubes were washed 10 to 15 times with PBST (0.1% Tween 20 in 1x PBS).

[0230] 1 mL of 100 mM Triethylamine (TEA) was added to each tube, and the tubes were incubated upright at room temperature for 10 minutes. The eluent was transferred from each tube to a new tube, 500 μL of 1 M Tris-HCl (pH 6.4) was added and mixed well, and the tubes were incubated overnight at 4°C (output phage). A small amount of output phage was diluted, and an appropriate amount was added to 500 μL of overnight XL1-blue culture medium. The mixture was then added to 4 mL of top agar and incubated on an agar plate. The phage titer was checked by incubating overnight at 37°C.

[0231] Day 2

[0232] For each library, 0.5 mL of O / N XL1-blue bacteria was inoculated into 50 mL of 2xYT (Yeast Extract) culture containing 10 μg / mL tetracycline and incubated at 37°C and 220 rpm until an OD600 value of 0.4–0.6 was reached. The output phage of each library was added to the corresponding culture and incubated at 37°C for 30 minutes. After incubating for another 30 minutes at 37°C and 220 rpm, 10 μM IPTG (isopropyl β) was added to 2xYT in each flask, and the mixture was incubated for an additional 3 hours while shaking. The culture medium was centrifuged at 7,000 rpm and 4°C for 15 minutes using a SORVALL RC6 plus (Thermo, Cat. No. 46910) and an F13S-14x50cy rotor. 36 mL of the supernatant from each library was transferred to a new 50 mL tube, 9 mL of 20% PEG (Polyethylene Glycol) and 3.5 M NH4OAC were added and mixed thoroughly, then left on ice for 30 minutes. Centrifugation was performed at 7,500 rpm at 4°C for 30 minutes, the supernatant was discarded, and the sample was centrifuged briefly once more. The liquid remaining in the tube was removed by aspiration, and the phage was completely resuspended in an appropriate amount of 1x PBS. After centrifugation at 12,000 xg for 3 minutes using a benchtop centrifuge, the supernatant was transferred to a new tube (Input phage). The titration of the input phage was verified.

[0233]

[0234] 1.1.2 Experimental Results

[0235] HDB001 and HDB002 libraries were constructed by artificially expressing the antibody library retained by phages following an immune response to TSPAN1. The input phage is the set of phages initially used in the experiment and consists of initial phages expressing a specific protein or peptide. The output phage is the set of phages selected after interaction and consists of phages bound to a specific target protein. A higher output / input value indicates that the corresponding peptide has formed a strong binding to TSPAN1.

[0236] The experimental results above are shown in Figures 1a and 1b. Figure 1a shows the results for the fabricated HDB001 Phage Library, and Figure 1b shows the results for the HDB002 Phage Library.

[0237]

[0238] 1.2 Filter lift assay

[0239] 1.2.1 Experimental Method

[0240] Approximately 300 plaques were cultured on a single plate. The membrane was coated with an anti-kapp antibody, placed on a phage plate, and incubated overnight. The membrane was treated with a biotin-conjugated anti-HA antibody. The membrane was cultured with Neutravidin-AP. Positive clones were detected using NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolyl-phosphate) chromogenic substrates. The same procedure was applied to the experiment, divided into Batch 1 and Batch 2 groups.

[0241]

[0242] 1.2.2 Experimental Results

[0243] In both cases, 94 clones were detected in the fabricated Fab library. Among them, 8 valid clones were derived from HDB001 and 40 from HDB002. Based on these results, the results were verified once more through a phage ELISA experiment (Fig. 2).

[0244]

[0245] 1.3 SPE ELISA

[0246] 1.3.1 Experimental Method

[0247]

[0248] Day 0

[0249] TSPAN1 antigen was coated onto ELISA plates at a concentration of 2 μg / ml, and 50 μL was added to each well and incubated at 4°C for one day. (For each sample clone, one well coated with TSPAN1 antigen and one well coated with a control antigen were prepared.) To prepare high-titer (HT) phages, 20 μL of phages in elution buffer were dispensed into 800 μL of LB (Luria Bertani) medium, 10% XL1-blue cells and 10 μg / mL TET were added, and the mixture was incubated at 37°C with shaking at 220 rpm for one day.

[0250]

[0251] Day 1

[0252] The culture medium was centrifuged at 3,000 rpm for 30 minutes at 4°C. An appropriate amount of the supernatant obtained after centrifugation was transferred to a new U-bottom 96-well plate, and HT phages were blocked (to prevent non-specific binding) in 1x PBS containing 0.1% BSA at room temperature for 1 hour. The ELISA plate was washed three times with 1x PBS. The plate was blocked with 200 μL / well of 1% casein solution (diluted in 1x PBS) and incubated at room temperature for 1 hour. The plate was washed three times with 1x PBS. Blocked phages (50 μL / well) were transferred to the plate and incubated at room temperature for 2 hours. The plate was washed 10 times with 1x PBST. Approximately 0.2 μg / mL of anti-M13-HRP (50 μL / well) was added and incubated at room temperature for 1 hour. The plate was washed 10 times with 1x PBST. TMB (50 μL / well) was added and color development was allowed for 5 minutes. The reaction was stopped by adding 2M H2SO4 (50 μL / well). The absorbance was read at 450 nm using a plate reader.

[0253]

[0254] 1.3.2 Experimental Results

[0255] The results of the above phage ELISA experiments are shown in Figures 3 to 6. Figures 3a and 3b illustrate (indicated by stars) clones with high binding affinity to TSPAN1 relative to IgG-Fc in Batch 1 for HDB001. Among these, six clones are unique clones according to sequence analysis. Figures 4a and 4b illustrate (indicated by stars) clones with high binding affinity to TSPAN1 relative to IgG-Fc in Batch 2 for HDB001. Among these, five clones exhibited high binding affinity.

[0256] Figures 5a and 5b illustrate (starred) clones with high binding affinity to TSPAN1 relative to IgG-Fc in Batch 1 for HDB002. Of these, 24 clones are unique clones according to sequence analysis. Figures 6a and 6b illustrate (starred) clones with high binding affinity to TSPAN1 relative to IgG-Fc in Batch 2 for HDB002. Of these, 40 clones exhibited high binding affinity.

[0257] Among the effective clones detected in Figures 3 to 6, a total of 29 hits selected in order of highest binding affinity with TSPAN1 were divided into Expression Batch 1, 2, and 3 and are shown in Figure 7.

[0258]

[0259] 2. Antibody binding target, i.e., the antigen name and the sequence information of the recognition site (epitope) on the antigen

[0260] The antibody recognition site and sequence information of the TSPAN1 antigen was investigated through Uniprot (a database that provides basic information such as protein sequences, gene names, descriptions of sequences, phylogenetic classification, and citation information, as well as clear annotation information based on biological existence, biological classification, cross-references, experimental data, and computer data).

[0261] The sequence information of the TSPAN1 antigen is shown in Figure 8. Since the topological domain corresponding to the 110-211 position of sequence number 1 is the longest and is exposed to the extracellular region for easy access by antibodies, it was expected that targeting this recognition site, designated as 'Tsbs', would be advantageous. Therefore, the following was used as an antigen for phage display.

[0262]

[0263] 3. Amino acid sequence information of each of the complementation determining regions (CDR1, 2, 3) of the variable heavy chain region and the complementation determining regions (CDR1, 2, 3) of the variable light chain region of the antibody

[0264] Amino acid sequence analysis was performed on a total of 29 hits selected in order of high binding affinity to the above TSPAN1, and the results are shown in Figures 9a and 9b.

[0265]

[0266] 4. Measurement of antigen-antibody binding affinity

[0267] Hit substances were selected to measure whether they bind well to the corresponding targets. ELISA (Enzyme-linked immunosorbent assay), Surface plasmon resonance (SPR), and FACS (Fluorescence Activated Cell Sorting) methods were primarily used.

[0268] ELISA is a method that measures the presence and extent of antigen-antibody reactions using enzymes as markers. Through this method, antibodies favorable for new drug development were screened among various antibodies. SPR analysis is a technique that observes intermolecular binding, interactions, and dissociation rates in real time. This allows for the determination of binding rate constants and dissociation rate constants, and can be usefully applied to the study of protein interactions. FACS is an experimental technique that can classify cell mixtures into groups of cells with various characteristics and traits through fluorescent staining of the cell surface and interior.

[0269] 4.1 ELISA

[0270] 4.1.1 Experimental Method

[0271] TSPAN1 was coated onto an ELISA plate at a concentration of 2 µg / ml at a volume of 50 µl / well and incubated at 4°C for one day. On the next day, the ELISA plate was washed three times with PBS. To prevent non-specific binding, 1% casein was dispensed at a volume of 200 µl / well and left at room temperature for one hour. Subsequently, to prevent non-specific binding of the antibodies, each antibody was diluted with 0.1% BSA and left for one hour. After washing the ELISA plate three times with PBS, 50 µl of antibody was added and incubated at room temperature for two hours. The ELISA plate was washed ten times with PBST (0.05% Tween 20 in Phosphate-Buffered Saline), 50 µl of anti-human-IgG1-Fc HRP was added, and the plate was incubated for one hour. After washing the ELISA plate 10 times with PBST solution, 50 µl of TMB (3,3´,5,5'-tetramethylbenzidine) buffer was added and developed. Finally, 50 µl of H2SO4 was added to stop the reaction.

[0272]

[0273] 4.1.2 ELISA Experiment Results

[0274] ELISA experiments were conducted on the 29 hits obtained from '1.3.2 Experimental Results' above. The results are shown in Figure 10. EC 50 (The dose or concentration required to obtain 50% of the maximum effect in vivo) The antibodies with the lowest levels were T147, T403, T454, and T458. The above EC 50 Antibodies with low levels can increase the therapeutic dose of the drug.

[0275]

[0276] 4.2 SPR

[0277] 4.2.1 SPR Experimental Method

[0278] 4.2.1.1 Buffer Preparation

[0279] This is the running buffer required for SPR operation. 100 ml of 10x PBS, 5 ml of 10% Tween20, and 895 ml of ddH2O were mixed uniformly. The solution was passed through a vacuum filter, and the pH was adjusted to 7.4.

[0280]

[0281] 4.2.1.2 CM5 Chip Preparation

[0282] The CM5 chip was preheated to room temperature before being placed in the SPR measuring machine. The surface was primed with running buffer.

[0283]

[0284] 4.2.1.3 Immobilization of Anti-Human IgG (Fc) Antibody

[0285] A 0.5 mg / ml solution of Anti-Human IgG (Fc) was diluted with 10 nM sodium acetate (pH 5.0), an immobilization buffer, to prepare a final solution of 10 µg / ml. The sensor surface was activated with a mixture of EDC (N-ethyl-N′) and NHS (N-hydroxysuccinimide) by passing it through the flow path at a flow rate of 10 µl / min for 420 seconds. Anti-Human IgG (Fc) was passed through at 5 µl / min for 450 seconds. Then, ethanolamine was passed through at 10 µl / min for 7 minutes. The chip was equilibrated for 1 hour.

[0286]

[0287] 4.2.1.4 TSPAN1 Preparation

[0288] TSPAN1 protein was diluted to a concentration of 250 nM in running buffer. It was diluted twofold six times consecutively at a concentration of 250 nM. To remove foam from the protein solution, it was centrifuged at 1000 rpm for 5 minutes and transferred to an analysis plate.

[0289]

[0290] 4.2.1.5 SPR Analysis Program Settings

[0291] The sample compartment and chip temperature were set to 25℃. The instrument was operated 3 times with running buffer. A TSPAN1 protein dose curve was plotted under conditions of a contact time of 180 seconds, a separation time of 1200 seconds, and a flow rate of 30 ul / min.

[0292]

[0293] 4.2.2 SPR Experiment Results

[0294] EC 50 SPR analysis was performed on selected low IgG, and the results are shown in Figure 11.

[0295] The dissociation rate constants (Kd) of T147, T403, T454, T458, T358, T437, T470, T368, T304, and T384 are summarized in the table below.

[0296] IgGT147T403T454T458T358T437T470T368T304T384Kd (nM)0.8414.83.1420.612.89.7988.74.8410.52.21

[0297] A lower Kd value indicates a stronger target binding affinity.

[0298] 4.3 FACS

[0299] 4.3.1 Experimental Method

[0300] AsPC-1, U87MG, and SH-SY5Y cells were collected and resuspended in 1×DPBS containing 2% FBS to adjust the cell concentration to 1×10^6 cells / ml. Subsequently, the cells were incubated with test antibodies of different concentrations at 4°C for 30 minutes. After washing the cells twice with DPBS, a secondary antibody at a 1:1000 dilution was added and incubated at 4°C for 30 minutes. After washing the cells twice with DPBS, 200 μl of DAPI diluted at a 1:1000 ratio was added, and the cells were stained at 4°C for 10 minutes. Afterward, the samples were read using a flow cytometer.

[0301]

[0302] 4.3.2 Experimental Results

[0303] The above results are shown in Fig. 12, where T147, T403, T454, T458, T368, and T384 bound to cells, and among them, T147, T403, T454, and T458 showed particularly good binding characteristics to cells. This result demonstrates that the antibodies of the present invention can be used as antibody-drug conjugates.

Claims

1. An antibody or antibody fragment that specifically binds to TSPAN1, wherein the antibody or antibody fragment specifically binds to a region comprising amino acid fragments 110 to 211 of the TSPAN1 amino acid sequence represented by SEQ ID NO.

1.

2. In claim 1, the antibody or antibody fragment thereof comprises a CDR (Complementary Determining Regions) 1 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 2 to 4; CDR2 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 6; and An antibody or antibody fragment comprising a VH domain comprising a CDR3 selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs 7 to 35.

3. In paragraph 2, the antibody or its antibody fragment, CDR1 comprising the amino acid sequence of SEQ ID NO. 36 or SEQ ID NO. 37 (wherein SEQ ID NO. 36 is RAS Xa1 Xa2 Xa3 and Xa1 is Q, R or E, Xa2 is GINN, GIGG, GIRN, GISN, GIGT, GIAN, GISS, GIST, GISR, SISS, SISI, SVSR, SVSS, SVNS, NIRH, TITT, DISR, PINN, or NYNY, and Xa3 is YLG, WLA, NLA, YLN, YVN, HLN, FVN, or YLA); A CDR2 comprising an amino acid sequence of any one of SEQ ID NOs 38 to 44 (wherein SEQ ID NO 38 is AAS Aa1 Aa2, where Aa1 is S, T, or N, and Aa2 is LES, LQS, LPD, SQS, or LQR; SEQ ID NO 39 is GAS Ba1 Ba2, where Ba1 is S, N, R, or T, and Ba2 is LHS, LQS, VHS, LQS, LRS, LDS, or RAT; and SEQ ID NO 40 is Ca1 ASS Ca2, where Ca1 is S, K, or D, and Ca2 is LQS or LES); and An antibody or antibody fragment comprising a VK domain comprising a CDR3 having any one of the amino acid sequences of SEQ ID NOs 45 to 49 (wherein SEQ ID NO 45 is QQ Da1 Da2, where Da1 is SYST, SYSP, SYSS, GYSS, GYSL, YWSY, SYGT, SHSS, YNSW, ASSL, ASSF, GYIA, YYRS, YHSY, ANSF, ANSL, YGSS, AHSS, ANDF, or AHSF, and Da2 is PLT, PIT, PFT, PPT, PLS, PWT, or QLS).

4. In paragraph 2, the antibody or its antibody fragment, A VH domain comprising CDR 1 having the amino acid sequence of SEQ ID NO. 2, CDR 2 having the amino acid sequence of SEQ ID NO. 6, and CDR 3 having the amino acid sequence of SEQ ID NO. 7, or A VH domain comprising a CDR 1 having the amino acid sequence of SEQ ID NO. 3, a CDR 2 having the amino acid sequence of SEQ ID NO. 5, and a CDR 3 having any one selected from the group consisting of the amino acid sequences of SEQ ID NOs. 8 to 17, 19 to 23, 25, and 27 to 35, or An antibody or antibody fragment comprising a VH domain comprising a CDR 1 having the amino acid sequence of SEQ ID NO. 4, a CDR 2 having the amino acid sequence of SEQ ID NO. 5, and a CDR 3 having any one selected from the group consisting of the amino acid sequences of SEQ ID NO. 18, 24, and 26.

5. In paragraph 3, the above antibody or antibody fragment thereof, CDR 1 containing the amino acid sequence of SEQ ID NO. 36 (wherein SEQ ID NO. 36 is RAS Xa1 Xa2 Xa3, where Xa1 is Q, Xa2 is GINN, GIGG, GIRN, GISN, GIGT, GIAN, GISS, GIST, GISR, SISS, SISI, SVSR, SVSS, SVNS, TITT, DISR, PINN, or NYNY, and Xa3 is YLG, WLA, NLA, YLN, YVN, HLN, FVN, or YLA); A CDR2 comprising an amino acid sequence of any one of SEQ ID NOs 38 to 40 (wherein SEQ ID NO 38 is AAS Aa1 Aa2, where Aa1 is S or T and Aa2 is LES, LQS, LPD, or LQR; SEQ ID NO 39 is GAS Ba1 Ba2, where Ba1 is S or N and Ba2 is LHS, LQS, or LRS; and SEQ ID NO 40 is Ca1 ASS Ca2, where Ca1 is S, K, or D and Ca2 is LQS or LES); and An antibody or antibody fragment comprising a VK domain, comprising a CDR3 having any one of the amino acid sequences of SEQ ID NOs 45 to 49 (wherein SEQ ID NO 45 is QQ Da1 Da2, where Da1 is SYST, SYSP, SYSS, GYSS, GYSL, SYGT, SHSS, ASSL, ASSF, GYIA, YYRS, YHSY, ANSF, ANSL, YGSS, AHSS, ANDF, or AHSF, and Da2 is PLT, PIT, PFT, PPT, PLS, PWT, or QLS).

6. In paragraph 3, the above antibody or antibody fragment thereof, CDR1 containing sequence number 36 (wherein sequence number 36 is RAS Xa1 Xa2 Xa3, where Xa1 is Q, Xa2 is GINN, GIRN, GISN, GIGT, GISS, or SISI, and Xa3 is WLA or YLG); A CDR2 comprising SEQ NO 38, SEQ NO 39, or SEQ NO 40 (wherein SEQ NO 38 is AAS Aa1 Aa2, where Aa1 is S or N and Aa2 is LES or LQS; SEQ NO 39 is GAS Ba1 Ba2, where Ba1 is S and Ba2 is LHS or RAT; and SEQ NO 40 is Ca1 ASS Ca2, where Ca1 is D and Ca2 is LQS or LES); and An antibody or antibody fragment thereof comprising a VK domain, comprising CDR3 comprising SEQ ID NO.

45.

7. In any one of claims 1 to 6, the antibody or its antibody fragment, A VH domain comprising CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 6, and CDR3 of SEQ ID NO. 7, and A VK domain comprising CDR1 of SEQ ID NO. 50, CDR2 of SEQ ID NO. 51, and CDR3 of SEQ ID NO. 52; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 123, and A VK domain comprising CDR1 of SEQ ID NO. 124, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 126; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 9, and A VK domain comprising CDR1 of SEQ ID NO. 128, CDR2 of SEQ ID NO. 129, and CDR3 of SEQ ID NO. 46; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 10, and A VK domain comprising CDR1 of SEQ ID NO. 130, CDR2 of SEQ ID NO. 131, and CDR3 of SEQ ID NO. 132; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 11, and A VK domain comprising CDR1 of SEQ ID NO. 133, CDR2 of SEQ ID NO. 129, and CDR3 of SEQ ID NO. 134; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 12, and A VK domain comprising CDR1 of SEQ ID NO. 135, CDR2 of SEQ ID NO. 42, and CDR3 of SEQ ID NO. 47; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 13, and A VK domain comprising CDR1 of SEQ ID NO. 136, CDR2 of SEQ ID NO. 129, and CDR3 of SEQ ID NO. 137; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 14, and A VK domain comprising CDR1 of SEQ ID NO. 138, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 139; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 15, and A VK domain comprising CDR1 of SEQ ID NO. 140, CDR2 of SEQ ID NO. 141, and CDR3 of SEQ ID NO. 142; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 16, and A VK domain comprising CDR1 of SEQ ID NO. 136, CDR2 of SEQ ID NO. 143, and CDR3 of SEQ ID NO. 144; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 17, and A VK domain comprising CDR1 of SEQ ID NO. 145, CDR2 of SEQ ID NO. 146, and CDR3 of SEQ ID NO. 147; A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 18, and A VK domain comprising CDR1 of SEQ ID NO. 148, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 48; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 19, and A VK domain comprising CDR1 of SEQ ID NO. 149, CDR2 of SEQ ID NO. 41, and CDR3 of SEQ ID NO. 150; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 20, and A VK domain comprising CDR1 of SEQ ID NO. 151, CDR2 of SEQ ID NO. 152, and CDR3 of SEQ ID NO. 153; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 21, and A VK domain comprising CDR1 of SEQ ID NO. 154, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 56; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 22, and A VK domain comprising CDR1 of SEQ ID NO. 155, CDR2 of SEQ ID NO. 156, and CDR3 of SEQ ID NO. 157; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 23, and A VK domain comprising CDR1 of SEQ ID NO. 158, CDR2 of SEQ ID NO. 43, and CDR3 of SEQ ID NO. 159; A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 24, and A VK domain comprising CDR1 of SEQ ID NO. 160, CDR2 of SEQ ID NO. 55, and CDR3 of SEQ ID NO. 161; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 25, and A VK domain comprising CDR1 of SEQ ID NO. 162, CDR2 of SEQ ID NO. 163, and CDR3 of SEQ ID NO. 164; A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 26, and A VK domain comprising CDR1 of SEQ ID NO. 54, CDR2 of SEQ ID NO. 55, and CDR3 of SEQ ID NO. 56; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 27, and A VK domain comprising CDR1 of SEQ ID NO. 37, CDR2 of SEQ ID NO. 165, and CDR3 of SEQ ID NO. 139; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 28, and A VK domain comprising CDR1 of SEQ ID NO. 166, CDR2 of SEQ ID NO. 167, and CDR3 of SEQ ID NO. 49; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 29, and A VK domain comprising CDR1 of SEQ ID NO. 158, CDR2 of SEQ ID NO. 168, and CDR3 of SEQ ID NO. 169; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 30, and A VK domain comprising CDR1 of SEQ ID NO. 170, CDR2 of SEQ ID NO. 125, and CDR3 of SEQ ID NO. 171; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 31, and A VK domain comprising CDR1 of SEQ ID NO. 172, CDR2 of SEQ ID NO. 173, and CDR3 of SEQ ID NO. 174; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 57, and A VK domain comprising CDR1 of SEQ ID NO. 58, CDR2 of SEQ ID NO. 59, and CDR3 of SEQ ID NO. 60; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 61, and A VK domain comprising CDR1 of SEQ ID NO. 62, CDR2 of SEQ ID NO. 63, and CDR3 of SEQ ID NO. 64; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 28, and A VK domain comprising CDR1 of SEQ ID NO. 166, CDR2 of SEQ ID NO. 167, and CDR3 of SEQ ID NO. 49; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 35, and An antibody or antibody fragment thereof comprising a VK domain including CDR1 of SEQ ID NO. 175, CDR2 of SEQ ID NO. 176, and CDR3 of SEQ ID NO.

177.

8. In paragraph 7, the above antibody or antibody fragment thereof, A VH domain comprising CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 6, and CDR3 of SEQ ID NO. 7, and A VK domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 51, and CDR3 of SEQ ID NO. 52; A VH domain comprising CDR1 of SEQ ID NO. 4, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 26, and A VK domain comprising CDR1 of SEQ ID NO. 54, CDR2 of SEQ ID NO. 55, and CDR3 of SEQ ID NO. 56; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 57, and A VK domain comprising CDR1 of SEQ ID NO. 58, CDR2 of SEQ ID NO. 59, and CDR3 of SEQ ID NO. 60; A VH domain comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 61, and An antibody or antibody fragment comprising a VK domain including CDR1 of SEQ ID NO. 62, CDR2 of SEQ ID NO. 63, and CDR3 of SEQ ID NO.

64.

9. In any one of claims 1 to 6, the antibody or its antibody fragment, Includes the VH domain sequence of SEQ ID NO. 65 and the VK domain sequence of SEQ ID NO. 94, Includes the VH domain sequence of SEQ ID NO. 66 and the VK domain sequence of SEQ ID NO. 95, or Includes the VH domain sequence of SEQ ID NO. 67 and the VK domain sequence of SEQ ID NO. 96, or Includes the VH domain sequence of SEQ ID NO. 68 and the VK domain sequence of SEQ ID NO. 97, Includes the VH domain sequence of SEQ ID NO. 69 and the VK domain sequence of SEQ ID NO. 98, or Includes the VH domain sequence of SEQ ID NO. 70 and the VK domain sequence of SEQ ID NO. 99, or Includes the VH domain sequence of SEQ ID NO. 71 and the VK domain sequence of SEQ ID NO. 100, or Includes the VH domain sequence of SEQ ID NO. 72 and the VK domain sequence of SEQ ID NO. 101, or Includes the VH domain sequence of SEQ ID NO. 73 and the VK domain sequence of SEQ ID NO. 102, or Includes the VH domain sequence of SEQ ID NO. 74 and the VK domain sequence of SEQ ID NO. 103, or Includes the VH domain sequence of SEQ ID NO. 75 and the VK domain sequence of SEQ ID NO. 104, or Includes the VH domain sequence of SEQ ID NO. 76 and the VK domain sequence of SEQ ID NO. 105, or Includes the VH domain sequence of SEQ ID NO. 77 and the VK domain sequence of SEQ ID NO. 106, or Includes the VH domain sequence of SEQ ID NO. 78 and the VK domain sequence of SEQ ID NO. 107, or Includes the VH domain sequence of SEQ ID NO. 79 and the VK domain sequence of SEQ ID NO. 108, or Includes the VH domain sequence of SEQ ID NO. 80 and the VK domain sequence of SEQ ID NO. 109, or Includes the VH domain sequence of SEQ ID NO. 81 and the VK domain sequence of SEQ ID NO. 110, or Includes the VH domain sequence of SEQ ID NO. 82 and the VK domain sequence of SEQ ID NO. 111, or Includes the VH domain sequence of SEQ ID NO. 83 and the VK domain sequence of SEQ ID NO. 112, or Includes the VH domain sequence of SEQ ID NO. 84 and the VK domain sequence of SEQ ID NO. 113, or Includes the VH domain sequence of SEQ ID NO. 85 and the VK domain sequence of SEQ ID NO. 114, Includes the VH domain sequence of SEQ ID NO. 86 and the VK domain sequence of SEQ ID NO. 115, or Includes the VH domain sequence of SEQ ID NO. 87 and the VK domain sequence of SEQ ID NO. 116, or Includes the VH domain sequence of SEQ ID NO. 88 and the VK domain sequence of SEQ ID NO. 117, or Includes the VH domain sequence of SEQ ID NO. 89 and the VK domain sequence of SEQ ID NO. 118, or Includes the VH domain sequence of SEQ ID NO. 90 and the VK domain sequence of SEQ ID NO. 119, Includes the VH domain sequence of SEQ ID NO. 91 and the VK domain sequence of SEQ ID NO. 120, or Includes the VH domain sequence of SEQ ID NO. 92 and the VK domain sequence of SEQ ID NO. 121, or An antibody or antibody fragment comprising the VH domain sequence of SEQ ID NO. 93 and the VK domain sequence of SEQ ID NO.

122.

10. A pharmaceutical composition for the treatment or prevention of cancer or prevention of cancer metastasis, comprising an antibody or a fragment of the antibody according to any one of claims 1 to 6.

11. A composition according to claim 10, wherein the cancer is gastric cancer, uterine cancer, breast cancer, colorectal cancer, ovarian cancer, brain tumor, or pancreatic cancer.

12. A polynucleotide encoding an antibody or a fragment of the antibody according to any one of claims 1 to 6.

13. In claim 12, the polynucleotide is a polynucleotide that is any one of SEQ ID NOs 178 to 206.

14. An expression vector comprising the polynucleotide of claim 13.

15. A host cell containing the expression vector of claim 14.

16. A method for producing an antibody or an antibody fragment according to any one of claims 1 to 6, comprising culturing the host cells of claim 15 to produce an antibody by nucleic acid expression.

17. An antibody-drug conjugate comprising an antibody according to any one of claims 1 to 6 or a fragment of the antibody thereof, to which a drug is conjugated, wherein the drug is any one selected from the group consisting of toxins, chemotherapy agents, anticancer agents, antibiotics, ADP-ribosyltransferase, radioisotopes, and nucleases.

18. A composition for diagnosing cancer and / or cancer metastasis, comprising an antibody or a fragment of the antibody according to any one of claims 1 to 6.

19. A cancer and / or cancer metastasis diagnostic kit comprising the diagnostic composition of claim 18.

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