KR101906558B1 - Novel Antibody Specific For TSPAN8 and Uses Thereof - Google Patents

Abstract

The present invention relates to novel antibodies that specifically bind to TSPAN8 (tetraspanin 8) and uses thereof. More specifically, the present invention relates to an antibody that specifically binds to a large extracellular loop (LEL) of TSPAN8, a method for producing the antibody, a composition comprising the antibody and a composition for inhibiting cancer metastasis, A method for preventing or treating cancer and / or cancer metastasis by administering the above antibody or composition, a method for preventing or treating cancer and / or cancer metastasis, A cancer diagnostic kit comprising the composition, a method for diagnosing cancer using the composition, a cancer including a substance suppressing the expression of TSPAN8, and a method for inhibiting cancer metastasis A cancer and / or cancer metastasis kit comprising the composition, a method of inhibiting cancer and / or cancer metastasis using the composition, a method of treating cancer, In / or epitopes for antibodies that inhibit cancer metastasis to the use of the TSPAN8 LEL.

Description

Novel Antibody Specific For TSPAN8 and Uses Thereof < RTI ID = 0.0 >

The present invention relates to novel antibodies that specifically bind to TSPAN8 (tetraspanin 8) and uses thereof. More specifically, the present invention relates to an antibody that specifically binds to a large extracellular loop (LEL) of TSPAN8, a method of producing an antibody, a composition for inhibiting cancer and / or cancer metastasis comprising an antibody, A method for preventing or treating cancer and / or cancer metastasis by administering a composition, antibody or composition for preventing or treating cancer and / or cancer metastasis comprising an antibody, A cancer diagnostic kit comprising the composition, a method of diagnosing cancer using the composition, a method of inhibiting cancer and / or cancer metastasis, including a substance for inhibiting the expression of TSPAN8, A method for inhibiting or treating cancer and / or cancer metastasis using the cancer and / or cancer metastatic use kit, composition comprising the composition for cancer and / or cancer metastasis, For antibodies for inhibition it relates to the use of using a TSPAN8 LEL to the epitope.

Metastasis is a major cause of cancer mortality and mortality worldwide. Over the past few decades, there has been a significant improvement in cancer treatment through the rapid development of small compounds and therapeutic antibodies. However, the current method has not successfully overcome tumor metastasis. Thus, a reasonable method for increasing therapeutic efficacy depends on the discovery of novel therapeutic targets associated with tumor metastasis and the subsequent development of therapeutics that specifically target these factors.

The tetraspanin superfamily consists of members comprising two extracellular loops including four membrane-pore domains, a small extracellular loop (SEL) and a large extracellular loop (LEL), and a cytosolic domain of three.

To date, evidence suggests that tetraspanin is closely related to tumor progression. Tetrapanin CD20 is predominantly overexpressed in malignant human B cells. Rituximab, a recombinant mouse / human chimeric monoclonal antibody to CD20, has been used clinically to treat patients with non-Hodgkin's B-cell lymphoma, and several additional therapeutic antibodies to CD20 are currently under clinical development. Another tetraspanin, CD81, is overexpressed in hepatocellular carcinoma and plays an important role as a receptor for hepatitis C virus-induced liver cancer. Overexpression of tetraspanin CD151 is also observed in several cancer types, including invasive breast cancer, prostate cancer, and squamous cell carcinoma of the skin. In the case of breast cancer in particular, CD151 has been identified as potentially a novel prognostic marker and thus provides a target for treatment.

TSPAN8 (CO-029) is also a member of the tetraspanin superfamily. Previous studies have suggested the biological function of human TSPAN8 with about 70% sequence homology with the murine homolog of D6.1A. D6.1A has been classified as a metastatic-related tetraspanin molecule. It has been reported that the association of D6.1A and [alpha] 6 [beta] 4 integrins is important for the formation of cell motility and liver metastasis. More recently, several reports have shown that TSPAN8 is directly involved in tumor metastasis.

Upregulation of TSPAN8 was observed in hepatocellular carcinoma, colorectal cancer, and pancreatic adenocarcinoma. Showed that TSPAN8 is involved in the metastatic interstitial transformation of liver cancer cells through orthotropic implantation of non-metastatic HCC cell lines containing stably transfected TSPAN8.

Under these technical backgrounds, the inventors of the present application confirmed the clinical significance of TSPAN8 in tumor progression and identified antibodies that specifically bind to TSPAN8 that can effectively inhibit cancer and / or cancer metastasis.

The information disclosed in the technical background section is intended to enhance the understanding of the background of the present invention and may therefore include information that does not constitute prior art already known to those skilled in the art.

The inventors of the present application confirmed the importance of TSPAN8 in regulating the invasion of cancer cells without affecting proliferation or binding to endothelial cells, using clinical samples and cancer cell lines.

Also, using phage display technology, the inventors of the present application developed a recombinant human antibody specific for TSPAN-LEL and confirmed that they can down-regulate the cell surface TSPAN8 on cancer cells to specifically inhibit tumor invasion. The efficacy was evaluated.

Finally, using the animal metastasis model, the inventors of the present application have demonstrated the in vivo efficacy of antibodies developed in relation to tumor metastasis inhibition. In summary, it provides evidence for further understanding of TSAPN8-LEL as a therapeutic target for tumor metastasis, as well as information to support the diagnostic and therapeutic potential of human antibodies developed in this study.

In order to achieve this object, the present invention provides an antibody that specifically binds to TSPAN8 (tetraspanin 8) and specifically binds to the LSP (large extracellular loop) of TSPAN8.

The present invention also provides an antibody that specifically binds to TSPAN8 and specifically binds to a site comprising amino acid fragments 110 to 205 of the TSPAN8 amino acid sequence represented by SEQ ID NO: 7.

The present invention also provides an antibody that specifically binds to TSPAN8 and specifically binds to a region comprising amino acid fragments 31 to 96 of the amino acid sequence of LEL in TSPAN8 represented by SEQ ID NO: 9.

The present invention also provides a nucleic acid encoding said antibody.

The present invention also provides a vector comprising said nucleic acid.

The present invention also provides a host cell comprising said vector or nucleic acid.

The present invention also provides a method for producing an antibody, comprising culturing the host cell to produce an antibody by nucleic acid expression.

The present invention also provides an antibody-drug conjugate comprising said antibody to which the drug is conjugated.

The present invention also provides a pharmaceutical composition for preventing or treating cancer or cancer metastasis comprising the antibody.

The invention also provides the use of said antibody for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer and / or cancer metastasis.

The present invention also provides an antibody for use in the prevention or treatment of cancer and / or cancer metastasis.

The present invention also provides a method of preventing or treating cancer and / or cancer metastasis, comprising administering the antibody to a subject in need thereof.

The present invention also provides a composition for inhibiting cancer and / or cancer metastasis comprising the antibody.

The present invention also provides the use of said antibody for the manufacture of a composition for inhibiting cancer and / or cancer metastasis.

The present invention also provides an antibody for use in the suppression of cancer and / or cancer metastasis.

The present invention also provides a method of inhibiting cancer and / or cancer metastasis comprising administering the antibody or the pharmaceutical composition to a subject in need thereof.

The present invention also provides a composition for diagnosing cancer and / or cancer metastasis comprising the antibody.

The present invention also provides the use of said antibody for the production of a diagnostic composition for cancer and / or cancer metastasis.

The present invention also provides antibodies for in-vitro diagnosis of cancer and / or cancer metastasis.

The present invention also provides a diagnostic kit for cancer and / or cancer metastasis comprising the diagnostic composition.

The present invention also provides a method for diagnosing cancer and / or cancer metastasis, comprising detecting TSPAN8 through an antigen-antibody complex in a biological sample isolated from cancer and / or cancer metastatic suspected individuals.

The present invention also provides polypeptides comprising the isolated TSPAN8 LEL that function as an epitope capable of inducing the production of antibodies that inhibit cancer and / or cancer metastasis.

The present invention also provides polypeptides comprising an N-terminal or C-terminal end of a TSPAN8 LEL isolated as an epitope capable of inducing the production of an antibody that inhibits cancer and / or cancer metastasis.

The present invention also provides a polypeptide comprising amino acid fragments 110 to 205 of the LEL of TSPAN8 as an epitope capable of inducing the production of an antibody that inhibits cancer and / or cancer metastasis.

The present invention also provides a method of producing an antibody that specifically binds to TSPAN8 comprising biopanning using the polypeptide as an epitope.

The present invention also provides a composition for inhibiting cancer and / or cancer metastasis comprising a substance that inhibits the expression of TSPAN8.

The present invention also provides a kit for inhibiting cancer and / or cancer metastasis comprising the composition.

The present invention also provides a method for inhibiting cancer and / or cancer metastasis, comprising administering the composition to a subject in need thereof.

The present invention also provides a method of preventing or treating cancer and / or cancer metastasis comprising administering the composition to a subject in need thereof.

The present invention also provides the use of a substance that inhibits the expression of TSPAN8 for the production of said cancer and / or cancer metastatic inhibiting composition.

The present invention also provides a substance that inhibits the expression of TSPAN8 for use in inhibiting cancer and / or cancer metastasis.

The present invention also provides the use of the LEL and Fc fusion proteins of TSPAN8 for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer and / or cancer metastasis.

The present invention also provides LEL and Fc fusion proteins of TSPAN8 for use in the manufacture of a pharmaceutical composition for the prevention or treatment of cancer and / or cancer metastasis.

Other configurations and embodiments of the present invention will become more apparent from the following detailed description and the appended claims.

Based on the novel finding that LSP of TSPAN8 plays an important role in cancer and metastasis, the present invention has confirmed the function of a novel antibody against LEL of TSPAN8 in cancer and / or cancer metastasis inhibition. Therefore, the present invention provides a novel target region capable of inhibiting cancer and / or cancer metastasis. In addition, the antibody of the present invention may be effective for preventing or treating cancer and / or cancer metastasis.

Figures 1A-1D show that TSPAN8 is overexpressed in colorectal cancer. Immunohistochemical staining with anti-TSPAN8 antibody was performed and the expression of TSPAN8 (N = 59) was measured in normal (N = 59) and colorectal (N = 59) samples. (FIGS. 1A and 1B) Representative immunohistochemical staining of TSPAN8 in normal and colorectal tissues. The bar graph shows the percentage of positive staining for TSPAN8. (Fig. 1c and Fig. 1d) Representative immunohistochemical staining of TSPAN8 in normal and color 1-4 cancer samples. The bar graph shows the percentage of positive staining for TSPAN8. Expression of TSPAN8 was stained brown by DAB. The square represents an area of 20 X magnification. Data are expressed as mean ± SEM. * P < 0.05.
Figures 2A-B show that overexpression of TSPAN8 promotes infiltration of COS-7 cells without affecting the proliferation and adhesion of HUVEC. 2a: Western blot analysis of lysates of COS-7 cells transfected with mock or TSPAN8 using anti-TSPAN8 antibody. 2b: Infiltration activity was measured in COS-7 cells transfected with TSPAN8 using a Matrigel-coated transwell chamber as described in Materials and Methods. The image shows cells invading in the membrane. The bar graph shows the results as a percentage of the control. Figure 2c: Cell counting kit-8 (CCK-8) assay of TSPAN8 transfected COS-7 cell proliferation after 48 h incubation. Figure 2d: TSPAN8 transfected COS-7 cells attached to human umbilical vein endothelial cells (HUVEC). Data are expressed as mean ± SEM. * P < 0.05.
Figures 3a-3d show that knockdown of TSPAN8 reduced invasion of HCT-8 colon cancer cells without affecting the proliferation and adhesion of HUVEC. Figure 3a: Western blot analysis of lysates of TSPAN8 siRNA transfected HCT-8 cells using anti-TSPAN8 antibody. Figure 3b: Matrix gel coated transwell chamber as described in Materials and Methods was used to measure invasion activity in TSPAN8 siRNA transfected HCT-8 cells. The image shows cells invading in the membrane. The bar graph shows the results as a percentage of the control. Figure 3c: Cell Counting Kit-8 (CCK-8) assay of HCT-8 cell proliferation transfected with TSPAN8 siRNA after 48 hours of incubation. Figure 3d: TSPAN8 siRNA transfected HCT-8 cells attached to human umbilical vein endothelial cells (HUVEC). Data are expressed as mean ± SEM. * P < 0.05.
Figures 4A-4C show the functional roles of TSPAN8-LEL in HCT-8 cell infiltration and characteristics of human antibody (scFv-FC) specific for TSPAN8-LEL. Figure 4a: Invasion assay was performed with HCT-8 colon cancer cells in the presence of purified TSPAN1-LEL-FC, TSPAN8-SEL-FC, TSPAN8-LEL-FC, or control Fc. The image shows cells invading in the membrane. Results were expressed as percentage of control. Data are expressed as mean ± SEM. * P < 0.05. Figure 4b: The binding specificity of the TSPAN8 scFv-FC clone was determined by indirect ELISA using Fc, TSPAN1-LEL-FC, TSPAN8-SEL-FC and TSPAN8-LEL-FC. Figure 4c: Western blot analysis of TSPAN8 scFv-FC clones bound to TSPAN8-LEL in non-reducing conditions.
Figures 5A-5C show that TSPAN8 scFv-FC inhibits the invasion of metastatic colorectal cell lines. Figure 5a: Downregulation of the < RTI ID = 0.0 &gt; P120 < / RTI &gt; Western blot analysis of lysates of HCT-8 cells transfected with p120 catenin siRNA using anti-p120 Cathode Antibody. Figure 5b: Infiltration activity was measured in HCT-8 cells transfected with p120 catenin siRNA after treatment of control scFv-FC or TSPAN8 scFv-FC. Infiltration was measured using a Martwell gel coated transwell chamber. Figure 5c: HCT116, LoVo and SW480 cells were treated with one of the control scFv-FC or TSPAN8 scFv-FC and infiltration assays were performed using a Martwell gel coated transwell chamber. The image shows cells invading in the membrane. Results were expressed as percentage of control. Data are expressed as mean ± SEM. * P < 0.05.
Figure 6 shows that TSPAN8 scFv-FC inhibits the invasion of other metastatic cancer cells, including U87 and PANC-1. Infiltration analysis was performed in metastatic U87 brain glioma and PANC-1 pancreatic cancer cell line to determine the effect of TSPAN8 scFv-FC (100 [mu] g / ml). The image shows cells invading in the membrane. Results were expressed as percentage of control. Data are expressed as mean ± SEM. * P &lt; 0.05.
Figure 7 shows that TSPAN8 IgG inhibits the invasion of metastatic HCT116 colon cancer cells. Infiltration analysis was performed in metastatic colorectal HCT116 cells to determine the effect of TSPAN8 IgG (20 [mu] g / ml) or Erbitux (20 [mu] g / ml). The image shows cells invading in the membrane. The bar graph was measured and the results were expressed as a percentage of the control. Data are expressed as mean ± SEM. * P &lt; 0.05.
Figure 8 shows enhanced TSPAN8 expression in ovarian cancer. Immunohistochemical staining was performed using anti-TSPAN8 antibody in normal (N = 5) and ovarian cancer tissues (N = 55) to predict the clinical significance of TSPAN8 expression in ovarian cancer progression. Typical examples of staining intensity are shown in various tissue samples. Blue represents nucleus, and brown represents TSPAN8 expression. Values were measured from various tissue samples and expressed as mean ± SEM. * P &lt; 0.05
Figure 9 shows enhanced TSPAN8 expression in ovarian cancer. Immunohistochemical staining was performed using anti-TSPAN8 antibody in normal (N = 5) and ovarian cancer tissues (N = 55) to predict the clinical significance of TSPAN8 expression in ovarian cancer progression. Relative expression of TSPAN8 is expressed as a percentage of the control (normal). Values were measured from various tissue samples and expressed as mean ± SEM. * P &lt; 0.05
Figures 10a-b show the inhibitory effect of TSPAN8 knockdown on cell invasion. Figure 10a: SK-OV-3 cell lysates transfected with scrambled or TSPAN8 siRNA were analyzed by immunoblotting with anti-TSPAN8 or anti-beta-actin antibody. Fig. 10b: Cell infiltration of SK-OV-3 cells transfected with scrambled or TSPAN8 siRNA was investigated. After 22 hours of transfection, images were taken. Left is the cell infiltration analysis photograph. The right side shows the number of infiltrating cells as a percentage. Values are expressed as the mean ± SEM measured three times from one of three independent experiments.
Figures 11A-11B show the inhibitory effect of TSPAN8 antibody on cell invasion. 11a: Ovarian cancer cell infiltration was measured in a modified Boyden chamber equipped with a Matrigel-coated membrane filter insert. SK-OV-3 cells treated with control IgG on the left. SK-OV-3 cells (100 X magnification) treated with TSPAN8 IgG on the right. Figure llb: Cellular infiltration is quantified by cell counts and expressed as a percentage of control in any three fields in one of three independent experiments.
Figure 12 shows that TSPAN8 IgG induces the internalization of cell surface TSPAN8 in colon cancer and ovarian cancer cell lines. FACS analysis of HCT116 or SK-OV-3 cells treated with TSPAN8 IgG in the presence or absence of defined conditions. The dashed line represents the unstained control and the solid line represents DyLight-488 TSPAN8 IgG.
Figures 13a-13c show the inhibitory effect of TSPAN8 antibody on in vivo metastasis of SK-OV-3 cells. Figure 13a: Schematic representation of the production of an upper, ovarian cancer xenograft model. Bottom, Abdominal injection schedule of TSPAN8 antibody. A black arrow indicates the day of antibody injection. Figure 13b: Biomolecular imaging of luciferase signaling in SK-OV-3 cells. The tumor expressing luciferase was intraperitoneally injected into the peritoneum of Balb / c nude mice to create xenotransplantation animals. Figure 13c: Molecular imaging of the luciferase signal in the peritoneum surrounding the colon, rectum, liver, kidney and ovary.
Figures 14a-14c show the effect of TSPAN8-LEL IgG on TSPAN8-mediated infiltration of epithelial ovarian cancer cell lines. 14A: Immunoblot analysis showing TSPAN8 expression in epithelial ovarian cancer cell lines. 14b: The number of invasive epithelial ovarian cancer cell lines was measured in the presence of control IgG or TSPAN8-LEL IgG and expressed as a percentage of control infiltration. All values are expressed as mean ± SEM of triplicate from one of three independent experiments.
15a-15c show confirmation of the binding site of TSPAN8-LEL IgG in TSPAN8-LEL. 15A: Schematic diagram of N- or C-terminal deletion mutants of TSPAN8-LEL (TSPAN8-LEL-N1, -N2, -N3 and -C1) such as wild type and FC fusion proteins. The cysteine residues involved in the disulfide bridges to form the steric structure are shown as C42, C43, C62, C64, C71 and C84. 15b: TSPAN8-LEL, TSPAN8-LEL-N1, TSPAN8-LEL-N2, TSPAN8-LEL-N3 and TSPAN8 LEL-C1 were coated onto 96-well microtiter plates. The binding specificity of TSPAN8-LEL IgG to these proteins was measured by ELISA. Values are expressed as mean ± SEM of triplicate from one of three independent experiments. 15c: Binding of the antibody to this purified protein was confirmed by immunoblot analysis.
Figures 16a-16c show the effect of TSPAN8-LEL IgG on the internalization and downregulation of TSPAN8 in SK-0V3 cells. Figure 16a: Down-regulation of TSPAN8 in SK-0V3 cells in the presence of control IgG () or TSPAN8-LEL IgG () was measured by cell ELISA in a time-dependent manner. Values are expressed as mean ± SEM of triplicate from one of three independent experiments. Figure 16b: Immunoblot analysis showing the effect of control IgG or TSPAN8-LEL IgG on down-regulation of TSPAN8 in SK-0V3 cells (top plate). Band intensity is shown in the graph bar (bottom panel). Figure 16c: SKOV3 cells were fixed and stained with lysotracker after treatment with FITC-labeled TSPAN8-LEL IgG. The location of TSPAN8-LEL IgG was examined using confocal microscopy. The results represent three separate experiments.
17a-17c show the effect of TSPAN8-LEL IgG on the frequency of SK-OV3 cell metastasis. Figure 17a: Schematic diagram of control IgG or TSPAN8-LEL IgG treatment protocol. Figure 17b: Peritoneal transfer mouse model of human ovarian cancer was made by injecting SK-OV3-luc. After antibody treatment, the frequency of SK-OV3-luc metastasis in the control IgG- or TSPAN8-LEL IgG treated groups was monitored by bioluminescence imaging. 17c: The frequency of SK-OV3-luc transposition is expressed as the number of mice in which the luminescent signal is detected in isolated organs and is expressed as a percentage of the control frequency of the SK-OV3-luc transposition.

In one aspect, the invention provides an antibody that specifically binds TSPAN8 (tetraspanin 8). Preferably, the antibody may be an antibody that specifically binds to the LSP (large extracellular loop) of TSPAN8.

Tumor metastasis is a complex process that is regulated by many regulatory signaling molecules. Thus, the discovery of new therapeutic targets for tumor therapy is essential. Here we first identified the clinical significance of TSPAN8 by comparing the expression of TSPAN8 in normal and colorectal cancers. Using the ectopic expression or knockdown of TSPAN8, the present inventors have demonstrated that TSPAN8 is important for colorectal cancer cell infiltration but not for endothelial cell proliferation or adhesion. More specifically, the present inventors have demonstrated that the large extracellular loop (LEL; amino acids 110-205) of TSPAN8 plays an important role in the invasion of colon cancer cells.

Next, recombinant human antibodies specific for LEL of TSPAN8 were screened from human synthetic antibody libraries using phage display technology and confirmed to more strongly inhibit the invasion of metastatic colorectal cancer cells than invasion of non-metastatic colorectal cancer cells . The inventors of the present application then performed immunohistochemical staining to verify the clinical relevance of TSPAN8 on ovarian cancer.

The inventors of the present application also confirmed the functional relevance of TSPAN8 to ovarian cancer cell infiltration using the knockdown of TSPAN8 or anti-TSPAN8 antibodies. Interestingly, the results of flow cytometry showed that the anti-TSPAN8 antibody down-regulates cell surface TSPAN8 in colon cancer and ovarian cancer cells through internalization.

Finally, the present inventors have used an ovarian tumor metastasis animal model to confirm that the anti-TSPAN8 antibody significantly reduces the metastasis of ovarian cancer cells. Taken together, these results suggest that the LEL of TSPAN8 may be a target for therapy to alter the progression of metastatic colorectal cancer and ovarian cancer, and that human antibodies targeting this domain have therapeutic and diagnostic potential.

The term "antibody" of the present invention means a protein molecule comprising an immunoglobulin molecule that immunologically reacts to a specific antigen that functions as a receptor that specifically recognizes an antigen, and includes a polyclonal antibody, a monoclonal antibody, Quot; means a protein molecule intended to comprise an antibody fragment. In addition, chimeric antibodies (e. G., Humanized murine antibodies), bifunctional or bispecific molecules (e. G. Bispecific antibodies), diabodies, triabodies and tetrabodies are within the scope of antibodies useful in the present invention . The whole antibody consists of a disulfide bond between the light and heavy chains and two full-length light chains and two full-length heavy chains. Mammals have five antibody isotypes known as IgA, IgD, IgE, IgM and IgG, and IgG is further divided into four subtypes, IgG1, IgG2, IgG3 and IgG4. In the present invention, "antibody fragment" means a fragment having at least an antigen-binding function, and may include Fab, F (ab ') 2, F (ab') 2 and Fv. Fab is an antigen binding site, consisting of a variable region of heavy and light chains, a constant region of light chain, and a first constant region (CH1) of heavy chain, respectively. Fab 'differs from Fab in that it contains one or more cysteine residues at the C-terminus of the CH1 domain of the heavy chain. F (ab ') 2 consists of two Fab' molecules with disulfide bonds between the cysteine residues in the hinge region. The FV (variable fragment) consisting of one variable region of each of the heavy and light chains is the minimal antibody fragment containing the original specificity of the parent immunoglobulin. Disulfide-stabilized FV (hsFv) is formed by connecting a variable region of a heavy chain to a variable region of a light chain through a disulfide bond. Single-chain FV (scFv) is an FV in which each variable region of the heavy chain and the light chain is linked by a covalent bond by a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be separated into papain or pepsin to obtain Fab or F (ab '), respectively), preferably produced by recombinant techniques .

Typically, antibody screening has been performed using the entire extracellular site of the target protein, resulting in problems in the isolation of the antibody. In the present invention, antibody screening using the functional domain of the target protein has been improved in order to produce antibodies more rapidly. In one embodiment of the invention, the LEL of TSPAN8 has been shown to play an important role in cancer and / or cancer metastasis and has been used in antibody screening. The process of antibody screening is schematically illustrated in Fig.

The term "monoclonal antibody" of the present invention means an antibody molecule of uniform molecular composition obtained from a substantially identical population of antibodies exhibiting binding specificity and affinity for a single epitope.

Generally, immunoglobulins have basic structural units consisting of two heavy chains and two light chains. Each heavy chain contains one variable region (also referred to as a "region") and three constant regions, while each light chain is comprised of one variable region and one constant region. Each variable region of the light and heavy chains contains three complementarity determining regions (CDRs) and four framework regions. The CDRs act to bind to the epitope of the antibody. The CDRs in each chain start at the N-terminus and are sequentially positioned with CDR1, CDR2 and CDR3. These are identified by the chain in which they are deployed.

The term "human antibody" of the present invention is a molecule consisting entirely of the amino acid sequence of all components of human immunoglobulins, including CDRs, framework regions, and the like. In the treatment of human diseases, human antibodies have at least three potential advantages. First, more preferably, the human antibody interacts with a human immune system that more effectively destroys the target cell by, for example, complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Another advantage is that the human immune system does not recognize human antibodies as external molecules. In addition, they are similar to those of naturally occurring antibodies in the human circulatory system, even when administered at smaller doses or less frequently. Thus, an antibody according to the invention may be a human monoclonal antibody, which may be useful for the treatment of cancer and / or cancer metastasis related diseases or cancers, as it has a strong affinity for TSPAN8, and TSPAN8-mediated TSPAN8-LEL expressed in human endothelial cells effectively inhibiting cancer or cancer metastasis is preferred.

The term "TSPAN8 " of the present invention means a member of the tetraspanin phase. TSPAN8 consists of members comprising two extracellular loops, including four transmembrane domains, a small extracellular loop (SEL) and a large extracellular loop (LEL), and three cytoplasmic domains. Information on TSPAN8 can be obtained from public databases such as NCBI's GenBank. For example, the human gene TSPAN8 may be, but is not limited to, Gene ID No 7103. Preferably, TSPAN8 can be obtained from a human, chimpanzee or mouse.

In the present invention, "large extracellular loop (LEL) of TSPAN8" is interchangeable with "TSPAN8-LEL"

In another aspect, the present invention provides isolated polypeptides of TSPAN8-LEL as epitopes capable of inhibiting cancer and / or cancer metastasis, and in particular, the present invention provides methods for inducing the production of antibodies that inhibit cancer and / or cancer metastasis The present invention provides isolated polypeptides comprising amino acid fragments 110 to 205 of TSPAN8 having an amino acid sequence represented by SEQ ID NO: More specifically, the present invention provides isolated polypeptides comprising amino acid fragments 31 to 96 of the LEL amino acid sequence in TSPAN8 represented by SEQ ID NO: 9. The present inventors first confirmed that LEL, that is, domains 110 to 205 of the amino acid sequence of TSPAN8, particularly amino acid fragments 31 to 96 of the LEL amino acid sequence in TSPAN8, plays an important role in cancer and / or cancer metastasis.

The term "epitope" in the present invention refers to a portion of an antibody that determines antigen specificity and can be used to interchange an antigenic determinant or an antigenic determinant site. For purposes of the present invention, the epitope refers to amino acid fragments 31 to 96 of the LEL amino acid sequence in TSPAN8, particularly LEL having amino acids 110 to 205 in TSPAN8, particularly TSPAN8 that can be used to inhibit cancer and / or cancer metastasis, Or a polypeptide having the same function as LEL. Thus, additional regions of the LEL may be included. Polypeptides having an amino acid sequence homology of 80%, 85%, 90%, 95%, 98% or 99% or more can be used as an epitope, as long as they play the same role in LEL. Sequences 110 to 205 of the TSPAN8 amino acid sequence, which is the LEL amino acid sequence of human, mouse and chimpanzee, are shown in Table 1 and are represented by SEQ ID NO: 7 (amino acid sequence of TSPAN8) and SEQ ID NO: 9 (amino acid sequence of LEL), respectively. The present inventors first confirmed that the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 9 can be used as epitopes used to produce antibodies that inhibit cancer and / or cancer metastasis.

Also, in one preferred embodiment, it has been suggested that the N-terminus or C-terminus of LEL may serve as an epitope that can be used to produce antibodies that inhibit cancer and / or cancer metastasis. Preferably, the region comprising amino acid fragments 31 to 96 of the amino acid sequence in the LEL may be an epitope for the antibody.

The antibody may specifically bind to TSPAN8-LEL or an effective fragment thereof for inhibiting cancer and / or cancer metastasis, and the present inventors have suggested that TSPAN8-LEL is a unique region that regulates cancer and / or cancer metastasis.

Tumor metastasis is a major cause of cancer deaths worldwide. Tumor migration and invasion into the peripheral organs, and mechanisms for tumor metastasis pathways have been attracting interest as promising targets for cancer therapy; However, tumor metastasis is complex and results in the coordination of many signaling molecules. This study confirmed the biological role of TSPAN8-LEL in tumor cell infiltration. Then, a recombinant human antibody targeting TSPAN8-LEL was developed and its efficacy and mechanism of action were evaluated. TSPAN8-LEL is an important factor in tumor metastasis, and TSPAN8-LEL specific antibodies may have therapeutic potential.

Several reports have suggested that TSPAN8 plays an important role in regulating cell motility; However, the molecular mechanism underlying this function is not clearly known. The inventors of the present application suggest that TSPAN8-LEL (amino acids 110-205) can be a unique domain that regulates cancer cell infiltration during tumor metastasis. Several pieces of evidence support this concept. First, overexpression of TSPAN8 in COS-7 cells promotes cell invasion but does not promote cell proliferation or adhesion with HUVEC. Second, in the HCT-8 colon cancer cell line, TSPAN8 knockdown inhibited HCT-8 cell infiltration, whereas HUVEC did not affect proliferation and adhesion. Third, HCT-8 cell infiltration was inhibited by TSPAN8-LEL rather than by TSPAN8-SEL. Fourth, knockdown of TSPAN8 also inhibited SK-0V3 cell infiltration in SKOV3 ovarian cancer cell lines.

Over the past two decades, the rapid development of recombinant antibody technology has led to a remarkable increase in the therapeutic antibody market. Recently, recombinant therapeutic antibodies have been used clinically for the treatment of tumors, but many are still under development. Since many medical needs are not met, identification of novel therapeutic targets for improved tumor therapy in response to drug resistance and side effects is of paramount importance. In this study, the inventors provide some evidence to support the functional relevance of TSPAN8 as a therapeutic target during tumor metastasis. Its clinical significance was confirmed by immunohistochemical staining when TSPAN8 was overexpressed in colorectal and ovarian cancer. In colorectal cancer in particular, TSPAN8 overexpression appears to be clinically dependent. The biological function of cancer cell infiltration was confirmed by ectopic expression or gene silencing of TSPAN8. Human recombinant antibodies identified to target TSPAN8-LEL also inhibited the invasion of pancreatic cancer cell lines, ovarian and brain cell lines, including SK-0V3, U87 and PANC-1, as well as colorectal cancer cell lines HCT-8, HCT116, LoVo and SW480 Respectively. In addition, in vivo efficacy studies, the antibody inhibited the metastasis of SKOV3 ovarian cancer cells. These results suggest that TSPAN8 may be a suitable therapeutic target for tumor therapy.

Tetraspanin plays an important role as a molecular promoter to form molecular webs to combine large molecule complexes and to promote its efficient function. Depending on the interacting molecule, tetraspanin is involved in many other cellular functions such as adhesion, motility and cell fusion. Previous studies have shown that TSPAN8 complexes with several binding partners, including EpCAM, claudin-7, and / or CD44v6, and modulates cancer cell motility. In this study, human antibodies targeting TSPAN8-LEL down-regulated TSPAN8 on the cell surface through an internalization process, suggesting that this process may be suitable as a therapeutic antibody-drug complex. Based on current evidence, the inventors of the present application contemplate that the mechanism of action of the anti-TSPAN8 antibody involves cross-linking of TSPAN8, leading to rapid internalization and down-regulation of the TSPAN8 signaling complex in cancer cells. This effect results in the inhibition of TSPAN8 signaling-mediated cancer cell infiltration. Although recent studies have focused on the internalization process induced by anti-TSPAN8 IgG1 antibodies, the present inventors have not ruled out the possibility that these antibodies also induce antibody-dependent cytotoxicity and / or complement-dependent cytotoxicity.

Based on the currently available evidence, the present inventors have proposed a functional role for TSPAN8-LEL in cancer cell infiltration during tumor metastasis. The TSPAN8-LEL antibody developed by the inventors of the present application specifically down-regulates cell-surface TSPAN8 in cancer cells through internalization to effectively inhibit cancer cell infiltration.

In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12;

A heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 13 to SEQ ID NO: 16; And

A heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: 17 to SEQ ID NO: 20; And

The light chain variable region comprises a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 29 to SEQ ID NO: 32;

A light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 33 to SEQ ID NO: 35; And

But is not limited to, the light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 36 to SEQ ID NO: 39.

In another embodiment, the antibody is a group consisting of an antibody comprising a heavy chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 21 to SEQ ID NO: 24; And an antibody comprising a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43.

In a preferred embodiment, an antibody of the invention comprises a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: A light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 33, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: . &Lt; / RTI &gt;

The antibody of the present invention may include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 40, but is not limited thereto. In one embodiment of the invention, the human monoclonal antibody comprising the heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and the light chain variable region having the amino acid sequence of SEQ ID NO: 40 is designated as clone 1. The nucleic acid encoding the antibody may include, but is not limited to, a heavy chain variable region having a nucleic acid sequence of SEQ ID NO: 25 and a light chain variable region having a nucleic acid sequence of SEQ ID NO: 44.

In a preferred embodiment, the antibody of the invention comprises a heavy chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 14, and a CDR3 defined by the amino acid sequence of SEQ ID NO: And includes a light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 37 can do.

Tetraspanin plays an important role as a molecular promoter to form molecular webs to combine large molecule complexes and to promote its efficient function. Depending on the interacting molecule, tetraspanin is involved in many other cellular functions such as adhesion, motility and cell fusion. Previous studies have shown that TSPAN8 complexes with several binding partners, including EpCAM, claudin-7, and / or CD44v6, and modulates cancer cell motility. In this study, human antibodies targeting TSPAN8-LEL down-regulated TSPAN8 on the cell surface through an internalization process, suggesting that this process may be suitable as a therapeutic antibody-drug complex. Based on current evidence, the inventors of the present application contemplate that the mechanism of action of the anti-TSPAN8 antibody involves cross-linking of TSPAN8, leading to rapid internalization and down-regulation of the TSPAN8 signaling complex in cancer cells. This effect results in the inhibition of TSPAN8 signaling-mediated cancer cell infiltration. Although recent studies have focused on the internalization process induced by anti-TSPAN8 IgG1 antibodies, the present inventors have not ruled out the possibility that these antibodies also induce antibody-dependent cytotoxicity and / or complement-dependent cytotoxicity.

Based on the currently available evidence, the present inventors have proposed a functional role for TSPAN8-LEL in cancer cell infiltration during tumor metastasis. The TSPAN8-LEL antibody developed by the inventors of the present application specifically down-regulates cell-surface TSPAN8 in cancer cells through internalization to effectively inhibit cancer cell infiltration.

In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12;

A heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 13 to SEQ ID NO: 16; And

A heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: 17 to SEQ ID NO: 20; And

The light chain variable region comprises a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 29 to SEQ ID NO: 32;

A light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 33 to SEQ ID NO: 35; And

But is not limited to, the light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 36 to SEQ ID NO: 39.

In another embodiment, the antibody is a group consisting of an antibody comprising a heavy chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 21 to SEQ ID NO: 24; And an antibody comprising a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43.

In a preferred embodiment, an antibody of the invention comprises a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: A light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 33, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: . &Lt; / RTI &gt;

The antibody of the present invention may include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 40, but is not limited thereto. In one embodiment of the invention, the human monoclonal antibody comprising the heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and the light chain variable region having the amino acid sequence of SEQ ID NO: 40 is designated as clone 1. The nucleic acid encoding the antibody may include, but is not limited to, a heavy chain variable region having a nucleic acid sequence of SEQ ID NO: 25 and a light chain variable region having a nucleic acid sequence of SEQ ID NO: 44.

In a preferred embodiment, the antibody of the invention comprises a heavy chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 14, and a CDR3 defined by the amino acid sequence of SEQ ID NO: And includes a light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 37 can do.

The antibody of the present invention may include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 41, but is not limited thereto. In one embodiment of the present invention, the human monoclonal antibody comprising the heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and the light chain variable region having the amino acid sequence of SEQ ID NO: 41 is designated as clone 2. The nucleic acid encoding the antibody may include, but is not limited to, a heavy chain variable region having the nucleic acid sequence of SEQ ID NO: 26 and a light chain variable region having the nucleic acid sequence of SEQ ID NO: 45.

In a preferred embodiment, the antibody of the invention comprises a heavy chain CDRl defined by the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 15, and a heavy chain variable comprising the heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: And a light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 38.

The antibody of the present invention may include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 42, but is not limited thereto. In one embodiment of the invention, the human monoclonal antibody comprising the heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and the light chain variable region having the amino acid sequence of SEQ ID NO: 42 is designated as clone 3. The nucleic acid encoding the antibody may include, but is not limited to, a heavy chain variable region having the nucleic acid sequence of SEQ ID NO: 27 and a light chain variable region having the nucleic acid sequence of SEQ ID NO: 46.

In a preferred embodiment, the antibody of the present invention comprises a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 16, and a heavy chain variable comprising the heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: And a light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 32, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 39.

The antibody of the present invention may include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 43, but is not limited thereto. In one embodiment of the present invention, the human monoclonal antibody comprising the heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and the light chain variable region having the amino acid sequence of SEQ ID NO: 43 is designated as clone 4. The nucleic acid encoding the antibody may include, but is not limited to, a heavy chain variable region having the nucleic acid sequence of SEQ ID NO: 28 and a light chain variable region having the nucleic acid sequence of SEQ ID NO: 47.

Thus, antibodies constructed with heterologous sequences were found to inhibit cancer and / or cancer metastasis as long as TSPAN8-LEL is specifically recognized. Thus, it has been confirmed that they can be effectively applied for prevention or treatment of cancer and / or cancer metastasis.

When the antibody of the present invention comprises a constant region, it can be derived from IgG, IgA, IgD, IgE, IgM or a combination or hybrid thereof.

In the present invention, "combination" means that a polypeptide encoding a short-chain immunoglobulin Fc fragment of the same origin is linked to a short-chain polypeptide of different origin to form a dimer or multimer. That is, the dimer or multimer may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE and IgM.

The term "hybrid" of the present invention means that sequences encoding two or more heavy chain constant regions of different origin are present in the short chain immunoglobulin heavy chain constant region. For example, the domain hybrid may be composed of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG, IgA, IgD, IgE and IgM. In addition, the binding or hybrid can be made from heavy chain constant regions of the subtypes IgG1, IgG2, IgG3 and IgG4 of IgG. Combinations and hybrids are defined as above.

Furthermore, when the antibody of the present invention further comprises a light chain constant region, it may be derived from a lambda (貫) or kappa (kappa) light chain.

Preferably, the antibody is a human monoclonal antibody that specifically binds to human TSPAN8-LEL as well as murine TSPAN8-LEL and inhibits cancer and / or cancer metastasis. The ability of human antibodies to function in both humans and mice, that is, the cross-reactivity, offers the advantage of rendering human antibodies that can be applied to preclinical studies in mice.

In another aspect, the present invention provides a vector comprising the nucleic acid and a host cell comprising the vector or the nucleic acid.

In yet another aspect, the invention provides a method of producing an antibody that specifically binds to TSPAN8, preferably human TSPAN8-LEL or human and murine TSPAN8-LEL. Preferably, the antibody may be a human monoclonal antibody. Methods of making antibodies can include biopanning using functional domains. The present invention also provides a method for producing an antibody comprising the host cell culture in which a nucleic acid is expressed to produce the antibody.

The monoclonal antibodies of the present invention can be readily prepared using known techniques. For example, the production of monoclonal antibodies can be achieved by hybridomas consisting of B lymphocytes from immunized animals (Koeher and Milstein, 1976, Nature, 256: 495) or phage display technology, no.

Preferably, the production of the monoclonal antibodies of the invention can be implemented using phage display technology. The method of the present invention is described, for example, in Barbas et al. (METHODS: A Companion to Methods in Enzymology 2: 119, 1991 and J. Virol. 2001 Jul; 75 (14): 6692-9) and Winter et al. (Ann. Rev. Immunol. 12: 433, 1994). The phage useful for constructing the antibody library may be, but is not limited to, fd, M13, f1, If1, Ike, Zj / Z, Ff as well as Xf, Pf1 and Pf3. Examples of vectors that can be used to display the agonistic gene on the surface of the mitochondrial phage include phage vectors such as fUSE5, fAFF1, fdCAT1 and fdtetDOG, or phagemid vectors such as pHEN1, pComb3, pComb8 and pSEX, no. Helper phage is used to provide a wild-type version of the coat protein required for successful reinfection of the recombinant phage for amplification, such as, but not limited to, M13K07 and VSCM13.

Polynucleotides encoding hybridoma-derived monoclonal antibodies or phage display clones according to the present invention can be readily isolated and sequenced using conventional procedures. For example, oligonucleotide primers specifically designed to amplify heavy and light chain coding regions from hybridomas and phage DNA templates can be used. Once isolated, the polynucleotide can be inserted into an expression vector that can be introduced into the host cell. As a result, host cells (e. G., Transformants) can produce target monoclonal antibodies. Thus, a method of producing an antibody of the invention may comprise amplifying an expression vector carrying a polynucleotide encoding said antibody. Preferably, recombinant antibodies can be prepared by pre-treating Fc binders from human scFv libraries and performing biopanning techniques with functional domains to select specific clones.

In another aspect, the present invention provides a polynucleotide encoding an epitope or an antibody, an expression vector carrying a polynucleotide, and a transformant into which a vector is introduced.

The antibody is as described above.

Expression vectors carrying epitopes or antibodies according to the present invention may be used in mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse, etc.), plant cells, yeast, insect cells and bacterial cells , &Lt; / RTI &gt; Escherichia coli, Escherichia coli, E. coli) or a prokaryotic cell. Preferably, the vector has an appropriate promoter operably linked to the polynucleotide to induce expression of the gene of interest and at least one selection marker. For example, polynucleotides can be introduced into phage, plasmid, cosmid, mini-chromosome, virus or retroviral vector.

The expression vector into which the polynucleotide encoding the antibody has been introduced may be a combination of an expression vector into which a polynucleotide encoding a heavy chain and a light chain of an antibody are respectively introduced or a polynucleotide encoding a heavy chain and a light chain of an antibody may be introduced Expression vector.

Examples of the transformant formed by the introduction of the expression vector according to the present invention include bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium, yeast, fungi such as Pichia pastoris, fruit fly, insect cells such as Spodoptera Sf9 , Mouse hamster ovary cells (CHO), SP2 / 0 (mouse myeloma), human lymphocyte cells, COS, NSO (mouse myeloma), 293T, melanoma cells, HT-1080, baby hamster kidney cells (BHK) Human embryonic kidney cells) and animal cells and plant cells such as PERC.6 (human retinal cells).

The term "introduction" of the present invention refers to the delivery of a polynucleotide encoding an epitope or antibody into a cell. Introduction employs a variety of methods known in the art, including calcium phosphate DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine transfection and protoplast fusion . &Lt; / RTI &gt; Transduction refers to the process of transferring foreign DNA to other cells via viral vectors based on infection. In addition, the delivery of the vector into the host cell can be accomplished by gene bombardment. In the present invention, introduction can be used interchangeably with transformation.

In another aspect, the invention includes a composition comprising an antibody for preventing or treating cancer and / or cancer metastasis.

Since the antibody of the present invention is capable of effectively inhibiting cancer and / or cancer metastasis, a composition comprising the antibody as an active ingredient can inhibit cancer and / or cancer metastasis and further prevent or treat cancer and / or cancer metastasis Can be used effectively.

The term "inhibiting cancer and / or cancer metastasis" of the present invention has the general meaning in the art and refers to inhibiting the formation or growth of cancer and / or inhibiting the growth of tumors from one organs or parts to other non- .

For purposes of the present invention, cancer and / or cancer metastasis inhibition is achieved by inhibiting the expression of TSPAN8, particularly TSPAN8 LEL.

The term "cancer and / or cancer metastasis " of the present invention means a disease involving cancer and / or cancer metastasis in its development or progression. As long as it can be treated with an antibody, any disease within the cancer and / or cancer metastasis-related diseases is not limited. Examples of cancer include cancer of the esophagus, gastric cancer, colon cancer, rectal cancer, oral cancer, pancreatic cancer, larynx cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, Cancer, ovarian cancer, brain cancer, pancreatic cancer, and preferably cancer such as colon cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma and multiple myeloid blood cancer, Brain cancer, or pancreatic cancer.

The term " prevention "or" disease prevention "of the present invention means any action which results in delay or inhibition of onset or metastasis by administration of an antibody or composition according to the present invention. &Quot; Treatment "or" treatment "in the present invention means any action which results in improvement of symptoms or improvement of symptoms by administration of an antibody or composition according to the present invention.

The composition comprising the antibody of the present invention is preferably a pharmaceutical composition, and more preferably may comprise a suitable vehicle, excipient or diluent commonly used in the art.

A pharmaceutical composition comprising a pharmaceutically acceptable vehicle may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, nonaqueous solutions, suspensions, Oral or parenteral dosage forms. In this regard, the pharmaceutical compositions of the present invention may be formulated with diluents or excipients such as fillers, thickeners, binders, wetting agents, disintegrants, surfactants, and the like. Solid formulations for oral administration include tablets, pills, powders, granules and capsules. For such solid preparations, the compounds of the present invention are formulated with at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to simple diluents such as water or liquid paraffin, various additives such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in the liquid preparation. In addition, the pharmaceutical composition of the present invention may be in the form of a parenteral administration such as a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-drying agent, a suppository and the like. Injectable oils, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethyl oleate, may be suitable for non-aqueous applications and suspensions. The base materials of suppositories include witepsol, macrogol, tween 61, cacao paper, laurin, and glycerogelatin.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" of the present invention means the amount of the pharmaceutical composition which is capable of sufficiently treating the disease, at a reasonable benefit / risk ratio applied to any medical treatment. The effective amount will depend on the age and sex of the patient, the type of disease, the drug activity, the sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the duration of the treatment, the coadministration of the drug and other parameters well known in the art, including the severity of the disease And the like. The compositions of the present invention may be administered alone or in combination with other therapeutic agents. In this case, the administration can be carried out sequentially or simultaneously with conventional therapeutic agents. In addition, the composition may be administered in a single dose or in divided doses. Taking into account all of these factors, it is important to administer a minimal dose sufficient to cause maximum efficacy without side effects. Dosages may be determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention is not particularly limited, but depends on various factors including the health condition and the weight of the patient, the severity of the disease, the kind of the drug, the administration route and the administration time. The compositions may be administered in a single daily or multi-dose manner to mammals, including rats, livestock, humans, etc., via any conventional route, such as, for example, orally, rectally, intravenously, subcutaneously, intrauterine, .

In another aspect, the present invention provides a method for inhibiting cancer and / or cancer metastasis, comprising administering the antibody or the composition to a subject in need thereof.

The inhibition of antibodies, compositions and cancer and / or cancer metastasis has been described above.

Specifically, the method of inhibition of the present invention comprises administering to a subject in need of inhibition of cancer and / or cancer metastasis a pharmaceutical composition in a pharmaceutically effective amount. The subject may be, but is not limited to, mammals such as dogs, cows, horses, rabbits, mice, rats, chickens, humans. The pharmaceutical compositions may be administered by any suitable route, including by intramuscular injection, as needed for parenteral, subcutaneous, intraperitoneal, intrapulmonary or nasal or topical treatments. The preferred dosage of the pharmaceutical composition of the present invention will depend on various factors including the health and weight of the subject, the severity of the disease, the type of the drug, the route of administration and the time of administration, and may be determined by those skilled in the art.

In another aspect, the present invention provides a pharmaceutical composition comprising an antibody for preventing or treating cancer and / or cancer metastasis.

The terms "antibody "," prevention ", and "treatment"

The cancer is not particularly limited as long as it can be treated with the peptide of the present invention. Preferably TSPAN8 mediated tumor progression. Specifically, the onset or progression of cancer is prevented by inhibiting cancer and / or cancer metastasis with the antibody of the present invention. Examples of cancer include cancer of the esophagus, gastric cancer, colon cancer, rectal cancer, oral cancer, pancreatic cancer, larynx cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, , Osteosarcoma, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma and multiple myeloid hematocarcinoma, preferably colon cancer, ovarian cancer, brain cancer or pancreatic cancer no.

The antibodies of the present invention may also be used in combination with other antibodies or biologically active agents or agents for various purposes.

In one embodiment of the present invention, the antibodies of the present invention have been found to inhibit cancer and / or cancer metastasis and ultimately delay or prevent cancer development or progression. Therefore, the antibody of the present invention can be effectively applied for prevention and treatment of cancer.

In another aspect, the present invention provides a method for preventing or treating cancer and / or cancer metastasis, comprising administering the antibody or pharmaceutical composition to prevent or treat cancer in a subject in need thereof.

The antibodies, compositions and cancers are as described above.

In a method of preventing or treating cancer and / or cancer metastasis, the pharmaceutical composition is administered in a pharmaceutically effective amount to a suspected subject. The subject may be, but is not limited to, mammals such as dogs, cows, horses, rabbits, mice, rats, chickens and humans. The pharmaceutical composition may be administered by any suitable method including, but not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, or intranasal injection if necessary for nasal or topical treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on various factors including the health condition and weight of the subject, the severity of the disease, the kind of the drug, the route of administration, and the time of administration, and can be determined by those skilled in the art.

In another aspect, the present invention provides a cancer diagnostic composition comprising the antibody.

Antibodies and cancers are as described above.

Preferably, TSPAN8 is a cancer specifically expressed.

The term "diagnosis" of the present invention means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine the occurrence of cancer or cancer metastasis.

In another aspect, the present invention provides a diagnostic kit for cancer and / or cancer metastasis comprising a diagnostic composition.

The kit of the present invention can detect LEL of TSPAN8, preferably TSPAN8, as a marker for cancer and / or cancer metastasis. The detection kits of the present invention may include one or more compositions, solutions or devices suitable for the assay method as well as antibodies that selectively recognize the marker.

Preferably, the diagnostic kit may comprise a secondary antibody labeled with a matrix, a suitable buffer solution, a chromogenic enzyme or a fluorescent substance, a chromogenic substrate or those for 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 can be used. As the chromogenic enzyme, peroxidase and alkaline phosphatase can be used. As a fluorescent substrate, FITC and RITC can be used. As the chromogenic substrate solution, ABTS (2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), OPD benzidine) may be used.

In another aspect, the present invention provides a method of diagnosing cancer and / or cancer metastasis, comprising detecting TSPAN8, preferably TSPAN8-LEL, detected in an antigen-antibody complex in a biological sample separated from the subject.

The antibody, cancer and diagnosis are as described above.

More specifically, protein separation in a biological sample can be performed using known procedures.

The term "biological sample" of the present invention includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine showing differences in the level of expression of the cancer marker TSPAN8, more preferably TSPAN8-LEL , But is not limited thereto.

By detecting the expression levels of TSPAN8, more preferably TSPAN8-LEL in normal control and patients, cancer development can be diagnosed. That is, the expression level of the marker of the present invention in the patient's suspected cells is compared with that of normal cells. If a significant increase in the level of expression of the marker in the patient's suspected cells is observed, the risk of cancer or cancer metastasis can be diagnosed.

Immunoprecipitation assays, complement fixation assays, FACS, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, And protein chip analysis. By comparing the amount of antigen-antibody complex formed by the assay method with that of the normal control group and the suspected cancer, the patient is diagnosed as suspicious of cancer by evaluating a marked increase in the amount of protein expression of the cancer marker gene.

The term "antigen-antibody complex" of the present invention means that a cancer marker protein product binds to this specific antibody. The amount of the formed antigen-antibody complex can be quantitatively determined by measuring the signal intensity of the detection label.

The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, and a radioactive isotope, but the present invention is not limited to these examples. Examples of enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urase, peroxidase or alkaline phosphatase , Acetylcholine esterase, glucose oxidase, hexokinase and GDPase, RNase, glucose, oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, But are not limited to phoenolpyruvate dicarboxylase and? -Lactamase. Examples of the fluorescent substance include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoerythrine, picocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. Examples of ligands include, but are not limited to, biotin derivatives. Examples of luminescent materials include, but are not limited to, acridinium esters, luciferin, luciferase. Examples of microparticles include, but are not limited to, colloidal gold and colored latexes. Les Examples of redox molecules are ferrocene, ruthenium complexes, rain to Hagen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3 ] ²⁺ , [RU (bpy) ₃ ] ^{2 +} , and [MO (CN) ₈ ] ^{4 -} . Examples of the radioactive isotope is ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and including, ¹⁸⁶ Re, In But is not limited thereto.

Preferably, the level of protein expression is measured by ELISA. Examples of ELISAs include direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled antibody recognizing a capture antibody that complexes with an antigen attached to a solid support, an antigen- Antibody complexes were prepared by direct sandwich ELISA using another labeled antibody and a second labeled antibody that reacts with another labeled antibody that recognizes the antigen in the antigen-antibody complex attached to the solid support and then recognizes another labeled antibody As well as indirect sandwich ELISA. More preferably, the protein expression level is detected by a sandwich ELISA in which the sample reacts with an antibody attached to a solid support, and the resultant antigen-antibody complex is detected by enzyme-labeled antibody by adding an antigen-specific labeled antibody , Or a secondary antibody specific for an antibody recognizing an antigen of an antigen-antibody complex, followed by enzyme development and detection. The frequency of cancer can be diagnosed by measuring the degree of complex formation of cancer marker proteins and antibodies thereto.

Also preferably, the level of protein expression is measured by Western blot using one or more antibodies against the cancer marker. The total proteins separated from the sample are electrophoresed to be distinguishable by size and migrate to the nitrocellulose membrane to react with the antibody. The amount of protein produced by gene expression is determined by measuring the amount of antigen-antibody complex produced using the labeled antibody, thus diagnosing the onset of cancer. The detection method consists of examining the level of expression of the marker gene in the control group and the cancer-producing cells. The level of mRNA or protein may be expressed as the absolute (e.g.,: [mu] g / ml) or relative (e.g., the relative intensity of the signal) of the amount of marker protein.

In addition, it is preferable that the protein expression level is measured by immunohistochemistry using one or more antibodies against a cancer marker. Normal epithelial tissues and suspected cancerous tissues were collected and fixed, and paraffin blocks were prepared according to well-known methods. The block was cut into small pieces (several microns in thickness) and attached to a glass slide to react with one or more selected from antibodies according to known methods.

The unreacted antibody was then washed, and the reacted antibody was observed with the following microscope labeled as selected from the above-mentioned detection markers.

It is also desirable to analyze protein levels using protein chips in which one or more antibodies to the cancer marker are located and immobilized at a high density in a determined location on the substrate. In this regard, the protein is isolated from the sample and hybridized with a protein chip that forms an antigen-antibody complex, and then read to examine the presence or expression level of the protein of interest, thereby diagnosing cancer development.

In another aspect, the present invention provides a composition for inhibiting cancer and / or cancer metastasis comprising a substance that inhibits the expression of TSPAN8. Preferably, the substance may be a substance for inhibiting LEL expression of TSPAN8.

In one embodiment of the invention, the LEL of TSPAN8 has been found to play an important role in cancer and cancer infiltration. These results suggest that substances that inhibit the expression of TSPAN8, preferably TSPAN8-LEL, may inhibit cancer and / or cancer metastasis.

Materials that inhibit the expression of TSPAN8, preferably TSPAN8-LEL, include antisense oligonucleotides, siRNA oligonucleotides, antibodies, aptamers, single chain variable region fragments, peptides, low molecular weight compounds and natural extracts, no.

The substance that inhibits the expression of TSPAN8, preferably TSPAN8-LEL, is an antisense oligonucleotide or siRNA oligonucleotide that specifically binds to a substance that inhibits the expression of TSPAN8, preferably the TSPAN8-LEL gene represented by SEQ ID NO: 10 .

The term "antisense oligonucleotide " in the present invention means a derivative thereof having a nucleic acid sequence complementary to the sequence of DNA or RNA or a specific mRNA, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into a protein. The antisense oligonucleotide sequence may be a DNA or RNA sequence complementary to the mRNA of TSPAN8, preferably TSPAN8-LEL, capable of binding to the mRNA of TSPAN8, preferably TSPAN8-LEL, and may be a TSPAN8, preferably TSPAN8 -LEL mRNA translation, cytoplasmic migration or maturation, or any other activity essential for total biological function. Antisense oligonucleotides have 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases in length.

Antisense oligonucleotides can be modified at one or more positions of the base, sugar or backbone to have an improved effect (De Mesmaeker et al., Curr Opin Struct Biol., 5 (3): 343-55 (1995) ). Oligonucleotide backbones can be modified using, for example, phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl, cycloalkyl or short chain heteroatomic or heterocyclic intersugar bonds. In addition, the antisense oligonucleotides may have one or more substituted sugar moieties. Antisense oligonucleotides may also have modified bases. Examples of modified bases include, but are not limited to, hypoxanthine, 6-methyladenine, 5-methylpyrimidine (especially 5-methylcytosine), 5-hydroxymethyl cytosine (HMC), glycosyl HMC, Adenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, Purine. In addition, the antisense oligonucleotides of the present invention may be chemically coupled to a complex that enhances the activity and cellular uptake of one or more residues or antisense oligonucleotides. For example, a liphophilic residue can be a cholesterol residue, a cholesteryl residue, a cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine chain, a polyethylene glycol chain, an adamantacetic acid, a palmityl residue, an octadecylamine residue, -Carbonyl-oxycholesterol residues. &Lt; / RTI &gt; Methods for preparing oligonucleotides containing lipid residues are well known in the art (US Patents 5,138,045, 5,218,105 and 5,459,255). Modified oligonucleotides can be improved in stability in the presence of nuclease and the binding affinity for the target mRNA can be improved.

Antisense oligonucleotides can be synthesized in vitro in a conventional manner and administered to the human body or synthesized in vivo. A method for synthesizing an antisense oligonucleotide in vitro is to use RNA polymerase I. The method of synthesis of in vivo antisense RNA involves performing antisense RNA transcription using a vector comprising a reverse multiple cloning site (MCS). These antisense RNAs preferably contain a translation termination codon in the sequence to block translation into the peptide sequence.

The design of the antisense oligonucleotides useful in the present invention can be found in TSPAN8, preferably TSPAN8-LEL (Weiss, B. (ed.): Antisense Oligodeoxynucleotides and Antisense RNAs: Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, See, for example, Weiss, B., et al., Antisense RNA gene therapy for studying and modulating biological processes, Cell. Mol. Life Sci., 55: 334-358 (1999) Can be easily performed.

The term "siRNA" of the present invention can mediate RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914) &Lt; / RTI &gt; Since siRNA can inhibit expression of a target gene, it provides an effective method of gene knockdown or gene therapy. First, siRNAs found in plants, insects, fruit flies and parasites have recently been developed and are being used to study mammalian cells.

For the siRNA molecule used in the present invention, its sense strand (sequence corresponding to TSPAN8, preferably the sequence corresponding to the TSPAN8-LEL mRNA sequence) and its antisense strand (sequence complementary to TSPAN8, preferably TSPAN8-LEL mRNA) And may have a structure for forming the double strand. Alternatively, it may have a single stranded structure with self-complementary sense and antisense strands.

siRNAs are not limited to double-stranded RNA portions constituting the entire pair, but include mismatches such as mismatch (the corresponding bases are not complementary), bulge (no corresponding base in one chain), and the like. The total length of the siRNA may be 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.

The end of the siRNA can be blunt or cohesive as long as it can inhibit the expression of TSPAN8, preferably TSPAN8-LEL, through RNA interference (RNAi). The cohesive may be either 3'- or 5'-cohesive.

In the present invention, the siRNA molecule may have a short nucleotide sequence inserted between self-complementary sense and antisense strands (for example, about 5 to 15 nucleotides). In this case, the siRNA molecule formed from the expression of the nucleotide sequence forms a hairpin structure through intramolecular hybridization, resulting in the entire stem-and-loop structure. The stem-and-loop structure is an activated siRNA molecule that can mediate RNAi via in vitro or in vivo processes.

The term "aptamer " of the present invention refers to a nucleic acid molecule having a binding affinity for a particular target molecule. An aptamer can also bind to a specific target molecule to inhibit the activity of a specific target molecule. The aptamers of the present invention may be RNA, DNA, modified nucleic acids or mixtures thereof. The aptamers of the present invention can be either linear or circular. The aptamers of the present invention are not particularly limited to lengths. Generally, it can have a length of about 15 to 200 nucleotides, for example about 100 nucleotides or less, preferably about 80 nucleotides or less, more preferably about 45 nucleotides or less Lt; / RTI &gt; The aptamers of the invention may have a length of about 18, 20, or 25 nucleotides or more. If the total number of nucleotides is small, chemical synthesis and mass production will be easy and the main advantage is cost. Chemical modification is easy, human stability is high, and toxicity is low.

The aptamers of the invention can be prepared using the SELEX method or an improved version thereof (e.g., Ellington et al., Nature, 1990 346, 818-822; Tuerk et al., Science, 1990 249, 505-510) have. The SELEX method is a method of selecting an oligonucleotide that specifically binds to a target molecule in an oligonucleotide pool having 10 to 14 different base sequences. The oligonucleotides used have an arbitrary sequence of about 40 residues that are located by the primer sequence. This oligonucleotide pool can be mixed with the target molecule, and only RNA bound to the target molecule is collected using a filter or the like. The oligonucleotides collected are amplified by RT-PCR, which is used as a template for the next round. By repeating this operation 10 times, an aptamer that specifically binds to a target molecule can be obtained. By increasing the number of rounds or by using competing materials, the aptamers, which indicate the potential for binding to the target molecule, are enriched and selected. Thus, aptamers with different binding forces or modes of attachment, and the same binding or binding modes, but with different base sequences, can be obtained in some cases, by adjusting the number of rounds of SELEX and / or changing the competition state. The SELEX method involves amplification by PCR; In the process, it is possible to perform SELEX with high diversity by causing mutation using manganese ions or the like.

In addition to the known SELEX methods, aptamers can be obtained using the Cell-SELEX method for complex targets, living cells and tissues (Guo et al. Int. J. Mol. Sci., 9 (4): 668, 2008) , And the Cell-SELEX method has the advantage of directly selecting an aptamer for the disease without prior knowledge of the target molecule on the surface. In addition, the Cell-SELEX method is advantageous over the conventional SELEX method, which allows a functional approach to the target protein in physiological state during the selection process, since it may not exhibit inherent properties when isolated.

On the other hand, aptamers bind to target molecules in a wide variety of binding modes such as ionic bonds based on the negative charge of phosphate groups, hydrophobic bonds based on ribose, hydrogen bonds and stacking interactions based on nucleic acid bases. In particular, the ionic bond based on the negative charge of the phosphate groups present in the same number as the number of constituent nucleotides is strong and binds to lysine and arginine present on the surface of the positive charge of the protein. For this reason, nucleic acid bases that do not participate in direct binding to the target molecule may be substituted. In particular, the sites of the stem structure have already formed base pairs, and because the internal structure of the double helix structure is opposite, nucleic acid bases are unlikely to bind directly to the target molecule. Thus, even when a base pair is replaced by a different base pair, the activity of the aptamer often does not decrease. In a structure in which no base pair is formed, such as a loop structure, base substitution is possible when the nucleotide base is not involved in binding directly to a target molecule. For example, in the 2'-position of ribose, the hydroxyl group is replaced by an atom or a group. Examples of the atom or group include a hydrogen atom, a fluorine atom or an -O-alkyl group (for example, -O-CH 3) -O-acyl group (for example, -O-CHO) and an amino group ). If a functional group involved in direct binding to a target molecule is not substituted or deleted, the aptamer often retains its activity.

In addition, aptamers can easily be modified because they allow chemical synthesis. For the aptamer, it is possible to predict the extent to which a nucleic acid can be substituted or deleted by predicting a secondary structure using the MFOLD program, or by predicting the steric structure by X-ray analysis or NMR analysis, As shown in Fig. The aptamers predicted with the new sequence can be chemically synthesized easily and can be used to determine whether to maintain activity using an existing assay system.

The aptamers of the present invention may be one in which the sugar residue (e.g., ribose) of each nucleotide is modified to increase binding activity, stability, drug delivery capacity, or the like. As a variation on the sugar residue, the substitution of the oxygen atom at the 2'-position 3'-position and / or the 4'-position or the like of the sugar residue having another atom can be mentioned. O-alkylation (e.g. O-methylation, O-ethylation), Oarylation, S-alkylation (e.g. S-methylation, S-ethylation), S-arylation And amination (e. G., -NH) can be mentioned. Such changes in sugar residues can be carried out by methods known per se (eg, Sproat et al., Nucle. Acid. Res. 1991 19, 733-738; Cotton et al., Nucl. 19, 2629-2635; Hobbs et al., Biochemistry 1973 12, 5138-5145).

The aptamers of the present invention may have a nucleic acid base (e. G., Purine or pyrimidine) or may be variable (e. G., By chemical substitution) to increase binding activity. Examples of such variants include pyrimidine modifications at the 5-position, purine modifications at the 6- and / or 8-positions, modifications to the extracyclic amines, modification with 4- thioureidines and 5-bromo or 5-iodo- Substitution can be mentioned.

The phosphate group contained in the aptamer of the present invention can be modified to impart tolerance to nuclease hydrolysis. For example, the P (O) O group can be replaced by P (O) S (thioate), P (S) S (dithioate) ) OR ', CO or CH2 (formacetal) or 3'-amine (-NH-CH2-CH2-), wherein each R or R' unit is independently H or substituted or unsubstituted alkyl )]. &Lt; / RTI &gt; For example, the linking group may be -O-, -S-, or -N-, and the nucleotide may be attached to the adjacent nucleotide through such a linking group.

Deformation may also include deformation such as capping at 3 'and 5'. Modifications may also be carried out at the ends with the addition of polyethylene glycol, amino acids, peptides, inverted DT, nucleic acids, nucleosides, myristoyl, lithocolic-oleyl, docosanyl, lauroyl, stearoyl, palmitoyl, oleyl, , Steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer agents, toxins, enzymes, radioactive substances, biotin. See, for example, U.S. Patent Nos. 5,660,985 and 5,756,703 for such modifications.

In addition, aptamers are attached to the surface of liposomes or nanoparticles that carry anti-cancer agents, toxins, tumor suppressor genes and liposomes or small interfering RNAs (siRNAs) encapsulated in nanoparticles into the target cells.

In the present invention, the substance which inhibits the expression of TSPAN8, preferably TSPAN8-LEL is particularly preferably a substance which inhibits the activity of the substance by binding to an antibody, a peptide, a low molecular weight compound or TSPAN8, preferably TSPAN8- It is preferably an extract.

Antibodies that specifically bind to TSPAN8, preferably TSPAN8-LEL, are described above.

Peptides that specifically bind to TSPAN8, preferably TSPAN8-LEL, to inhibit its activity can be obtained by conventional methods known in the art, such as, for example, phage display (Smith GP, "Filamentous fusion phage: quot; antigens on the virion surface ", Science 228 (4705): 1315-1317 (1985), Smith GP, Petrenko VA, Phage display. Chem. Rev. 97 (2): 391-410 .

Preferably, the composition for inhibiting cancer and / or cancer metastasis can inhibit cancer and / or cancer metastasis by inhibiting cancer and / or cancer metastasis.

In another aspect, the present invention provides a kit for inhibiting cancer and / or cancer metastasis comprising the composition, wherein the composition comprises a substance that inhibits the expression of TSPAN8, preferably TSPAN8-LEL.

In another aspect, the present invention provides a method for inhibiting cancer and / or cancer metastasis, comprising administering the composition to a subject in need thereof, wherein the composition comprises TSPAN8, preferably TSPAN8-LEL Lt; RTI ID = 0.0 &gt; expression. &Lt; / RTI &gt; In another aspect, the present invention provides a method of treating cancer comprising administering the composition to a subject in need thereof, wherein the composition comprises a substance that inhibits the expression of TSPAN8, preferably TSPAN8-LEL do.

In yet another aspect, the present invention provides an antibody-drug conjugate comprising the antibody of any one of claims 1 to 17 linked to a drug. The drug may be any one selected from the group consisting of a toxin, a chemotherapeutic agent, an anticancer agent, an antibiotic, an ADP-ribosyl transferase, a radioactive isotope and a nucleic acid degrading enzyme, but is not limited thereto.

Preferably, the antibody-drug conjugate can be internalized into the cell. More preferably, the cell may be a cancer cell. In one embodiment of the invention, TSPAN8-LEL IgG can be internalized into cells expressing TSPAN8, such as cancer cells.

In another aspect, the present invention provides the use of said fusion protein of LEL of TSPAN8 and Fc to produce a pharmaceutical composition for preventing or treating cancer and / or cancer metastasis.

In another aspect, the present invention provides a fusion protein of the LEL of TSPAN8 and Fc for use in the manufacture of a pharmaceutical composition for preventing or treating cancer and / or cancer metastasis.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by the working examples.

Example  1: Cell culture and transfection

The human colon cancer cell line (HCT-8, HCT116, SW480 and LoVo), pancreatic cancer cell line (PANC-1), glioblastoma cell line (U87), ovarian cancer cell line (SK- (Korea Cell Line Bank). Cell lines were grown in RPMI 1640 (Gibco, Grand Island, NY, USA) or Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin / streptomycin ) Culture medium. HUVECs (Lonza, Baltimore, MD, USA) were maintained in EGM-2 (endothelial growth medium-2). All cells were maintained in a 37 ° C incubator (Sanyo, Panasonic Healthcare Company, Wood Dale, IL, USA) supplied with CO ₂ (5%). Expi293 cells were cultured in a Freestyle ™ expression medium (Invitrogen, Carlsbad, CA, USA) in a Multitron shaking incubator (Infors HT, Switzerland) at 37 ° C with 8% CO ₂ .

Twenty-four hours prior to transfection, the cells were cultured at 50-80% and the ON-TARGET plus Smart (TM) specific to TSPAN8 (Thermo scientific, Pittsburgh, Pa., USA) was assayed using Lipofectamine 2000 transfection reagent (Invitrogen) pool siRNA and TSPAN8-plasmid were transiently transfected. To examine TSPAN8 knockdown and overexpression, cells were harvested 2 days after transfection and analyzed by western blot.

Cells were resuspended in RIPA lysis buffer (150 mM NaCl, 50 mM Tris / HCl, 2 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X- [GenDEPOT, Barker, TX, USA]) at 4 ° C for 30 minutes and centrifuged at 13,000 rpm for 39 minutes. The lysate was loaded into each well of a 12% polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes using a wet transfer system (GE Healthcare Life Sciences, Pittsburgh, Pa., USA). The membranes were reacted for 1 hour at room temperature in TBST (10 mM Tris / HCl, pH 7.5; 150 mM NaCl; and 0.05% Tween 20) containing 5% skim milk and incubated in TBST containing 5% (1: 1,000; BD Biosciences, San Jose, Calif., USA) and a mouse anti-p120 catenin monoclonal antibody (2 ug / ml; R & D Systems, Inc., Minneapolis, Mouse anti-beta-actin monoclonal antibody (1: 5,000; Applied Biological Materials Inc., Richmond, BC, Canada) was reacted overnight at 4 占 폚. Membranes were washed with TBST and resuspended in horseradish peroxidase (HRP) -conjugated Goat anti-rat IgG (1: 3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and HRP- 3,000; Santa Cruz Biotechnology, Inc.) was diluted in TBST containing 5% skim milk and reacted at room temperature for 1 hour. The protein bands were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill., USA) following several washes with TBST and according to the manufacturer's instructions.

Formalin and the sections embedded with paraffin were treated with 0.01 M citrate buffer pH 6.0 for 5 minutes at 700 W in a microwave oven and then treated at 300 W for 5 minutes. Endogenous alkaline phosphatase activity was inhibited by treatment with 3% hydrogen peroxide for 6 minutes. Slides were blocked for 5 minutes and then reacted with anti-TSPAN8 antibody (Abcam, Cambridge, Mass., USA) diluted 1: 200 at room temperature for 1 hour. After complete rinsing with PBS, the slides were incubated for 30 minutes with a non-ohy- licrated secondary anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) diluted 1: 200 and then incubated with avidin- The reaction was continued for 30 min. For the color reaction, the slides were reacted with DAB (3.3'-diaminobenzidine tetrahydrochloride) solution for 2 minutes. All samples were lightly counterstained with Meyer's hematoxylin for 10 seconds before dehydration and mounting. Samples were observed with a phase contrast microscope (BX51, Olympus, Tokyo, Japan).

Example  2: Fc  Design and manufacture of fusion protein

(SEQ ID NO: 2) and 5'-TCGCGGCCGGCCTGGCCATTTTTTGCCAAGAAGTC-3 '(SEQ ID NO: 2) were amplified using the following primers: 5'-TCGCGGCCCAGGCGGCCAAATCTAAGTCTGATCGCATTG-3' . The TSPAN8-SEL (amino acids 34-57) structure was generated using the following primers: 5'-TCGCGGCCCAGGCGGCCCGAGTAAGCAATGACTCTCAAG-3 '(SEQ ID NO: 3) and 5'-TCGCGGCCGGCCTGGCCGTCCACAGCAACGTAGGAG-3' (SEQ ID NO: 4). The TSPAN1-LEL (amino acid 110-211) structure was generated using the following primers: 5'-TCGCGGCCCAGGCGGCCAAATCTAAGTCTGATCGCATTG-3 '(SEQ ID NO: 5) and 5'-TCGCGGCCGGCCTGGCCATTTTTTGCCAAGAAGT-3' (SEQ ID NO: 6). All primers were designed to add Sfi I restriction sites at both 5 'and 3' ends. The PCR fragment was digested with Sfi I (NEB, Ipswich, MA, USA), transformed into a hinge at the 3 'end of the cloning site and a CH2-CH3 domain of human IgG1 Gt; pCEP4 &lt; / RTI &gt; mammalian expression vector (Invitrogen). The conjugated product was transformed into calcium chloride -complex Escherichia coli DH5a cells, and the plasmid DNA was isolated from these cells. HEK293F cells (6 × 10 ⁸ cells) were transfected with 0.75 mg of each DNA using 1.5 mg of polyethyleneimine (Polysciences, Inc., Warrington, PA, USA). Transfected cells were maintained in Freestyle ^TM expression medium. After 7 days, the culture medium was collected and the fusion protein was purified using Affinity Chromatography with Protein A Sepharose (GenScript, Piscataway, NJ, USA). Protein concentrations were quantified using a NanoDrop spectrophotometer (Wilmington, DE, USA) and dialyzed in PBS. The purity of the samples was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie brilliant blue staining.

The final fraction was subdivided and stored at -80 &lt; 0 &gt; C.

Type of sequence Detail The amino acid sequence of TSPAN8
(Human TSPAN8)
SEQ ID NO: 7 MAGVSACIKYSMFTFNFLFWLCGILILALAIWVRVSNDSQAIFGSEDVGSSSYVAVDILIAVGAIIMILGFLGCCGAIKESRCMLLLFFIGLLLILLLQVATGILGAVF KSKSDRIVNETLYENTKLLSATGESEKQFQEAIIVFQEEFKCCGLVNGAADWGNNFQHYPELCACLDKQRPCQSYNGKQVYKETCISFIKDFLAKN LIIVIGISFGLAVIEILGLVFSMVLYCQIGNK The nucleic acid sequence of TSPAN8
(Human TSPAN8)
SEQ ID NO: 8 ATGGCAGGTGTGAGTGCCTGTATAAAATATTCTATGTTTACCTTCAACTTCTTGTTCTGGCTATGTGGTATCTTGATCCTAGCATTAGCAATATGGGTACGAGTAAGCAATGACTCTCAAGCAATTTTTGGTTCTGAAGATGTAGGCTCTAGCTCCTACGTTGCTGTGGACATATTGATTGCTGTAGGTGCCATCATCATGATTCTGGGCTTCCTGGGATGCTGCGGTGCTATAAAAGAAAGTCGCTGCATGCTTCTGTTGTTTTTCATAGGCTTGCTTCTGATCCTGCTCCTGCAGGTGGCGACAGGTATCCTAGGAGCTGTTTTCA AATCTAAGTCTGATCGCATTGTGAATGAAACTCTCTATGAAAACACAAAGCTTTTGAGCGCCACAGGGGAAAGTGAAAAACAATTCCAGGAAGCCATAATTGTGTTTCAAGAAGAGTTTAAATGCTGCGGTTTGGTCAATGGAGCTGCTGATTGGGGAAATAATTTTCAACACTATCCTGAATTATGTGCCTGTCTAGATAAGCAGAGACCATGCCAAAGCTATAATGGAAAACAAGTTTACAAAGAGACCTGTATTTCTTTCATAAAAGACTTCTTGGCAAAAAAT TTGATTATAGTTATTGGAATATCATTTGGACTGGCAGTTATTGAGATACTGGGTTTGGTGTTTTCTATGGTCCTGTATTGCCAGATCGGGAACAAA Amino acid sequence of TSPAN8 LEL
(Human TSPAN8 LEL)
SEQ ID NO: 9 KSKSDRIVNETLYENTKLLSATGESEKQFQEAIIVFQEEFKCCGLVNGAADWGNNFQHYPELCACLDKQRPCQSYNGKQVYKETCISFIKDFLAKN Nucleotide sequence of TSPAN8 LEL
(Human TSPAN8 LEL)
SEQ ID NO: 10 AATCTAAGTCTGATCGCATTGTGAATGAAACTCTCTATGAAAACACAAAGCTTTTGAGCGCCACAGGGGAAAGTGAAAAACAATTCCAGGAAGCCATAATTGTGTTTCAAGAAGAGTTTAAATGCTGCGGTTTGGTCAATGGAGCTGCTGATTGGGGAAATAATTTTCAACACTATCCTGAATTATGTGCCTGTCTAGATAAGCAGAGACCATGCCAAAGCTATAATGGAAAACAAGTTTACAAAGAGACCTGTATTTCTTTCATAAAAGACTTCTTGGCAAAAAAT

Example 3: Selection of TSPAN8-LEL scFvs using phage display technology

Fc binder pre-clearing of the human synthetic scFv antibody library was performed as described above. For each round of bio-panning, magnetic beads (Dynabeads M-270 epoxy, Invitrogen) were coated with 4 ug of human TSPAN8-LEL-FC and the coated beads were subjected to six rounds of biopanning Respectively. After the final round of bio-panning, 96 individual phage clones expressing scFv selected arbitrarily from the colonies grown on the output plate were examined for their activity against human TSAN8-LEL-Fc using enzyme immunoassay. The DNA of the final scFv clone was analyzed for DNA sequences and classified into four scFv clones with different complementarity determining regions (CDRs).

The amino acid sequence of the VH domain of TSPAN8 LEL IgG FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Clone 1
(SEQ ID NO: 21) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYAMS
(SEQ ID NO: 11) WVRQAPGKGLEWVS GISPNSGNKYYADSVKG
(SEQ ID NO: 13) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RSTIFDY
(SEQ ID NO: 17) WGQGTLVTVSS Clone 2
(SEQ ID NO: 22) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYAMS
(SEQ ID NO: 11) WVRQAPGKGLEWVS AISPGSGNTYYADPVKG
(SEQ ID NO: 14) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RSSVFDY
(SEQ ID NO: 18) WGQGTLVTVSS Clone 3
(SEQ ID NO: 23) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYSMS
(SEQ ID NO: 12) WVRQAPGKGLEWVS AISPGSSNTYYADSVKG
(SEQ ID NO: 15) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR KHMPFDY
(SEQ ID NO: 19) WGQGTLVTVSS
Clone 4
(SEQ ID NO: 24) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYAMS
(SEQ ID NO: 11) WVRQAPGKGLEWVS AISPGSSNKYYADSVKG
(SEQ ID NO: 16) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR KHLPFDY
(SEQ ID NO: 20) WGQGTLVTVSS

The amino acid sequence of the V _L domain of TSPAN8 LEL IgG FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Clone 1
(SEQ ID NO: 40) QSVLTQPPSASGTPGQRVTISC TGSSSNIGSNDVS
(SEQ ID NO: 29) WYQQLPGTAPKLLIY DNSHRPS
(order
Number: 33) GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GAWDASLSGYV
(SEQ ID NO: 36) FGGGTKLTV Clone 2
(SEQ ID NO: 41) QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNDVT
(SEQ ID NO: 30) WYQQLPGTAPKLLIY SNSHRPS
(order
Number: 34) GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GTWDSSLSGYV
(SEQ ID NO: 37) FGGGTKLTV Clone 3
(SEQ ID NO: 42) QSVLTQPPSASGTPGQRVTISC SGSSSNIGNNSVY
(SEQ ID NO: 31) WYQQLPGTAPKLLIY SDSQRPS
(order
Number: 35) GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GTWDYSLNGYV
(SEQ ID NO: 38) FGGGTKLTV Clone 4
(SEQ ID NO: 43) QSVLTQPPSASGTPGQRVTISC SGSSSNIGNNDVY
(SEQ ID NO: 32) WYQQLPGTAPKLLIY SDSQRPS
(order
Number: 35) GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GSWDYSLSGYV
(SEQ ID NO: 39) FGGGTKLTV

The nucleotide sequence of the V _H domain of TSPAN8 LEL IgG FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Clone 1
(SEQ ID NO: 25) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAATTATGCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GGGATCTCTCCTAATAGTGGTAATAAATATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGA CGGAGTACGATTTTCGACTAC TGGGGCCAGGGTACACTGGTCACCGTGAGCTCA Clone 2
(SEQ ID NO: 26) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAATTATGCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GCGATCTCTCCTGGTAGTGGTAATACATATTACGCTGATCCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGA AGGTCTAGTGTTTTCGACTAC TGGGGCCAGGGTACACTGGTCACCGTGAGCTCA Clone 3
(SEQ ID NO: 27) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAGTTATTCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GCGATCTCTCCTGGTAGTAGTAATACATATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGA AAGCATATGCCGTTCGACTAC TGGGGCCAGGGTACACTGGTCACCGTGAGCTCA Clone 4
(SEQ ID NO: 28) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAATTATGCTATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GCGATCTCTCCTGGTAGTAGTAATAAATATTACGCTGATTCTGTAAAAGGT CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGA AAGCATTTGCCGTTCGACTAC TGGGGCCAGGGTACACTGGTCACCGTGAGCTCA

The nucleotide sequence of the V _L domain of TSPAN8 LEL IgG FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Clone 1
(SEQ ID NO: 44) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT ACTGGCTCTTCATCTAATATTGGCAGTAATGATGTCTCC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT GATAATAGTCATCGGCCAAGC &Lt; GGTGCTTGGGATGCTAGCCTGAGTGGTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTA Clone 2
(SEQ ID NO: 45) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCAGTAATGATGTCACC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT TCTAATAGTCATCGGCCAAGC &Lt; GGTACTTGGGATTCTAGCCTGAGTGGTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTA Clone 3
(SEQ ID NO: 46) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCAATAATTCTGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT TCTGATAGTCAGCGGCCAAGC &Lt; GGTACTTGGGATTATAGCCTGAATGGTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTA Clone 4 (SEQ ID NO: 47) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGT AGTGGCTCTTCATCTAATATTGGCAATAATGATGTCTAC TGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT TCTGATAGTCAGCGGCCGAGC &Lt; GGTTCTTGGGATTATAGCCTGAGTGGTTATGTC TTCGGCGGAGGCACCAAGCTGACGGTCCTA

Example  4: Cell infiltration analysis

Cell infiltration analysis was performed using the modified Boyden chamber method. A 24 well-cell culture plate (BD Biosciences, Bedford, Mass.) Was used with a transwell insert membrane coated with Matrigel with 8 占 퐉 pores as described above (for infiltration analysis). The introduction of Mock- or TSPAN8 transfected COS-7 cells (1 X 10 ⁵ cells / well), or RNA or TSPAN8 siRNA (Dharmacon, Chicago, IL , USA) the HCT-8 (1 X 10 ⁵ transfected cells / well ) Or SK-OV (5 X 10 ⁴ cells / well) cells were dispensed into the upper chamber with serum-free medium and the lower chamber filled with complete medium containing 10% FBS as chemoattractant. (1 × 10 ⁵ cells / well), LoVo (1 × 10 ⁵ cells / well), SW480 (1 × 10 ⁵ cells / well), and U87 (3 x 10 ⁴ cells / well) and PANC-1 (1 x 10 ⁵ cells / well) cell lines and control IgG or TSPAN8 IgG (20 μg / 1 × 10 ⁵ cells / well) and SK-OV-3 (5 × 10 ⁴ cells / well) cells. HCT-8 cells were incubated in an upper chamber in which Fc, TSPAN1-LEL-Fc, TSPAN8-SEL-Fc and TSPAN8-LEL-Fc were present in serum-free medium to test the inhibitory effect of TSPAN8- It was busy. After incubation in a 5% CO ₂ chamber at 37 ° C for 22 to 48 hours, the non-infiltrating cells were wiped off with a cotton swab on the upper surface of the membrane, the membrane was fixed with 4% paraformaldehyde for 15 minutes, 0.0 &gt; 0.2% &lt; / RTI &gt; crystal violet. The degree of cellular infiltration was quantified by counting cells migrating through the membrane in three random fields (X 200) per filter. The experiment was repeated three times in triplicate.

Example  5: Attachment analysis

Leukocyte adhesion analysis was performed as described above with some modifications. Briefly, 6 X 10 ⁴ HUVECs were pre-dispensed into 24-well plates. At the same time, mock- or TSPAN8 transduced COS-7 cells or scrambled RNA- or TSPAN8 siRNA transduced HCT-8 cells were transfected with 5,6-carboxy-fluorescein succimidyl ester (CFSE; Molecular Probes, USA), and 6 X 10 ⁴ , 1.2 X 10 ⁵ , 2.4 X 10 ⁵ or 4.8 X 10 ⁵ cells were added to each well. Cells labeled with CFSE were incubated with HUVEC for 1 h in a 5% CO ₂ incubator at 37 ° C. In order to remove unbound cells, the wells were washed five times with PBS containing 0.2 mM CaCl ₂ and 0.1 mM MgCl _2. Bound cells were desensitized by trypsin / EDTA (Gibco, Toronto, Ontario, Canada) for 5 min and then washed twice with DMEM containing 10% FBS. In flow cytometry, cells with fluorescence were counted as CFSE-labeled cells attached to HUVEC. The assay was performed at 488 nm using a FACS Calibur instrument (BD Biosciences) and the emission filter was set to a 530/30 bandpass. For each measurement, 10,000 cells were counted without gating. Results were expressed as a percentage of CFSE-positive cells counted among the total counted events.

Example  6: Measurement of cell survival rate

Approximately 6 X 10 ³ mock- or TSPAN8 transduced COS-7 cells or 2 X 10 ⁴ scramble RNA- or TSPAN8 siRNA transduced HCT-8 cells were dispensed into 96-well plates. After 2 days, cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Final absorbance was measured at 450 nm with a spectrophotometer (VICTOR ™ X4, Perkin Elmer, Mass., USA).

Example  7: Enzyme-Linked Immunosorbent Assay (ELISA)

To determine the specificity of the four selected clones, 100 占 퐇 of PBS containing 0.1 占 퐂 of the recombinant TSPAN8-LEL-Fc, TSPAN8-SEL-Fc, TSPAN1-LEL-Fc or Fc fragment of IgG1 was added to a 96 well microplate Respectively. After overnight incubation at 37 ° C and washing three times with PBST (PBS containing 0.05% Tween 20), the plates were incubated with PBS containing 2% skim milk for 1 hour at 37 ° C. The cells were then incubated with 0.1 μg of unrelated scFv-Fc or TSAN8-LEL scFv-Fcs in PBST containing 3% BSA at 37 ° C for 2 hours. After 2 hours, the cells were washed with PBST and reacted with HRP-conjugated anti-human lambda light chain antibody (1: 5,000; Bethyl Laboratories Inc., Montgomery, TX, USA) in PBST containing 3% BSA. Washed three times with PBST, and 100 占 퐇 of 3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate solution (BD Biosciences) was added to each well. An equal amount of 1N H ₂ SO ₄ was added to the microplate to terminate the reaction. The optical density was measured at 450 nm using a micro-reader (VICTOR ™ X4, Perkin Elmer).

Example  8: Flow cell  analysis

Cells were harvested and fixed with 4% formaldehyde (PFA) for 30 min at room temperature. After washing, the cells were stained with 20 μg / ml TSPAN8 IgG in PBS staining buffer containing 1% BSA for 1 hour at room temperature. (1: 1,000; Jackson Immunoresearch Laboratory Inc., West Grove, PA, USA) in DyeLight-488 labeled buffer at room temperature Lt; / RTI &gt; for 1 hour. Cells were collected with a flow cytometer (BD FACSCalibur, BD Bioscience) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Example  9: TSPAN8 - LEL IgG  Production and production of antibodies

scFv clones four variable heavy chain gene was selected amplified using primers 5'-CGGGAATTCGCCGCCACCATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCTCTCCGAGGTGCAGCTGTTGGAGTCTG-3 '(SEQ ID NO: 48) and 5'-GGCGGGCCCTTG GTGGAGGCTGAGCTCACGGTGACCAGTGTACCCTG-3' (SEQ ID NO: 49). The variable light chain gene of the clone was amplified using the primers 5'-CCCAAGCTTGCCGCCACCATGGAGACACATTCTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGAAGGACCAGTCTGTGCTGACTCAGCC-3 '(SEQ ID NO: 50) and 5'-GGCCGTACGTAGGACCGTCAGCTTGGTGCCTCCGCC-3' (SEQ ID NO: 51). The variable heavy primer was designed to add EcoR I and Apa I restriction sites at both 5 'and 3' ends. The variable light chain primers were designed to add HindIII and BsiWI restriction sites at both 5 ' and 3 ' ends. The PCR fragment was digested with appropriate restriction enzymes (NEB) and cloned into a bicistronic mammalian expression vector pCDNA3.1 (Invitrogen) encoding the hinge and CH2-CH3 domain of human IgG1 3 'in the variable heavy chain cloning site. After producing TSPAN8-LEL IgG, the antibodies were purified using affinity column chromatography using Ultra Protein A resin (GeneScript USA Inc., Piscataway, NJ, USA). After dialysis in PBS, proteins were quantified using NanoDrop 2000 (Thermo Fisher Scientific, Boston, Mass., USA) and the antibody was subdivided and stored at -80 ° C.

Example  10: Immunoblot  analysis

30 쨉 g of a human epithelial ovarian cancer cell line or a lysate of a control IgG- or TSPAN8-LEL IgG-treated SK-OV3 cells was placed in each well of a polyacrylamide gel, and the cells were transferred to a wet transfer system (GE Healthcare Life Sciences , Pittsburgh, Pa., USA) to transfer to a nitrocellulose membrane. After blocking with TTBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% (v / v) Tween 20) containing 5% (w / v) skim milk, the membranes were incubated with rabbit anti-TSPAN8 poly (1: 1,000; Abcam) or mouse anti-β-actin monoclonal antibody (1: 5,000; Applied Biological Materials Inc., Richmond, BC, Canada) overnight at 4 ° C. Then, horseradish peroxidase (HRP) -conjugated goat anti-ratbbit IgG or goat anti-mouse IgG antibody (1: 5,000; Santa Cruz Biotechnology, Inc.).

To verify the binding specificity of TSPAN8-LEL IgG to TSPAN8-LEL, 0.1 mu g of the purified wild type or deletion mutant of TSPAN8-LEL in its native or denatured state was separated by electrophoresis and transferred to nitrocellulose membrane. After blocking, the membrane was reacted with TSPAN8-LEL IgG at 20 占 퐂 / ml overnight at 4 占 폚 and HRP-conjugated mouse anti-human Fab (1: 1,000; Thermo Fisher Scientific, Inc., Waltham, ). After several washes with TTBS, protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill., USA) according to the manufacturer's instructions.

Example  11: Cell infiltration analysis

To evaluate the effect of TSPAN8-LEL IgG on the epithelial ovarian cancer cell line infiltration, 5 × 10 ⁴ SK-OV3 and SNU-251 or 1 × 10 ⁴ cells were incubated with 20 μg / ml of control IgG or TSPAN8- ⁵ cell infiltration of SNU-8 was individually confirmed. After removing the non-invading cells by wiping the upper surface of the membrane with a cotton swab, the membrane was fixed with 4% paraformaldehyde (PFA) and stained with 0.2% crystal violet. The degree of cell infiltration was quantified by counting the number of cells migrated through membranes in any three fields (X 200) per filter.

Example  12: Fc  As a fusion protein TSPAN8 - Of LEL  Preparation and preparation of N- or C-terminal deletion mutants

LEL-N1 (amino acids 11-96), TSPAN8-LEL-N2 (amino acids 21-96), TSPAN8-LEL-N3 (amino acids 31-96), and TSPAN8- 3 '(SEQ ID NO: 52) and 5'-TCGCGGCCGGCCTGGCCATTTTTTGCCAAGAAGTCTTTTATG-3' (SEQ ID NO: 53), TSPAN8-LEL-N1 for TSPAN8-LEL-N1 were amplified by PCR using the following primers: 5'-TCGCGGCCCAGGCGGCCCCTCGATGGACCAACACAAAG- (SEQ ID NO: 54) and 5'-TCGCGGCCGAGCCATAATTGTGTTTC-3 '(SEQ ID NO: 56) for TSPAN8-LEL-N3 for 5'-TCGCGGCCCAGGCGGCCGCCACAGGGGAAAGTGAAAAC-3' (SEQ ID NO: 54) and 5'- TCGCGGCCGGCCTGGCCATTTTTTGCCAAGAAGTCTTTTATG- And 5'-TCGCGGCCGGCCTGGCCATTTTTTGCCAAGAAGTCTTTTTGG-3 '(SEQ ID NO: 57) and 5'-TCGCGGCCCAGGCGGCCAAATCTAAGTCTGATCGCATTG-3' (SEQ ID NO: 58) and 5'-TCGCGGCCGGCCTGGCCCTGGAATTGTTTTTCACTTTC-3 '(SEQ ID NO: 59) for TSPAN8-LEL-C1. PCR products were subcloned into Sfi I site of the modified pCEP4 mammalian expression vector (Invitrogen) encoding the hinge and the CH2-CH3 domain of human IgG ₁ in the cloning site 3 'end. Fc fusion protein was prepared and the protein was purified using affinity column chromatography as in the antibody purification protocol.

Example  13: Cell ELISA

To test the effect of TSPAN8-LEL IgG downregulating TSPAN8 in SK-OV3 cells, 10 ⁴ SK-OV3 cells were plated on 96-well plates and cultured in the presence of 20 ug / ml of control IgG or TSPAN8-LEL IgG And cultured at 37 ° C for 0, 0.5, 1, 1.5, 2, 2.5, or 3 hours. The cells were then fixed with 4% PFA and reacted with 2 μg / ml of HRP-conjugated TSPAN8-LEL IgG to detect the expression of TSPAN8 in SK-OV3 cells. After washing three times with cold PBS, cells were cultured with TMB solution. The optical density was measured at 450 nm using a microtiter plate reader.

Example  14: Immunocytochemistry

To detect TSPAN8 internalization of TSPAN8-LEL IgG in SK-OV3 cells, cells were cultured in poly-L-lysine coated coverslip (SPL Lifesciences, Gyeonggi-Do, Korea) Lt; / RTI &gt; Cells were incubated with 20 μg / mL FITC-labeled TSPAN8-LEL IgG at 37 ° C for 30, 60, or 120 minutes. Before stimulation, LysoTracker Red DND-99 (Molecular Probes, Eugene, OR, USA) was added to the cells for 1 hour (final concentration 200 nM). Cells were washed twice with PBS. Images were acquired with an Olympus FV300 laser scanning confocal microscope (Olympus, Tokyo, Japan).

Example  15: In vivo  Efficacy test

Seven-week-old female BALB / c-nude mice (SLC Inc, Seoul, Korea) were housed under specific pathogenic conditions and adapted to laboratory conditions for at least one week prior to use. It was maintained at the animal facility of the National Cancer Center in accordance with AAALAC International Animal Care Policy (Authorized Unit of the National Cancer Center Research Institute, Unit Number, NCC-13-163B). Mice were intraperitoneally injected with 2 X 10 ⁶ SK-OV3-luc in 200 μl PBS. Before inoculation with SK-OV3-luc, 10 mg / kg of control IgG or TSPAN8-LEL IgG was intravenously injected twice a week up to 42 days.

Experimental Example  One: TSPAN8 - LEL scFv - Fc  Stronger inhibition of metastatic colon cancer cell line infiltration than invasion of non-metastatic colorectal cancer cell line

Abnormal expression of P120 &apos; s catenin is known to be involved in downregulating E-cardinal expression in cancer cells and tumor metastasis. Immunoblot analysis was performed after silencing the p120catenin gene by siRNA in HCT-8 colon cancer cells and found that the knockdown of p120-catenin down-regulates the expression of E-carderine (Fig. 5A). The neutralization effects of antibodies identified in HCT-8 cell infiltration in the presence or absence of TSPAN-8-LEL scFv-Fc were compared. Similar to Erbitux®, TSPAN-8-LEL scFv-Fc strongly inhibited the infiltration of HC1-8 cells transfected with p120-catenin siRNA, but less strongly inhibited the infiltration of scrambled RNA transfected HCT-8 cells 5B). This suggests that TSPAN-8-LEL scFv-Fc is involved in the suppression of metastatic colorectal cancer cell infiltration.

In order to directly examine the difference in inhibition of metastatic and non-metastatic colorectal cancer cell infiltration by TSPAN-8-LEL scFv-Fc, metastatic LoVo and HCT-116 cell lines, in the presence or absence of TSPAN- And non-metastatic SW480 cell lines. In this experiment, the TSPAN-8-LEL scFv-Fc inhibited the infiltration of metastatic LoVo and HCT-116 cell lines rather than infiltration of the non-metastatic SW480 cell line in a manner similar to the effect on non-metastatic HCT-8 cell infiltration (FIG. 5B) (Fig. 5C). Collectively, these results indicate that TSPAN-8-LEL scFv-Fc strongly inhibits the invasion of metastatic colorectal cancer cells.

Experimental Example  2: TSPAN8 - LEL scFv - Fc  U87 malicious Brain glioma  And PANC -1 Suppresses pancreatic cancer cell infiltration

To investigate the effect of TSPAN8-LEL scFv-Fc on other metastatic tumor cells, we examined the inhibitory effect of TSPAN8-LEL scFv-Fc on U87 malignant brain glioma and PANC-1 pancreatic cancer cell line infiltration. Indeed, TSPAN8 antibody significantly inhibited the invasion of both U87 malignant brain glioma and PANC-1 pancreatic cancer cell line.

Experimental Example  3: TSPAN8 IgG  Stronger inhibition of metastatic HCT-116 colon cancer cell line infiltration

After TSPAN8 IgG production, this antibody was overproduced and purified using affinity column chromatography. The purity was> 90% (data not shown). The inhibitory effect of purified TSPAN8 IgG was then confirmed in the infiltration of HCT116 colon cancer cell lines. TSPAN8 IgG had an inhibitory effect on metastatic HCT116 cell infiltration (Figure 7). Although TSPAN8 IgG has an effect corresponding to a concentration five times lower than that of TSPAN8 scFv-Fc, this effect is similar to that produced by TSPAN8 scFv-Fc (FIG. 5C).

Experimental Example  4: TSPAN8 Overexpressed in ovarian cancer

To confirm the progress of ovarian cancer and the clinical significance of TSPAN8, 60 clinical samples (normal, n = 5 and ovarian cancer, n = 55) were immunochemically stained with commercially available anti-TSPAN8 antibodies. The total signal strength was obtained by manually removing the noise signal using ImageJ software. The overall signal intensity of ovarian cancer was increased about 1.8 times as compared with the normal sample. Most distinguished signals are seen in certain types of ovarian cancer, including papillary serous cytoadenocarcinoma, serous adenocarcinoma, endometrial carcinoma and undifferentiated carcinoma, but signaling is detected in normal samples or other cancer types including clear cell carcinoma and fibrothema (Fig. 8-9). These results indicate that TSPAN8 is involved in ovarian cancer progression.

Experimental Example  5: knockdown or TSPAN8  Antibodies specifically inhibit the invasion of ovarian cancer cells

To investigate the biological role of TSPAN8 in ovarian cancer cell infiltration, scrambled RNA or TSPAN8 siRNA was transfected into the metastatic SK-OV3 cell line and the knockdown of TSPAN8 was confirmed by immunoblot analysis (Fig. 10A). We also measured the effect of TSPAN8 silencing on infiltration of this cell type. The knockdown of TSPAN8 specifically inhibited the infiltration of SK-OV3 ovarian cancer cells (Fig. 10B). In order to confirm the functional relevance of TSPAN8 in ovarian cancer cell infiltration, the presence or absence of TSPAN8-LEL scFv-Fc was performed. Here, TSPAN8-LEL scFv-Fc specifically inhibited SK-OV3 ovarian cancer cell infiltration (Fig. 11). In summary, these results suggest that TSPAN8 plays an important role in ovarian cancer cell invasion.

Experimental Example  6: TSPAN8 IgG  Down-regulation of cell-surface TSPAN8 in colorectal cancer and ovarian cancer cells through internalization

To understand the mechanism of inhibition of TSPAN8 antibody in tumor cell infiltration, HCT116 colon cancer and SK-OV3 ovarian cancer cell line were treated with TSPAN8-LEL scFv-Fc under basic or fixed conditions and cells were examined by flow cytometry. TSPAN8-LEL scFv-Fc down-regulated the cell-surface TSPAN8 of HCT116 colon cancer and SK-OV3 ovarian cancer cells under basic conditions, but under both fixed conditions, both cell lines had little effect on the expression level of surface TSPAN8 (Fig. 12). This suggests that down-regulation of cell-surface TSPAN8 is dependent on internalization.

Experimental Example  7: TSPAN8 IgG  In ovarian tumor metastasis model, SK-OV3 cells in vivo  Reduce metastasis

To examine the in vivo efficacy of TSPAN8 IgG during ovarian tumor metastasis, SK-OV3 cells were injected into the peritoneum of Balb / c nude mice to confirm ovarian tumor metastasis in animal models. Control IgG or TSPAN8 IgG was then intravenously injected twice a week up to 1 month (Figure 13A). No transduction was observed in 6 out of 9 mice treated with TSPAN8 IgG, although 2 out of 10 mice treated with control IgG showed no metastasis of SK-OV3 cells. This confirmed the inhibitory effect of TSPAN8 IgG on ovarian cancer cell metastasis (Fig. 13B). In addition, in the control IgG-treated group, the metastasis of SK-OV3 cells was observed in the diaphragm, as well as peritoneal organs such as colon, liver, kidney, and ovary, as well as terminal metastases. However, no end-to-diaphragm transition was observed in the group treated with TSPAN8 IgG (Fig. 13C). Taken together, these results indicate that TSPAN8 IgG can significantly inhibit the in vivo metastasis of ovarian cancer cells.

Experimental Example  8: TSPAN8 - LEL IgG  Epithelial ovarian cancer cell line TSPAN8 - Suppress median infiltration

To investigate the effect of TSPAN8-LEL IgG on epithelial ovarian cancer invasion, TSPAN8 expression was analyzed by immunoblot analysis in epithelial ovarian cancer cell lines including SNU-8 cystadenocarcinoma, SNU-251 endometrial carcinoma, or SK-OV3 adenocarcinoma cell line (Fig. 14A). As a result, remarkable expression of TSPAN8 was observed in these cell lines. Tumor cell infiltration analysis of these cell lines was then performed in the presence or absence of TSPAN8-LEL IgG (Fig. 14B). As a result, the antibodies showed significant inhibition of TSPAN8-mediated infiltration of all epithelial ovarian cancer cell lines, including SNU-8, SNU-251, and SK-OV3 cells. Thus, this result indicates that TSPAN8-LEL antibody significantly inhibits TSPAN8-mediated epithelial ovarian cancer cell invasion during tumor metastasis.

Experimental Example  9: TSPAN8 - LEL IgG  The antibody TSPAN8 - Of LEL  Binds to amino acids 31-96

In order to identify TSPAN8-LEL IgG antibody binding sites, fc of wild type and N- or C-terminal consecutive deletion mutants of TSPAN8-LEL identified by> 90% purity by SDS-PAGE and Kumasi brilliant blue staining (data not shown Fusion forms were constructed and produced (Figure 17A). The purified protein was then subjected to ELISA and immunoblot (Figs. 15B and 15C). As a result, the antibody was found to bind to amino acids 31-96 of TSPAN8-LEL.

Experimental Example  10: TSPAN8 - LEL IgG  Internalization in epithelial ovarian cancer TSPAN8  Down regulation

In order to investigate the effect of TSPAN8-LEL IgG on down-regulation of TSPAN8 in epithelial ovarian cancer, TSPAN8-LEL IgG was treated with SK-OV3 cells and ELISA using HRP-conjugated TSPAN8-LEL IgG Lt; RTI ID = 0.0 &gt; (Figure 16A). &Lt; / RTI &gt; Here, we compared the activity of TSPAN8-LEL IgG or HRP-conjugated TSPAN8-LEL IgG against TSPAN8-LEL using ELISA and found that the HRP conjugation had little effect on antibodies binding to TSPAN8-LEL (data not shown) . As a result, TSPAN8 is significantly down-regulated in a time-dependent manner by TSPAN8-LEL IgG, not by control IgG on the surface of SK-OV3 cells. To confirm down-regulation of TSPAN8 expression by TSPAN8-LEL IgG, immunoblots were performed under the same experimental conditions (Fig. 16B). As a result, TSPAN8 was down-regulated by TSPAN8-LEL IgG. In order to confirm that the antibody can induce the internalization, TSPAN8-LEL IgG was treated with SK-OV3 cells and then the internalization of the antibody was monitored by immunocytochemistry (FIG. 16C). Here, lysotracker was used to detect lysosomal localization of TSPAN8-LEL IgG. As a result, TSPAN8-LEL IgG rapidly co-localized with LAMP1 antibody in SK-OV3 cells and confirmed rapid internalization of TSPAN8-LEL IgG with lysosomes. In conclusion, this result can lead to rapid internalization of TSPAN8-LEL antibodies and down-regulation of TSPAN8 on the surface of epithelial ovarian cancer cells.

Experimental Example  11: TSPAN8 - LEL IgG  in in vivo  Suppression of epithelial ovarian cancer cell metastasis

In order to investigate the in vivo efficacy of TSPAN8-LEL IgG in epithelial ovarian cancer metastasis, luciferase-expressing SK-0V3 cells (SK-0V3-luc) were injected into the abdominal cavity of nude mice to induce epithelial ovarian cancer cell metastasis Model. Then, two times a week, control IgG and TSPAN8-LEL IgG were intravenously injected until day 42, and metastasis was observed using bioluminescence images (FIG. 17A). Here, the development of cell metastasis was indicated by the number of mice in which the luminescent signal was detected in the removed organs including the ovary, pancreas, colon, heart, liver, spleen, and kidney (data not shown). SK-OV3-luc metastasis occurred in 24 out of 31 mice in the control IgG treatment group, whereas cell migration in the TSPAN8-LEL IgG treatment group was about 36% in 15 out of 30 mice, and TSPAN8-LEL IgG (Fig. 17B and Fig. 17C). These results demonstrate that self-developed antibodies can inhibit TSPAN8-mediated epithelial ovarian cancer cell metastasis in vivo .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Scripps Korea Antibody Institute <120> NOVEL ANTIBODY SPECIFIC FOR TSPAN8 AND USES THEREOF <130> 1554 <150> 61 / 946,236 <151> 2014-02-28 <160> 59 <170> Kopatentin 2.0 <210> 1 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying TSPAN8-LEL (amino acids 110-205) <400> 1 tcgcggccca ggcggccaaa tctaagtctg atcgcattg 39 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying TSPAN8-LEL (amino acids 110-205) <400> 2 tcgcggccgg cctggccatt ttttgccaag aagtc 35 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying TSPAN8-SEL (amino acids 34-57) <400> 3 tcgcggccca ggcggcccga gtaagcaatg actctcaag 39 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying TSPAN8-SEL (amino acids 34-57) <400> 4 tcgcggccgg cctggccgtc cacagcaacg taggag 36 <210> 5 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying TSPAN1-LEL (amino acids 110-211) <400> 5 tcgcggccca ggcggccaaa tctaagtctg atcgcattg 39 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying TSPAN1-LEL (amino acids 110-211) <400> 6 tcgcggccgg cctggccatt ttttgccaag aagt 34 <210> 7 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of TSPAN8 <400> 7 Met Ala Gly Val Ser Ala Cys Ile Lys Tyr Ser Met Phe Thr Phe Asn   1 5 10 15 Phe Leu Phe Trp Leu Cys Gly Ile Leu Ile Leu Ala Leu Ala Ile Trp              20 25 30 Val Arg Val Ser Asn Asp Ser Gln Ala Ile Phe Gly Ser Glu Asp Val          35 40 45 Gly Ser Ser Ser Tyr Val Ala Val Asp Ile Leu Ile Ala Val Gly Ala      50 55 60 Ile Ile Met Ile Leu Gly Phe Leu Gly Cys Cys Gly Ala Ile Lys Glu  65 70 75 80 Ser Arg Cys Met Leu Leu Leu Phe Phe Ile Gly Leu Leu Leu Ile Leu                  85 90 95 Leu Leu Gln Val Ala Thr Gly Ile Leu Gly Ala Val Phe Lys Ser Lys             100 105 110 Ser Asp Arg Ile Val Asn Glu Thr Leu Tyr Glu Asn Thr Lys Leu Leu         115 120 125 Ser Ala Thr Gly Glu Ser Glu Lys Gln Phe Gln Glu Ala Ile Ile Val     130 135 140 Phe Gln Glu Glu Phe Lys Cys Cys Gly Leu Val Asn Gly Ala Ala Asp 145 150 155 160 Trp Gly Asn Asn Phe Gln His Tyr Pro Glu Leu Cys Ala Cys Leu Asp                 165 170 175 Lys Gln Arg Pro Cys Gln Ser Tyr Asn Gly Lys Gln Val Tyr Lys Glu             180 185 190 Thr Cys Ile Ser Phe Ile Lys Asp Phe Leu Ala Lys Asn Leu Ile Ile         195 200 205 Val Ile Gly Ile Le Ply Gly Leu     210 215 220 Val Phe Ser Met Val Leu Tyr Cys Gln Ile Gly Asn Lys 225 230 235 <210> 8 <211> 711 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of TSPAN8 <400> 8 atggcaggtg tgagtgcctg tataaaatat tctatgttta ccttcaactt cttgttctgg 60 ctatgtggta tcttgatcct agcattagca atatgggtac gagtaagcaa tgactctcaa 120 gcaatttttg gttctgaaga tgtaggctct agctcctacg ttgctgtgga catattgatt 180 gctgtaggtg ccatcatcat gattctgggc ttcctgggat gctgcggtgc tataaaagaa 240 agtcgctgca tgcttctgtt gtttttcata ggcttgcttc tgatcctgct cctgcaggtg 300 gcgacaggta tcctaggagc tgttttcaaa tctaagtctg atcgcattgt gaatgaaact 360 ctctatgaaa acacaaagct tttgagcgcc acaggggaaa gtgaaaaaca attccaggaa 420 gccataattg tgtttcaaga agagtttaaa tgctgcggtt tggtcaatgg agctgctgat 480 tggggaaata attttcaaca ctatcctgaa ttatgtgcct gtctagataa gcagagacca 540 tgccaaagct ataatggaaa acaagtttac aaagagacct gtatttcttt cataaaagac 600 ttcttggcaa aaaatttgat tatagttatt ggaatatcat ttggactggc agttattgag 660 atactgggtt tggtgttttc tatggtcctg tattgccaga tcgggaacaa a 711 <210> 9 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of TSPAN8 LEL <400> 9 Lys Ser Lys Ser Asp Arg Ile Val Asn Glu Thr Leu Tyr Glu Asn Thr   1 5 10 15 Lys Leu Leu Ser Ala Thr Gly Glu Ser Glu Lys Gln Phe Gln Glu Ala              20 25 30 Ile Ile Val Phe Gln Glu Glu Phe Lys Cys Cys Gly Leu Val Asn Gly          35 40 45 Ala Ala Asp Trp Gly Asn Asn Phe Gln His Tyr Pro Glu Leu Cys Ala      50 55 60 Cys Leu Asp Lys Gln Arg Pro Cys Gln Ser Tyr Asn Gly Lys Gln Val  65 70 75 80 Tyr Lys Glu Thr Cys Ile Ser Phe Ile Lys Asp Phe Leu Ala Lys Asn                  85 90 95 <210> 10 <211> 287 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of TSPAN8 LEL <400> 10 aatctaagtc tgatcgcatt gtgaatgaaa ctctctatga aaacacaaag cttttgagcg 60 ccacagggga aagtgaaaaa caattccagg aagccataat tgtgtttcaa gaagagttta 120 aatgctgcgg tttggtcaat ggagctgctg attggggaaa taattttcaa cactatcctg 180 aattaggtgc ctgtctagat aagcagagac catgccaaag ctataatgga aaacaagttt 240 acaaagagac ctgtatttct ttcataaaag acttcttggc aaaaaat 287 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 11 Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser   1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 12 Gly Phe Thr Phe Ser Ser Tyr Ser Ser Ser   1 5 10 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 13 Gly Ile Ser Pro Asn Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 14 Ala Ile Ser Pro Gly Ser Gly Asn Thr Tyr Tyr Ala Asp Pro Val Lys   1 5 10 15 Gly     <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 15 Ala Ile Ser Pro Gly Ser Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 16 Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 17 Arg Ser Thr Ile Phe Asp Tyr   1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 18 Arg Ser Ser Val Phe Asp Tyr   1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 19 Lys His Met Pro Phe Asp Tyr   1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 20 Lys His Leu Pro Phe Asp Tyr   1 5 <210> 21 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for Clone 1 <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Ser Pro Asn Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Arg Ser Thr Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 22 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for Clone 2 <400> 22 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Pro Gly Ser Gly Asn Thr Tyr Tyr Ala Asp Pro Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Arg Ser Ser Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 23 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for Clone 3 <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Pro Gly Ser Ser Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Lys His Met Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 24 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for Clone 4 <400> 24 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Lys His Leu Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 25 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for clone 1 <400> 25 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctctccta atagtggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagacggagt 300 acgattttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 26 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for clone 2 <400> 26 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcctg gtagtggtaa tacatattac 180 gctgatcctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaaggtct 300 agtgttttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 27 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for clone 3 <400> 27 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agttattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcctg gtagtagtaa tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaaagcat 300 atgccgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 28 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH domains of TSPAN8 LEL IgGs for clone 4 <400> 28 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcctg gtagtagtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaaagcat 300 ttgccgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 29 Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Ser Ser   1 5 10 <210> 30 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 30 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Thr   1 5 10 <210> 31 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 31 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ser Val Tyr   1 5 10 <210> 32 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 32 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val Tyr   1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 33 Asp Asn Ser His Arg Pro Ser   1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 34 Ser Asn Ser His Arg Pro Ser   1 5 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 35 Ser Asp Ser Gln Arg Pro Ser   1 5 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 36 Gly Ala Trp Asp Ala Ser Leu Ser Gly Tyr Val   1 5 10 <210> 37 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 37 Gly Thr Trp Asp Ser Ser Leu Ser Gly Tyr Val   1 5 10 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 38 Gly Thr Trp Asp Tyr Ser Leu Asn Gly Tyr Val   1 5 10 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 39 Gly Ser Trp Asp Tyr Ser Leu Ser Gly Tyr Val   1 5 10 <210> 40 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 1 <400> 40 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Asp Val Ser Trp Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Asp Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Ala Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val             100 105 <210> 41 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 2 <400> 41 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Asp Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val             100 105 <210> 42 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 3 <400> 42 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Ser Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Tyr Ser Leu                  85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val             100 105 <210> 43 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 4 <400> 43 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Asp Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val             100 105 <210> 44 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 1 <400> 44 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc agtaatgatg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gataatagtc atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt gcttgggatg ctagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 45 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 2 <400> 45 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc agtaatgatg tcacctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tctaatagtc atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt acttgggatt ctagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 46 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 3 <400> 46 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc aataattctg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tctgatagtc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt acttgggatt atagcctgaa tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 47 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL domain of TSPAN8 LEL IgG for clone 4 <400> 47 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc aataatgatg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tctgatagtc agcggccgag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt tcttgggatt atagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 48 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> primer for variable heavy chain gene <400> 48 cgggaattcg ccgccaccat ggaatggagc tgggtctttc tcttcttcct gtcagtaact 60 acaggtgtcc tctccgaggt gcagctgttg gagtctg 97 <210> 49 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for variable light chain gene <400> 49 ggcgggccct tggtggaggc tgagctcacg gtgaccagtg taccctg 47 <210> 50 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> primer for variable light chain gene <400> 50 cccaagcttg ccgccaccat ggagacacat tctcaggtct ttgtatacat gttgctgtgg 60 ttgtctggtg ttgaaggacc agtctgtgct gactcagcc 99 <210> 51 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer for variable light chain gene <400> 51 ggccgtacgt aggaccgtca gcttggtgcc tccgcc 36 <210> 52 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-N1 <400> 52 tcgcggccca ggcggccact ctctatgaaa acacaaag 38 <210> 53 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-N1 <400> 53 tcgcggccgg cctggccatt ttttgccaag aagtctttta tg 42 <210> 54 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-N2 <400> 54 tcgcggccca ggcggccgcc acaggggaaa gtgaaaaac 39 <210> 55 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-N2 <400> 55 tcgcggccgg cctggccatt ttttgccaag aagtctttta tg 42 <210> 56 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-N3 <400> 56 tcgcggccca ggcggccgaa gccataattg tgtttc 36 <210> 57 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-N3 <400> 57 tcgcggccgg cctggccatt ttttgccaag aagtctttta tg 42 <210> 58 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-C1 <400> 58 tcgcggccca ggcggccaaa tctaagtctg atcgcattg 39 <210> 59 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer for TSPAN8-LEL-C1 <400> 59 tcgcggccgg cctggccctg gaattgtttt tcactttc 38

Claims (22)

An antibody that specifically binds to the large extracellular loop (LEL) of TSPAN8 (tetraspanin 8) containing the following heavy chain variable region and light chain variable region,
(a) the heavy chain variable region comprises
i) a heavy chain variable region comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOs: 11, 13 and 17, respectively;
ii) a heavy chain variable region comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 11, 14 and 18, respectively;
iii) a heavy chain variable region comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOs: 12, 15 and 19, respectively; And
iV) heavy chain variable region comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOs: 11, 16 and 20, respectively;
, &Lt; / RTI &gt;
(b) the light chain variable region comprises
i) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOs: 29, 33 and 36, respectively;
ii) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 30, 34 and 37, respectively;
iii) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 31, 35 and 38, respectively; And
iv) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 32, 35 and 39, respectively;
&Lt; / RTI &gt;
The antibody according to claim 1, which specifically binds to a site including amino acid fragments 110 to 205 of the TSPAN8 amino acid sequence represented by SEQ ID NO: 7.
The antibody according to claim 1, which specifically binds to a region comprising amino acid fragments 31 to 96 of the amino acid sequence of LEL in TSPAN8 represented by SEQ ID NO: 9.
The method according to claim 1,
i) heavy chain variable regions comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 11, 13 and 17, respectively, and light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 29, A light chain variable region comprising;
ii) heavy chain variable regions comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOs: 11, 14 and 18, respectively, and light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOs: 30, A light chain variable region comprising;
iii) heavy chain variable regions comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 12, 15 and 19, respectively, and light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 31, A light chain variable region comprising; or
iv) heavy chain variable regions comprising the heavy chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 11, 16 and 20, respectively, and light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NOS: 32, A light chain variable region comprising;
&Lt; / RTI &gt;
The method according to claim 1,
A heavy chain variable region of an amino acid sequence selected from the group consisting of SEQ ID NOS: 21 to 24; And
Wherein the antibody comprises a light chain variable region of an amino acid sequence selected from the group consisting of SEQ ID NOS: 40-43.
A nucleic acid encoding the antibody of claim 1.
A vector comprising the nucleic acid of claim 6.
A host cell comprising the vector of claim 7 or the nucleic acid of claim 6.
9. A method for producing an antibody according to claim 1, comprising culturing the host cell of claim 8 to produce an antibody by nucleic acid expression.
An antibody-drug conjugate comprising the antibody of claim 1, wherein the drug is conjugated to a drug, wherein the drug is selected from the group consisting of toxin, a chemotherapeutic agent, an anticancer agent, an antibiotic, ADP-ribosyl transferase, a radioactive isotope and a nucleic acid degrading enzyme wherein the antibody-drug conjugate is one selected from the group consisting of nucleolytic enzymes.
A pharmaceutical composition for preventing or treating cancer or cancer metastasis comprising the antibody of claim 1.
12. The pharmaceutical composition according to claim 11, wherein the cancer is colon cancer, ovarian cancer, brain tumor or pancreatic cancer.
6. The method of claim 5,
A heavy chain variable region of the amino acid sequence of SEQ ID NO: 21 and a light chain variable region of the amino acid sequence of SEQ ID NO: 40;
A heavy chain variable region of the amino acid sequence of SEQ ID NO: 22 and a light chain variable region of the amino acid sequence of SEQ ID NO: 41;
A heavy chain variable region of the amino acid sequence of SEQ ID NO: 23 and a light chain variable region of the amino acid sequence of SEQ ID NO: 42; or
A heavy chain variable region of the amino acid sequence of SEQ ID NO: 24 and a light chain variable region of the amino acid sequence of SEQ ID NO: 43;
&Lt; / RTI &gt;
delete A composition for diagnosing cancer and / or cancer metastasis comprising the antibody of claim 1.
16. The diagnostic composition according to claim 15, wherein the cancer is colon cancer, ovarian cancer, brain tumor or pancreatic cancer.
delete A cancer and / or cancer metastasis diagnostic kit comprising the diagnostic composition of claim 15.
delete delete delete delete

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