KR20190132949A - Specific monoclonal antibody against autocatalytic loop portion of PRSS14/ST14 and use thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody for specifically recognizing an autocatalytic loop portion of PRSS14/ST14 and a use thereof and more specifically, to a monoclonal antibody mAb3F3 specifically combined with an autocatalytic loop portion of PRSS14/ST14, a humanization antibody thereof and a cancer metastasis diagnosing or inhibiting use. According to the present invention, it is confirmed that PRSS14/ST14 is decisively involved in lung metastasis of breast cancer in a mouse model, and epitope including the autocatalytic loop portion of the PRSS14/ST14 protein can be metastasis preventing vaccines. In a MMTV-PyMT mouse model, it is confirmed that a new monoclonal antibody specific for an autocatalytic loop recognizes epitope sequence and inhibits metastasis with a structure specific method. The antibody specific for the autocatalytic loop portion of the PRSS14/ST14 is expected to be usefully applied to cancer metastasis diagnosis and cancer metastasis treatment.

Description

Specific monoclonal antibody against autocatalytic loop portion of PRSS14 / ST14 and use approximately}

The present invention relates to a monoclonal antibody that specifically recognizes an autocatalytic loop portion of PRSS14 / ST14 and its use, wherein the monoclonal antibody mAb3F3 specifically binds to the autolytic ring portion of PRSS14 / ST14. , Humanized antibodies thereof and their use for diagnosing or inhibiting cancer metastasis.

Epithin, Matriptase, Membrane types serine protease 1 (MT-SP1) and Serine protease tumor associated differentially expressed gene 15 Prss14 (serine protease 14) / suppression of tumorigenicity 14 (also known as TADG-15) is a representative member of type II membrane serine proteases.

Several pathological analyzes using cancer patient samples showed consistently overexpressed Prss14 / ST14 in epithelial type advanced cancer. Surgical results of esophageal squamous cell carcinoma showed that the prognosis of disease-free survival was extremely poor when Prss14 / ST14 was expressed. High levels of Prss14 / ST14 were also detected in several human breast cancer cell lines and breast cancer tissues. Currently, the role of Prss14 / ST14 in breast cancer progression, metastasis and patient survival is very important. Recently, the inventors of the present inventors have systematically analyzed specific genes (ER, PR, HER2) and EMT genes according to breast cancer type using a gene expression public database. As a result, Prss14 / ST14 is ER ^- or triple negative breast cancer (triple negative; It is reported to be a good prognostic marker for TN) type.

The intracellular function of Prss14 / ST14 on cancer progression and metastasis is widely studied through high expressing cell and mouse models. 1) Ectodomain secreted from the cells into the culture medium induced angiogenesis such as endothelial cell migration, invasion and tube formation. The angiogenic process may be blocked by polyclonal specific antibodies. 2) Prss14 / ST14 are essential for EMT. TGFβ-induced EMT in thymoma cell line 427.1.86 was blocked when the Prss14 / ST14 message was knocked down. In addition, induced Prss14 / ST14 gene expression led to EMT in MDCK cells. 3) Prss14 / ST14 is critically involved in endothelial cell migration of cancer cells by upregulating Tie2 signaling in the endothelium and is critically involved in endothelial cell permeation migration of macrophages through IFNγ activation. 4) Prss14 / ST14 plays an important role in metastatic pulmonary nodule formation in 4T1 breast cancer cells.

Genetically modified mouse models exist which clearly show the phenotype of Prss14 / ST14 related cancers. When the K5 promoter is used for transgenic expression of Prss14 / ST14, skin adenoma spontaneously develops in mice that can be accelerated by tumor promoting compounds. These studies clearly demonstrate the tumorigenic function of Prss14 / ST14. The MMTV-PyMT mouse model was shown to live with longer breast cancer tumor burden than Prss14 / ST14 normal-expressing animals when crossed with a mattriptase hypomorphic mouse. This study also indicates that Prss14 / ST14 plays an important role in cMet signaling corresponding to HGF secreted from fibroblasts in the tumor microenvironment.

Several specific substrates for Prss14 / ST14 proteases are well known for their role in tumor progression and metastasis and can be classified into several families. Extracellular matrix proteins, including collagen and fibronectin, are degraded by Prss14 / ST14 protease activity, which is easily degraded by cell invasion during metastasis. In addition, proteins involved in tumor growth and proliferation, such as EGFR and PDGF-D, are also known as substrates. Proteins such as MSP-1 and Laminin 322 are involved in cell survival. In addition, Prss14 / ST14 protease can cleave PAR-2, uPA and MMP3 to sequentially activate several proteases. Prss14 / ST14 protease activity can induce signaling by cleaving and activating cellular receptors or ligands. The self-activating ability of the protease may be a major feature as an important part of regulatory targets to block tumor progression and metastasis.

The Prss14 / ST14 protein undergoes several processing steps prior to expressing protease function, which requires activation and separation from the cognate inhibitors HAI1 and HAI2. The first processing of the Prss14 / ST14 protein occurs at aa149 during biogenesis in the endoplasmic reticulum. When the protein is located on the cell membrane, more than one proteolytic cleavage occurs. When Prss14 / ST14 protein is shed, it is known that cleavage occurs near aa189 and aa204. The enzyme involved in the TGFβ or PMA induction process is TNF converting enzyme (TACE). However, serine proteases may also contribute to the shedding process. In the case of PMA induced shedding, filamin was found to be important for transporting proximal Prss14 / ST14 proteins via secretory enzymes and actin rearrangements. Like other serine proteases, the Prss14 / ST14 protein also requires activation through cleavage of the activation ring located at aa604. However, the detailed processing mechanism associated with it is still not clear.

Registered Korean Patent No. 10-1500670 (registered on March 3, 2015)

An object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to the autolytic ring portion of PRSS14 / ST14.

Another object of the present invention comprises a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid molecule, a cell transformed with the recombinant expression vector, and culturing the cell. Disclosed is a method for producing an antibody or antigen-binding fragment thereof that specifically binds to an autolytic ring portion of ST14.

Still another object of the present invention is to provide a cancer diagnostic composition or cancer diagnostic kit comprising the antibody or antigen-binding fragment thereof as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer metastasis, comprising the antibody or antigen-binding fragment thereof as an active ingredient, and a health functional food composition for preventing or improving cancer metastasis.

Another object of the present invention is to provide a composition for detecting PRSS14 / ST14 protein comprising the antibody or antigen-binding fragment thereof as an active ingredient, and a method for detecting PRSS14 / ST14 protein in a sample.

In order to achieve the above object, the present invention includes a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2 and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3 A light chain variable region; And a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, PRSS14 An antibody or antigen-binding fragment thereof that specifically binds to the autolytic ring portion of / ST14 is provided.

In addition, the present invention provides a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 7; And a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 8 or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9, or an antibody that specifically binds to an autolytic ring portion of PRSS14 / ST14; or Antigen binding fragment thereof.

The present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

The present invention also provides a recombinant expression vector comprising the nucleic acid molecule.

The present invention also provides a cell transformed with the recombinant expression vector.

The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to an autolytic ring portion of PRSS14 / ST14 comprising culturing the transformed cells.

The present invention also provides a hybridoma cell line producing the antibody or antigen-binding fragment thereof.

The present invention also provides a cancer diagnostic composition comprising the antibody or antigen-binding fragment thereof as an active ingredient.

In addition, the present invention is the antibody or antigen thereof.

It provides a diagnostic kit for cancer comprising the binding fragment as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating cancer metastasis, comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The present invention also provides a health functional food composition for preventing or improving cancer metastasis comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The present invention also provides a composition for detecting PRSS14 / ST14 protein comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The present invention also provides a method for detecting PRSS14 / ST14 protein in a sample using the antibody or antigen-binding fragment thereof.

The present invention relates to a monoclonal antibody that specifically recognizes an autocatalytic loop portion of PRSS14 / ST14 and its use, wherein the monoclonal antibody mAb3F3 specifically binds to the autolytic ring portion of PRSS14 / ST14. , Humanized antibodies thereof and their use for diagnosing or inhibiting cancer metastasis. In mouse models, it was confirmed that Prss14 / ST14 is critically involved in lung metastasis of breast cancer, epitopes containing autolytic ring portions of Prss14 / ST14 protein can function as metastasis prevention vaccines, and in MMTV-PyMT mouse model, It was found that new monoclonal antibodies specific for the autolytic ring can recognize the epitope sequence and inhibit metastasis in a structure specific manner. Accordingly, the antibody specific for the autolytic ring portion of the PRSS14 / ST14 of the present invention is expected to be useful for diagnosing cancer metastasis and treating cancer metastasis.

1 shows that PRSS14 / ST14 plays an important role in metastasis of E0771 breast cancer cells orally injected into 4T1 and C57BL / 6 mice orally injected into Balb / c mice. (A) Survival curves of host mice with 4T1 and 4T1 EpiKD cells are shown. 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ , 1 × 10 ^5, or 1 × 10 ⁶ cells (Con; solid line) or 4T1 EpiKD cells (EpiKD; dotted line) of the 4T1 control group in the 7th breast of Balb / c mice. It was implanted into a fat pad. The total cell number injected is shown in the graph. (B) Growth curves of 4T1 primary tumors are shown. Tumor size was calculated according to the following equation. (Length × width ² ) / 2. In order from top to bottom, 1 × 10 ⁴ , 1 × 10 ⁵ and 1 × 10 ⁶ cell numbers were injected. Solid line: 4T1con, dotted line: 4T1EpiKD. (C) 4T1 primary tumor weight at death and 4T1 lung metastasis number. Top panel: primary tumor weight, bottom panel: counted nodules. Black circle: 4T1con, White circle: 4T1EpiKD. Mean and standard error are shown. (D) Survival rate of C57BL / 6 mice implanted on 7th mammary fat pad (n = 10), E0771-EpiKD-C3 cells (dotted line) or E0771-Control-D11 cells (solid line) (1 × 10 ⁴ , 1 × 10 ⁵ , 1 × 10 ⁶ cells / mouse, darker lines are more dense). (E) E0771-EpiKD-C3 (dotted line) or E0771-Control-D11 (solid line). Growth curves of primary tumors are shown (tumor volume (mm ³ ) = (width x length ² ) / 2). (F) Comparison of E0771 primary tumor weight and E0771 lung weight of dead mice (E0771-EpiKD-C3 tumor (white circle) or E0771-Control-D11 (black circle)). P values were determined by unpaired t test in Prism 6 software.
2 shows antigen production and test results with cancer metastasis prevention vaccine functionality. (A) The amino acid sequence position and the position of the antigen selected as the test object are shown. (B) mouse and human sequences are shown. (C) The location and structure of the selected antigens are shown. (D) The autolysis ring Epi-SP antigen was tested with a cancer prophylactic vaccine in Balb / c with 4T1 cells. In the presence of CFA, 4T1 cells were injected after immunization with Epi-SP antigen. Survival and number of lung metastatic nodules in (E, F) mice were observed. Adj: Adjuvant, SP: Epi-SP. Mean values for metastasis of each group are shown. (G) The level of 4T1 metastasis reduction by Epi-SP immunization was comparable to Prss14 / ST14KD levels. 4T1C: 4T1 control cells, 4T1KD: 4T1 EpiKD. (H) The antigen antibody specificities of Epi-SP and Epi-Sc are shown. Antigens were immunized three times through prophylactic vaccine experiments and antibody responses to specific antigens were tested by ELISA. SP: EPi-SP, Sc: EPi-Sc. (I) Immunized mouse-derived antiserum blocks endothelial cell permeation migration. The overall schematic of the experiment is shown on the left, and the results of cell migration of MS1 endothelial cells are shown on the right. Mean and standard error are shown.
3 shows antigen production of stable self-activating loop structures and test results as metastasis prevention vaccines in C57BL / 6 mice. (A) The 604: 627 amino acid sequence of human PRSS14 comprising autoactivation cleavage site is well conserved. (B) The comparison results of the natural 604: 627 sequence structure (two left panels) and the synthesized 604: 627 sequence peptide structure (two right panels) in human and mouse Prss14 / ST14. The structure of 604: 627 is conserved as a ring structure in both humans and mice. The expected structure of the synthesized 604: 627 peptide did not retain the original ring structure (Linear). To create a ring structure similar to the original ring structure, cysteine was added to the 627th amino acid (Loop). Activated cleavage sites are indicated with white arrows or blue arrows. Single amino acids of the peptide are shown in white letters. (C) After immunization with SP, Loop, and Linear peptides with Immun Alum, 1 × 10 ⁶ E0771 cells were injected through the tail vein of C57BL / 6 mice. Percentage of metastasis area in lung derived from immunized C57BL / 6 mice. The transition region was measured by Image J program (transition region / total region * 100). (D) After immunization with SP, Loop and Linear peptides with MF59 adjuvant, 1 × 10 ⁶ E0771 cells were injected through the tail vein of C57BL / 6 mice. Nodule number and metastasis area were measured by ImageJ program. Lung weights were measured on sacrifice day. P values were determined by unpaired t test of Prism 6 software. (E) Representative images of metastatic lungs are shown. (F) Results of antibody production against SP and Loop peptides were shown, and its isotypes were tested by enzyme-linked immunosorbent assay (ELISA).
Figure 4 shows the results of the production and characterization of sequence and structure specific mAb in the self-activating ring of PRSS14. (A) BSA conjugation Shows ELISA results of mAbs that only bind Mat-Loop and BSA to the coated antigen. (B) Competitive ELISA results of human ring (Mat-Loop) and mouse ring (Epi-Loop) peptides binding to mAb3F3 are shown. Sc, scrambled sequence. (C) 3F3 Fab modeling results based on amino acid sequence via PIGS. Red; H-CDR1, yellow; H-CDR2, blue; H-CDR3, green; L-CDR1, purple; L-CDR2, gray; L-CDR3. (D) Docking modeling results of mAb3F3 for Mat-loop peptide are shown (lowest energy, -238). Turquoise; Cyclic peptides. (E) The amino acids interacting with mAb3F3 in the Mat-loop peptide are shown in cyan. White arrows indicate activated cleavage sites. (F to I) The results of immunoprecipitation and Western blotting to confirm the specific interaction of mAb3F3 to undenatured Prss14 / ST14 expressed in cells are shown. (F) Whole lysates of HEK293T cells transfected with full length human Prss14 / ST14 (Mat) and vector (Vec) were reacted with mAb3F3, respectively. (G) Total lysates of HEK293T cells transfected with EGFP-S805A and EGFP were reacted with mAb3F3, respectively. (H) Human breast cancer cells MCF7 were reacted with mAb3F3. (I) Results of mouse mammary tumor cells 4T1 are shown. (J) Flow cytometry results of human Prss14 / ST14 expressed in HEK293T cells are shown. Mat-Loop and Epi-Sc were used as competitors. (K) Specificity test results through two-dimensional flow cytometry analysis of CHO-s cells transiently transfected with CD8 or full length Prss14 / ST14.
5 shows the results of immunofluorescence detection for Prss14 / ST14 expression in several human and mouse cancer cells. (A) MCF7 breast cancer cell results stained with DAPI, actin and mAb3F3 are shown. (B) T47D breast cancer cells stained with mAb3F3 and DAPI are shown. (C) Several cancer cells stained with mAb3F3. MDA-MB-453: human triple negative breast cancer cell line, SNU216: human gastric adenocarcinoma cell line, MKN45: human gastric cancer cell line, PC3: human prostate cancer cell line, OE19: esophageal adenocarcinoma cell line, HCT116: human colon cancer, 427.1.86: mouse thymoma Cell line, 4T1: mouse breast cancer cell line.
FIG. 6 shows that mAb3F3 may reduce cancer cell mobility and reduce breast cancer metastasis in the MMTV-PyMT transgenic mouse model. (A) Results of wound healing migration assay in 4T1 cells after mAb3F3 treatment. (B) Transfection migration was shown when 5, 10, 20 μg of mAb3F3 and mouse monoclonal antibody mAb5 were added to MCF7 cells. Solid line: mAb5, dotted line: mAb3F3. (C) Cells were expressed after mAb3F3 was added to MCF7 cells cultured for 12 hours. (D) Cell cycle of MCF7 cells after mAb3F3 was added. (E) Tumor size growth curves of MMTV-PyMT mice injected with PBS, Taxol and mAb3F3 are shown. The first injection was performed on Day 0. Arrows indicate days of infusion of mAb3F3. (F) Graph of tumor volume for each group on Day 11. (G) Shows the number of metastatic nodules measured on sacrifice day. (H) Representative lung images of each group are shown.
FIG. 7 shows that humanized mAb3F3 binds to unmodified proteins that reduce transwell migration and inhibit activation in flow cytometry. (A) The treatment of mouse mAb3F3 and humanized mAb3F3 in human breast cancer cells MCF7 decreased transwell migration. mAb5 mouse antibody was used as a negative control. Black: mAb5, red: mouse mAb3F3, orange: humanized mAb3F3-35, green: humanized mAb3F3-37. (B) Flow cytometry showed that humanized mAb3F3 clones bound to S805A protein in HEK293TF cells co-transformed with GFP and unactivated protein (S805A) and tDTomato transfected HEK293TF cells. Blue: tdTomato +, red: GFP +

The present inventors confirmed that in mouse models, Prss14 / ST14 is critically involved in pulmonary metastasis of breast cancer, and epitopes containing autolytic ring portions of Prss14 / ST14 protein can function as metastasis prevention vaccines. In the MMTV-PyMT mouse model, it was confirmed that new monoclonal antibodies specific for the autolytic ring can recognize the epitope sequence and inhibit metastasis in a structure specific manner, thus completing the present invention.

The present invention provides a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, PRSS14 An antibody or antigen-binding fragment thereof that specifically binds to the autolytic ring portion of / ST14 is provided.

In addition, the present invention provides a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 7; And a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 8 or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9, or an antibody that specifically binds to an autolytic ring portion of PRSS14 / ST14; or Antigen binding fragment thereof. In detail, the antibody may be a humanized antibody.

Preferably, the autolytic ring portion of PRSS14 / ST14 may include an amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 11, but is not limited thereto.

On the other hand, light chain CDR1, CDR2 and CDR3 consisting of the amino acids represented by SEQ ID NO: 1 to SEQ ID NO: 3, and heavy chain CDR4, CDR5 and CDR6 consisting of the amino acids represented by SEQ ID NO: 4 to SEQ ID NO: 6 are listed in Table 1, the sequence A light chain variable region comprising the amino acid sequence represented by No. 7; And a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 8 or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9.

In addition, the amino acid sequence represented by SEQ ID NO: 10 is the autolytic ring portion (Mat-Loop) of human PRSS14 / ST14, and the amino acid sequence represented by SEQ ID NO: 11 is the autolytic ring portion (Epi-Loop) of mouse PRSS14 / ST14 to be.

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, for example, a monoclonal antibody, It may include both clone antibodies, full-length antibodies and antibody fragments. The term “antibody” may also include bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies, or tetrabodies.

As used herein, the term “monoclonal antibody” refers to an antibody molecule of monomolecular composition obtained from substantially the same antibody population, which monoclonal antibody, in contrast to a polyclonal antibody, may bind to several epitopes. Single binding and affinity are shown for epitopes. As used herein, the term “full length antibody” is a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected by a heavy chain and a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.

As used herein, the term "humanized antibody" refers to an antibody that is nonimmunogenic or reduced immunogenicity to humans. The humanized antibody is an altered amino acid sequence and the amino acid sequence of the antibody can be reconstituted for the desired purpose. These possible changes are numerous and vary from one or several amino acids to complete reconstitution of the variable and / or constant regions of the antibody. In general, the modification of the variable region is performed to increase the binding capacity and affinity of the antigen, whereas the modification in the constant region may cause intracellular actions such as fixation of complement, interaction with the membrane, and other functioning agents. To increase.

As used herein, the term “heavy chain” refers to a variable region VH comprising an amino acid sequence having sufficient variable region sequence for conferring specificity to an antigen and a full length heavy chain comprising three constant regions CH1, CH2 and CH3 and fragments thereof. It can contain everything. In addition, the term “light chain” in the present invention may include both the full-length light chain and the fragment thereof including the variable region VL and the constant region CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. have.

As used herein, the terms “fragment”, “antibody fragment” and “antigen binding fragment” are used interchangeably to refer to any fragment of an antibody of the invention that retains the antigen binding function of the antibody. Exemplary antigen binding fragments include, but are not limited to, Fab, Fab ', F (ab') 2, Fv, and the like.

The antibody or antigen-binding fragment thereof of the present invention may include not only the sequences of the antibodies described herein but also biological equivalents thereof within the range that can exhibit the ability to specifically bind to the autolytic ring portion of PRSS14 / ST14. have. For example, further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on this, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan, and tyrosine are biologically equivalent functions.

The present invention also provides nucleic acid molecules encoding antibodies or antigen binding fragments thereof.

As used herein, the term “nucleic acid molecule” has the meaning of encompassing DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic structural units in nucleic acid molecules are not only natural nucleotides but also sugar or base sites. It also includes modified analogs. The sequences of nucleic acid molecules encoding heavy and light chain variable regions of the invention can be modified, which modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides.

The present invention also provides a recombinant expression vector comprising the nucleic acid molecule.

In the present invention, "vector" refers to a DNA molecule that replicates itself that is used to carry a clone gene (or another piece of clone DNA).

In the present invention, an "expression vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, which corresponds to all genes that can distinguish transformed cells from non-transformed cells. Examples include antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, Chloramphenicol, and the like. It does not need to be, but can be appropriately selected by those skilled in the art.

To express the DNA sequences of the present invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTR of lactovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters. , A major operator and promoter region of phage lambda, a regulatory region of fd code protein, a promoter for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, yeast alpha-crossing system Other sequences of construction and induction known to modulate the expression of promoters and prokaryotic or eukaryotic cells or genes of their viruses may be included and various combinations thereof.

The vector expressing the antibody of the present invention may be a vector system in which the light and heavy chains are simultaneously expressed in one vector, or a system in which the light and heavy chains are expressed in separate vectors, respectively. In the latter case, both vectors are introduced into the host cell via co-transformation and targeted transformation. Simultaneous transformation is a method of introducing each vector DNA encoding a light chain and a heavy chain into a host cell at the same time, and then selecting cells expressing both the light chain and the heavy chain. Target transformation involves selecting the cells transformed with the vector containing the light (or heavy) chain and transforming the selected cells expressing the light chain with the vector containing the heavy (or light) chain to express both the light and heavy chains. This is the method of finally selecting the cells.

The present invention also provides a cell transformed with the recombinant expression vector.

Cells capable of stably and continuously cloning and expressing the vectors of the invention can be any host cell known in the art, for example Escherichia coli, Bacillus subtilis and Bacillus thuringin Bacillus genus strains such as cis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g. For example, prokaryotic host cells such as, but not limited to, Staphylocus carnosus.

The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to an autolytic ring portion of PRSS14 / ST14 comprising culturing the transformed cells.

Cultivation of the transformed cells in the method for producing the antibody or antigen-binding fragment thereof may be carried out according to appropriate media and culture conditions known in the art. This culturing process can be used by those skilled in the art can be easily adjusted according to the strain selected. Cell culture is divided into a batch culture, a fed-batch, and a continuous culture method according to the suspension culture and adhesion culture, the culture method according to the cell growth method. The medium used for culturing must adequately meet the requirements of the particular strain.

The present invention also provides hybridoma cell lines that produce antibodies or antigen binding fragments thereof.

The term "hybridoma cell line" of the present invention refers to a cell made by artificially fusion of two different cell types, which is a cell fusion such as polyethylene glycol or two or more homologous cells or heterologous cells using some kind of virus. A cell or cell line in which cells are fused to integrate different functions of different cells into one cell. In particular, hybrid cells in which myeloma cells and B cells, which are precursor cells of antibody-producing cells, are fused among myeloma cells and lymphocytes contained in the spleen or lymph nodes, are widely used in research and clinical practice. In addition, T cells producing lymphokines (physiologically active substances) and hybrid horses, which are tumor cells thereof, have been put into practical use.

The present invention also provides a composition for diagnosing cancer comprising an antibody or antigen-binding fragment thereof as an active ingredient.

Specifically, the cancer may be breast cancer, stomach cancer, prostate cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, blood cancer, bone cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, bronchial cancer, bladder cancer , Kidney cancer, gallbladder cancer, cervical cancer, malignant melanoma, lymphoma or bone marrow cancer, but is not limited thereto.

The present invention also provides a kit for diagnosing cancer, comprising an antibody or antigen-binding fragment thereof as an active ingredient.

The cancer diagnostic kit may include radioimmunoassay (RIA) kit, enzyme immunoassay (ELISA) kit, immunofluorescence kit, chemiluminescence binding kit, protein chip kit, chromatography kit, magnetic activated cell sorting, MACS) kit, etc., but is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating cancer metastasis, comprising the antibody or antigen-binding fragment thereof as an active ingredient.

Specifically, the cancer may be breast cancer, stomach cancer, prostate cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, blood cancer, bone cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, bronchial cancer, bladder cancer , Kidney cancer, gallbladder cancer, cervical cancer, malignant melanoma, lymphoma or bone marrow cancer, but is not limited thereto.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch , Acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stearic acid Magnesium acid and mineral oil, and the like, but is not limited thereto. The composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a humectant, a sweetener, a flavor, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration, etc. Can be administered. Upon oral administration, because the protein or peptide is digested, the oral composition can be formulated to coat the active agent or protect it from degradation in the stomach, and the compositions of the present invention can be any device capable of moving the active substance to target cells. It can be administered by.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or by incorporating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, suppositories, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.

The present invention also provides a health functional food composition for preventing or improving cancer metastasis comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The dietary supplement composition may be provided in the form of powder, granules, tablets, capsules, syrups, beverages or pills, wherein the health food composition is used with other foods or food additives in addition to the composition according to the present invention as an active ingredient, It can be suitably used according to the phosphorus method. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the antibody or antigen-binding fragment thereof contained in the dietary supplement composition may be used in accordance with the effective dose of the pharmaceutical composition, but for long-term intake for health and hygiene purposes or for health control purposes. It may be less than the above range, it is obvious that the active ingredient can be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic drinks, vitamin complexes, etc. are mentioned.

The present invention also provides a composition for detecting PRSS14 / ST14 protein comprising an antibody or an antigen-binding fragment thereof as an active ingredient.

In detail, the PRSS14 / ST14 protein may be in a native form, but is not limited thereto.

The present invention also provides a method for detecting PRSS14 / ST14 protein in a sample using the antibody or antigen-binding fragment thereof.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

1. Cell Culture

All cells were maintained in 10% fetal bovine serum (FBS; Welgene or Gibco), penicillin and streptomycin (Welgene) and Dulbecco's Modified Eagle's Media (DMEM; Welgene) with 4 mM L-glutamine (Welgene). I was. All attached cells were passaged every 2-3 days with trypsin-EDTA (Welgene).

2. Mouse Model

Balb / c and C57BL / 6 mice were purchased from Biolink. Mice were bred in animal facilities at Inha University molecular cell immunology laboratory according to the LMCI protocol appropriate for the use of experimental animals. In a breast cancer orthotopic mouse model, Avertin (2) dissolved in 2-Methylbutanol-2 (Sigma) was used for Balb / c female mice transplanted with 4T1 cells or C57BL / 6 female mice transplanted with E0771 cells. Anesthetized with 2,2-Tribromoethanol (Sigma). The incision site was closed with a wound clip, and primary tumor growth was observed by measuring tumor length (a) and width (b) twice a week. Tumor volume (V) was determined according to the Carlsson: V = (ab ² ) / 2 equation, with 'a' and 'b' representing the longest and smallest diameters of the tumor, respectively. After death, the primary tumor was weighed and the number of pulmonary nodules was checked. To confirm the effect of the antibody in the tumor model, 9-week-old female MMTV-PyMT mice were injected intraperitoneally with PBS, Taxol or mAb3F3 antibody in an amount of 50 μg / mouse. Treatment was started on five 9-week-old mice per group, injected twice a week until 14 weeks of age. Tumor size was measured via a caliper twice a week. Mice were euthanized at 15 weeks of age. The number and extent of pulmonary nodules were photographed and processed using imageJ.

3. Antigenicity Peptide  synthesis

Peptides were synthesized in Thermo or Abclone. Disulfide bond formation between both ends was confirmed by mass spectrometry (Abclone). Conjugation of keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) was performed in Youngin Frontier. Epi-Sc (EQGKGARDWPEWAVQGVNT), the same amino acid composition as the Epi-SP peptide (KQARVVGGTNADEGEWPWQ), was selected from the website (http://users.umassmed.edu/ian.york/Scramble.shtml) and synthesized at Youngin Frontier.

4. Protein structure modelling

Tertiary model structures of proteins and peptides were obtained from the I-TASSER server (http://zhanglab.ccmb.med.unich.edu/I-TASSER/). Prediction models were analyzed through chimera software (http://www.cgl.ucsf.edu/chimera/download.html). Structural modeling of fragment antigen-binding (Fab) of monoclonal antibodies was performed via a PIGS web server (http://www.biocomputing.it/pigs). Antigen-antibody docking modeling was obtained via a ClusPro 2.0 web server (http://cluspro.bu.edu).

5. Endothelial cell permeation migration ( Transendothelial  migration analysis

On Boyden Chamber (8 μm pore insert, Falcon), MS1 cells (5 × 10 ⁴ ) were incubated until confluent in DMEM with 10% FBS. 4T1 cells were stained with CFSE (Molecular Probes ™) and resuspended in DMEM containing 5% FBS and anti-SP sera. CFSE-stained 1 × 10 ⁵ 4T1 cells were added on MS1 cells and incubated for 4 hours. Cells were fixed with 3.7% formaldehyde and residual cells were scraped off and washed off. Images of CFSE-positive cells migrated to the opposite side were obtained through an IX-51 microscope (Olympus) equipped with AxioCam MRm (Zeiss), and the number of migrated cells was transferred to ImageJ (http://rsbweb.nih.gov/ij). Analyzed.

6. Transwell  move ( Transwell  migration analysis

5 × 10 ⁴ MCF7 cells were incubated with humanized mAb3F3 and serum-free culture on top of the Boyden Chamber (8 μm pore insert, Corning). Each sample was tested in triplicate volumes. After 4 hours, the culture solution containing 10% serum was added to the lower chamber. After 24 hours, the cells on the upper chamber membrane surface were removed with a cotton swab and the cells migrated to the lower chamber membrane surface were fixed for 10 minutes with 100% methanol. After staining for 5 minutes with 0.2% crystal violet, 10 areas were photographed by checking the microscope. The number of migrated cells was analyzed by ImageJ.

7. Monoclonal antibody  production

Female Balb / c mice were immunized into the peritoneum four times at three-week intervals with KLH conjugated peptide antigens mixed with Imject ^R Alum (Thermo). Three days prior to fusion, primed mice were boosted with final immunization. Upon fusion, single cell suspension collected from the spleen was fused with SP2 / 0-Ag14 myeloma using 50% polyethylene glycol (PEG) solution (Sigma) and 96 wells containing DMEM with 20% serum added. The plates were incubated. Hybridoma cells were selected from Hypoxanthine, Aminopterin, Thymidine (with HAT) (Gibco), and from Hyxoxanthine, Thymidine (with HAT). Maintained. Hybridoma clones were selected through two limiting dilutions.

8. Enzyme Bond Immunoprecipitation Assay (Enzyme-Linked Immunosorbent Assay; ELISA)

For conventional ELISA analysis, on wells of 96 well plates (SPL), 50 ng of peptides were coated with coating buffer (32 mM Na ₂ CO ₃ , 68 mM NaHCO ₃ , 0.1% NaN ₃ , pH 9.6) and dissolved in PBS. Blocked with% nonfat dry milk. After washing with 0.4 M Tris-Buffered Saline (pH7.4) containing 0.1% Tween20, primary antibodies were added and reacted at 37 ° C for 1 hour. After washing, anti-mouse horseradish peroxidase (HRP) conjugated antibody was added and reacted at 37 ° C. for 1 hour. Super AquaBlue ELISA substrate (eBioscience) was used to measure the optical density at 405 nm using an ELISA Reader (TECAN, Sunrise ^™ ). C-terminal coating ELISA was performed using a peptide coating kit according to the protocol provided by TaKaRa. In 96-well reaction plates, 225 ng of peptide was coated at 37 ° C. for 2 hours with 50 μl reaction buffer and 10 μl coupling reagent per well. The coated wells were blocked for 1 hour with blocking solution. Except for washing with distilled water, the rest of the process was the same as the general ELISA.

9. Flow cell  analysis

To test for binding of undenatured proteins, HEK293T cells transfected with constructs (full length human Prss14 / ST14, S805A mutant and EGFP-S805A) were washed twice with PBS and enzyme-free to float into single cells. Treated with PBS-based cell dissociation buffer (Gibco). For CHO-s cells and HEK293TF cells, cells were harvested without dissociation. 5 × 10 ⁵ cells were seeded in round bottom 96-well plates containing FACS buffer (0.1 mg / ml BSA in PBS). Cells were centrifuged at 4 ° C. for 2 minutes at 1500 rpm. To detect live cells, Fixable Viability dye eFluor 455UV (affymetrix) was treated in the dark at 4 ° C. for 30 minutes. After washing, cells were fixed with Fix & Perm kit (CALTAG) and permeated. To block Fc receptors, Human Fc receptor binding inhibitor (14-9161, affymetrix) was used. For mAbs used as primary antibodies, 3 μg / ml PE conjugated anti-mouse Kappa (PharMingen) was used as secondary antibody. HEK293TF cells were used to confirm binding of humanized mAb3F3 with undenatured proteins. Round bottom 96-C mixtures of HEK293TF cells co-transfected with 70% pcDNA3.1 / EGFP and pcDNA3.1 / matriptase (S805A) -TST, and HEK293TF cells co-transformed with 30% pcDNA3.1 and pCMV / tdTomato. Inoculate well plates. FreeStyle ^(TM) 293 Expression Medium, a culture of HEK293TF cells, was used for washing and dilution. The cells were washed by centrifugation at 4 ° C. at 1500 rpm for 2 minutes, and then treated with 10% normal rat serum, 10% normal hamster serum, and 70% 10 × 2.4G 2 for 5 minutes. Cells were fixed and permeabilized using Foxp3 / Transcription Factor Staining Buffer. After washing, 30 μg / ml of humanized mAb3F3 was reacted in the dark for 20 minutes. After washing, Alexa Fluor ^® 647 anti-human IgG Fc antibody was reacted as a secondary antibody in the dark for 15 minutes. Data was collected using a BD Accuri ^{( TM )} Flow Cytometer and analyzed in the Flowjo 10 program.

10. Immunoprecipitation and Weston Blotting

Cells were lysed in non-denaturing lysis buffer (IP buffer; 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 50 mM HEPES, pH7.4) with protease inhibitor cocktail and 4 ° C., 20 minutes in a stirrer. Reacted for a while. Lysed cells were collected with a scraper. After cleaving the genomic DNAs through a 1 ml syringe with a 26G needle, cell debris was pelleted by centrifugation at 15000 rpm for 15 minutes at 4 ° C. The cell lysates were reacted with monoclonal antibody at 4 ° C. overnight, and then rotated at 50% suspension of protein A / G agarose bead (Pierce) for 2 hours at room temperature. Protein A / G beads capture antibody-antigen complex was washed three times with PBS by centrifugation at 1500 rpm for 1 minute at 4 ℃, the supernatant was removed. To elute the antibody-antigen complex, the complex was mixed with SDS sample buffer and reacted at 99 ° C. for 5 minutes. The eluted supernatant was analyzed by Western blot using PVDF membrane (Pall, FluoroTrans). The membrane was reacted with 5% skim milk dissolved in 0.1% TritonX-100 in PBS (PBS-T) and reacted with polyclonal rabbit antibody IM1014 (Calbiochem) corresponding to human protein or polyclonal rabbit anti-epithin serum corresponding to mouse protein. I was. HRP-conjugated anti-rabbit IgG (1: 5000 dilution) was used with Luminol / Enhancer solution and Stable Peroxide solution (SuperSignal ^R West Pico, Thermo). Chemiluminescence was observed through LAS-4000mini (GE healthcare Life Sciences), and the signal of each band was digitized through ImageJ.

11. Immunofluorescence

Cells were inoculated in 0.1% gelatin coated coverslips and fixed at 4% paraformaldehyde at room temperature for 10 minutes. Cells were then permeated with 0.5% PBS-T and nonspecific binding was blocked for 30 minutes with 10% goat serum / 1% gelatin dissolved in 0.1% PBS-T. mAb3F3 was used as the primary antibody. FITC-conjugated anti-mouse IgG was used as a secondary antibody. Actin was visualized using phalloidin-iFluor647 (Abcam) and nuclei stained with 0.3 μM DAPI (Molecular Probes) diluted in Mowiol mounting media (Sigma). The stained cells were visualized by confocal microscopy using LSM510 meta (Zeiss) and processed with Photoshop.

< Example  1> In metastasis of 4T1 and E0771 breast cancer cells Prss14 / ST14 role

To confirm the role of PRSS14 / ST14 in breast cancer progression and metastasis, two mouse breast cancer cell lines, 4T1 and E0771, were used, and Balb / C and C57BL / 6 were used as syngeneic mouse hosts, respectively (FIG. One). PRSS14 / ST14 knockdown cells (EpiKD) and their control cells (con) were injected orthotopically into the mammary fat pad and followed for survival, tumor initiation, primary tumor growth and lung metastasis. 4T1 EpiKD increased survival in a dose dependent manner (FIG. 1A), slightly reduced primary tumor growth (FIGS. 1B and 1C), and significantly reduced the number of metastatic nodules in the lung (FIG. 1C). E0771 EpiKD cell lines were also prepared and tested in the same manner (FIGS. 1D-1F). Knockdown of PRSS14 / ST14 significantly increased survival (FIG. 1D), reduced tumor growth (FIG. 1E and FIG. 1F), and reduced lung metastasis (FIG. 1F). In the 4T1 breast cancer model, which is more metastatic than E0771, lung metastasis was clearly inhibited by knockdown. The results indicated that Prss14 / ST14 is essential for 4T1 tumor proliferation and metastasis.

< Example  2> specific structural antigens that can trigger antibody responses Peptide  making

Since Prss14 / ST14 plays an important role by activating several downstream substrates, we hypothesized that inhibition of this function through antibodies can block tumor metastasis and increase the survival of tumor patients. Accordingly, the inventors decided to produce antigenic epitopes showing high antigenicity, hydrophilicity, surface probability, evolutionary conservation, and avoiding protein modification sites such as glycosylation. Selected antigenic epitopes are regions associated with activation cleavage and are Epi-SP, an activation loop (19mer) in proteases (FIGS. 2A-2C). Epi-SP epitopes are located at appropriate sites as antigens in the model, so we decided to test their potential as anti-metastatic vaccines. Immunization of Epi-SP, a KLH conjugated mouse epitope peptide, produced antibodies that were readily detectable in Balb / c mice.

< Example  3> antigens that are effective in inhibiting 4T1 breast cancer metastasis Peptide  Immunization

To test the possibility of reducing cancer metastasis through epitope self-immunization, metastasis analysis via tail vein injection was performed after three immunizations with Complete Freund's adjuvant (FIG. 2D). 19 days after cell injection, metastatic nodules in the lungs were counted. Epi-SP statistically significant reduced the number of metastatic nodules in the lungs, indicating that autologous cancer immunization of the Prss14 / ST14 antigen can inhibit cancer metastasis.

Epi-Sc, an epi-SP sequence-scrambled peptide, was selected to verify sequence non-specific effects on blocking metastasis by antibodies. For tail vein variation analysis, 4T1 and 4T1 EpiKD cells were injected into mice immunized with two antigens and tested under the same conditions, clearly showing that metastasis was inhibited in 4 out of 5 mice immunized with Epi-SP peptide. Epi-Sc, on the other hand, failed to block metastasis (FIG. 2G). The level of metastasis reduction was similar to that of 4T1 EpiKD cells. The results indicate that PRSS14 / Epithin peptides reduce 4T1 cancer metastasis in a sequence specific manner. Each antiserum binds only to antigenic sequences with high specificity, with no cross-reactivity between the two antibodies (FIG. 2H).

To determine how Epi-SP peptide immunization can reduce 4T1 cancer metastasis, endothelial cell permeability migration was tested in the presence of mouse serum. 4T1 cancer cells inoculated on a confluent monolayer of MS1 endothelial cells migrated less in the presence of anti-Epi-SP serum (FIG. 2I). The results indicate that antibodies induced with Epi-SP peptides inhibit metastasis by blocking endothelial cell permeation migration of cancer cells.

< Example  4> an autolytic ring capable of inhibiting E0771 metastasis Autocatalytic  loop) peptide

We attempted to obtain monoclonal antibodies specific for the EPi-SP sequence, but were not successful. Such unsuccessful attempts led to the idea that the ephetope peptide might not be stable enough to screen good antibodies. The present inventors reconfirmed the structural model by measuring the distance of amino acids that can form a stabilizing ring. The peptide range of aa604 to aa627 appeared to be the closest terminus covering the autoactivating ring region. This region is highly conserved in three species (FIG. 3A). Only one amino acid showed a difference between human and two rat sequences. The present inventors added cysteine to the C terminus to make disulfide bonds by reduction. Structural modeling of autoactivating rings based on crystal structure analysis of human and mouse proteins as a whole showed a very similar structure compared to modeling of peptide sequences (FIG. 3B).

Three KLH-conjugated peptides, Epi-SP (KQARVVGGTNADEGEWPWQ), human derived linear; Mat-Linear; CGLRSFTRQARVVGGTDADEGEWP, and cysteine bonded loop; Mat-Loop; Immunization with (Alum adjuvant) showed a significant decrease in all C57BL / 6-derived E0771 metastasis assays, whereas the Epi-Sc antigen with random sequences did not (FIG. 3C). KLH conjugated antigens were tested with two adjuvants, Alum and MF59, which showed that Mat-Loop can significantly reduce the number and extent of metastatic nodules and lung weight, The statistical significance was shown to be beyond the impact (FIGS. 3D and 3E). On the other hand, it was confirmed that the antibodies generated under the above conditions are all IgG1 isotype (FIG. 3F).

< Example 5> To human protein  Specificity, mouse PRSS14 Cross-reactive to / ST14 ring mAb3F3 Hybridoma

Based on the successful metastatic blockade results in metastatic preventive vaccine experiments, we have developed monoclonal antibodies (mAbs) capable of recognizing self-activating ring structures. In order to construct a structure specific mAb that directly corresponds to the autolytic ring of PRSS14 / ST14, we performed hybridoma experiments with Mat-Loop peptides. After extensive screening, we isolated mAb3F3 clones and analyzed their binding specificities (FIG. 4). In ELISA coated with BSA conjugated Mat-Loop, mAb3F3 specifically bound to Mat-Loop peptide sequence, but not when tested with BSA alone (FIG. 4A). On ELISA plates, mAb3F3 binding to BSA conjugated Mat-Loop competed not only with Mat-Loop but also with Epi-Loop (FIG. 4B), which was found to exhibit cross-reactivity with human and mouse protein sequences.

As a result of sequencing of the hybridoma-derived mRNA, CDR1, 2, and 3 regions forming antigen binding pockets in both the heavy and light chains were identified (Table 1). The structure of the mAb3F3 Fab region was then predicted based on the amino acid sequence (FIG. 4C) and applied to docking modeling (FIG. 4D). As shown in the modeling results, the antigen rings derived from the whole protein model appeared to fit well with the antibody binding pocket. CDRs of mAb3F3 included an activating cleavage site (FIG. 4E). SPR sensorgrams determined the affinity of mAb3F3 for the BSA conjugated peptide antigen. MAb3F3 affinity for both the human chain (Mat-Loop) and mouse ring (Epi-Loop) peptide appeared to nanomolar levels ^{_{(human: K D = 5.333 × 10 -9}} , ^{_{mouse: K D = 7.814 × 10 -}} 9 (Table 2). The linear form showed 2 logs low affinity and did not bind any sequence at all.

< Example 6> human and  In mouse breast cancer cells PRSS14 Specificity for the unmodified form of / ST14 protein mAb3F3

When peptide antigens are used to produce monoclonal antibodies, whether mAb3F3 can recognize undenatured protein structure is an important issue. Therefore, we confirmed whether mAb3F3 binds to PRSS14 / ST14 expressed in cells. HEK293T cells were transfected with full length human Prss14 / ST14 (Mat) and immunoprecipitation assays were performed (FIGS. 4F-4H). As shown in FIG. 4, transfecting HEK293T cells with full-length constructs revealed four different processed forms of major bands in Western blotting results, with mAb3F3 strongly immunoprecipitated in two or more bands (FIG. 4F). ). Transfection of EGFP-S805A, a construct of recombinant EGFP fusion Prss14 / ST14 protein with S805 mutation, confirmed the immunocomplex with mAb3F3 (FIG. 4G). No detectable band could be identified in the control group transfected with EGFP. Next, the inventors confirmed that mAb3F3 binds to endogenous Prss14 / ST14 expressed in human MCF7 (FIG. 4H). mAb3F3 could easily detect mouse proteins of 4T1 breast cancer cells (FIG. 4I). The results indicated that mAb3F3 specifically binds to mouse and human undenatured proteins. To further confirm by other assays whether mAb3F3 can bind to undenatured Prss14 / ST14, we performed flow cytometry (FIGS. 4J and 4K). mAb3F3 was able to detect Prss14 / ST14 protein expressed in cells. In a sequence specific competition mode, only its own peptides could competitively bind, and peptides with any sequence did not bind, so sequence specificity was clearly confirmed. In addition, binding specificity was also verified through two-color analysis, with cells expressing CD8 protein as a negative population.

< Example  7> expressed in many cancer cells PRSS14 Capable of detecting / ST14 mAb3F3

We also identified the possibility of mAb3F3 as a diagnostic tool. MAb3F3 was applied to immunocytochemical staining of several cancer cell lines (FIG. 5). Specific Prss14 / ST14 staining in the cytoplasm, cell attachment and membrane was detected in human breast cancer cells MCF7 and T47D (FIGS. 5A and 5B). Nuclear staining was not apparent. Interestingly, Prss14 / ST14 staining was only seen in subpopulations and not in all MCF7 cells (FIG. 5A). Through expanded application in human and mouse cancer cell lines expressing Prss14 / ST14, MDA-MB-453 triple negative breast cancer cell line, SNU216 and MKN45 human gastric adenocarcinoma cell line, PC3 human prostate cancer cell line, OE19 human esophageal adenocarcinoma cell line, HCT116 human It was confirmed that mAb3F3 can stain the membrane region or cell attachment region of colorectal cancer, 427.1.86 mouse thymoma and 4T1 mouse breast cancer cell line (FIG. 5C). All cell lines used in the present invention were found to express Prss14 / ST14. Thus, mAb3F3 can be utilized as a new and unique diagnostic reagent for detecting the inactivated form of Prss14 / ST14 protein with an intact uncut activating ring structure.

< Example  8> MMTV - PyMT  Reduce metastasis of breast cancer mAb3F3

We first identified the biological effect of mAb3F3 on whether mAb3F3 could block (self) activation, ie, cleavage of the ring resulting in a 30 kD band in denaturing SDS-PAGE. To induce activation, treatment of MCF7 cells with pH 6 buffer resulted in an increase in the amount of the cleaved activated protease domain (aMat-S) as well as the Press 14 / ST14 long shed form (Mat-S ') in the medium. . Interestingly, leupeptin, a serine protease inhibitor, also increased in both shed and activated forms. The inventors concluded that shedding also affects the system, and determined that a clear system for performing activation experiments was needed. In vitro migration and invasion assays using mouse 427.1.86 cells and human MCF7 cells confirmed a statistically significant increase in invasion or migration through transwells when mAb3F3 was added to both cells.

In spite of unexpected in vitro experiments, the inventors conducted in vivo experiments with antibodies purified from the MMTV-PyMT model (FIG. 6). MMTV-PyMT mice were treated three times with mAb3F3 antibody at 6 weeks of age or with Taxol as a control. With the exception of some size reductions observed in mAb3F3 treated mice on day11, no significant difference in primary tumor volume increase was found between the three groups (FIGS. 6E and 6F). Advanced primary tumors maintained Prss14 / ST14 expression. However, on day 14, the number of tumor nodules decreased, leading to a significant reduction in lung metastases (Figures 6G and 6H). The results indicate that mAb3F3 can inhibit cancer metastasis in MMTV-PyMT mouse model. From these results, it was confirmed that mAb3F3 could be used as a candidate group of prophylactic / therapeutic antibodies targeting metastasis.

Example 9 Humanized mAb3F3 Antibody Clones Inhibiting Cell Mobility and Showing Specificity to Inactivated PRSS14 / ST14 Proteins

To verify the effect on the cell mobility of the humanized antibody, the function of moving to the opposite side of the transwell was confirmed (FIG. 7A). MAb5 binding to denatured epitin protein did not inhibit cell migration up to 30 μg / ml when added to the culture, and mouse hybridoma antibody mAb3F3 showed 70% mobility inhibition up to 3 μg / ml. In this case, humanized antibody h3F3-37 exhibited about 50% mobility inhibition. h3F3-35 showed a similar degree to mAb3F3. In order to confirm the specificity of the humanized antibody, the specificity of antigen-specificity was confirmed by staining the cells in which the protease-functional mutant MatriptaseS805A with EGFP and the cells with only TdTomato were stained together (FIG. 7B). It was confirmed that h3G3-37 was able to stain cells specifically with h3F3-35 within the GFP + range as compared to the case of only the secondary antibody on the left. The amino acid sequences of h3F3-35 and h3F3-37 obtained through the humanization process are shown in Table 3.

VH VL Hu3F3-35 EVQLVESGGGLVQPGGSLRLSCAASGYTFSIYWLEWVRQAPGKGLEWIGEILPGSGNANYNEKFKGRFTFSADTSKNTLYLQMNSLRAEDTAVYYCARSGTDWGQGTLVTVSS
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLEWFQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQGSHVPFTFGPGTKVDIK
Hu3F3-37 QVQLVQSGAEVKKPGASVKVSCKASGYTFSIYWLEWVRQAPGQGLEWMGEILPGSGNANYNEKFKGRVTMTADTSTSTVYMELSSLRSEDTAVYYCARSGTDWGQGTLVTVSS
Same as above

As described above in detail certain parts of the present invention, it is apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Specific monoclonal antibody against autocatalytic loop portion          of PRSS14 / ST14 and use <130> ADP-2019-0198 <150> KR 10-2018-0057630 <151> 2018-05-21 <160> 11 <170> KopatentIn 2.0 <210> 1 <211> 16 <212> PRT <213> mouse <400> 1 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Phe Leu Glu   1 5 10 15 <210> 2 <211> 7 <212> PRT <213> mouse <400> 2 Lys Val Ser Asn Arg Phe Ser   1 5 <210> 3 <211> 9 <212> PRT <213> mouse <400> 3 Phe Gln Gly Ser His Val Pro Phe Thr   1 5 <210> 4 <211> 10 <212> PRT <213> mouse <400> 4 Gly Tyr Thr Phe Ser Ile Tyr Trp Leu Glu   1 5 10 <210> 5 <211> 17 <212> PRT <213> mouse <400> 5 Glu Ile Leu Pro Gly Ser Gly Asn Ala Asn Tyr Asn Glu Lys Phe Lys   1 5 10 15 Gly     <210> 6 <211> 4 <212> PRT <213> mouse <400> 6 Ser Gly Thr Asp   One <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody VL <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Ser              20 25 30 Asn Gly Asn Thr Phe Leu Glu Trp Phe Gln Gln Lys Pro Gly Lys Ala          35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro      50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile  65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Phe Gln Gly                  85 90 95 Ser His Val Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys             100 105 110 <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody VH <400> 8 Glu Val Gln Leu Val 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 Tyr Thr Phe Ser Ile Tyr              20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Asn Ala Asn Tyr Asn Glu Lys Phe      50 55 60 Lys Gly Arg Phe Thr Phe Ser Ala Asp Thr 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 Ser Gly Thr Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser             100 105 110 Ser     <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized antibody VH <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ile Tyr              20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Asn Ala Asn Tyr Asn Glu Lys Phe      50 55 60 Lys Gly Arg Val Thr Met Met Ala Asp Thr Ser Thr Ser Thr Val Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ser Gly Thr Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser             100 105 110 Ser     <210> 10 <211> 24 <212> PRT <213> Human <400> 10 Cys Gly Leu Arg Ser Phe Thr Arg Gln Ala Arg Val Val Gly Gly Thr   1 5 10 15 Asp Ala Asp Glu Gly Glu Trp Pro              20 <210> 11 <211> 24 <212> PRT <213> mouse <400> 11 Cys Gly Leu Arg Ser Phe Thr Lys Gln Ala Arg Val Val Gly Gly Thr   1 5 10 15 Asn Ala Asp Glu Gly Glu Trp Pro              20

Claims (18)

A light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, PRSS14 An antibody or antigen-binding fragment thereof that specifically binds to the autolytic ring portion of / ST14. A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7; And a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 8 or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9, or an antibody that specifically binds to an autolytic ring portion of PRSS14 / ST14; or Antigen binding fragments thereof. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody specifically binds to an autolytic ring portion of PRSS14 / ST14, wherein the antibody is a humanized antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the autolytic ring portion of PRSS14 / ST14 comprises an amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 11. A nucleic acid molecule encoding the antibody of any one of claims 1 to 3 or an antigen binding fragment thereof. A recombinant expression vector comprising the nucleic acid molecule of claim 5. A cell transformed with the recombinant expression vector of claim 6. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to an autolytic ring portion of PRSS14 / ST14 comprising culturing the transformed cells of claim 7. A hybridoma cell line producing the antibody or antigen-binding fragment thereof of any one of claims 1 to 3. A cancer diagnostic composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3 as an active ingredient. The method of claim 10, wherein the cancer is breast cancer, stomach cancer, prostate cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, blood cancer, bone cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, bronchial cancer , Bladder cancer, kidney cancer, gallbladder cancer, cervical cancer, malignant melanoma, lymphoma or bone marrow cancer composition for cancer diagnosis. A cancer diagnostic kit comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient. A pharmaceutical composition for preventing or treating cancer metastasis, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient. The method of claim 13, wherein the cancer is breast cancer, stomach cancer, prostate cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, blood cancer, bone cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, bronchial cancer , Bladder cancer, kidney cancer, gallbladder cancer, cervical cancer, malignant melanoma, lymphoma or bone marrow cancer pharmaceutical composition for preventing or treating cancer metastasis. A health functional food composition for preventing or improving cancer metastasis comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3 as an active ingredient. A composition for detecting a PRSS14 / ST14 protein comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient. The method of claim 16, wherein the PRSS14 / ST14 protein is a composition for detecting PRSS14 / ST14 protein, characterized in that the native (native) form. A method for detecting PRSS14 / ST14 protein in a sample using the antibody of claim 1 or an antigen binding fragment thereof.

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