Anti-TM4SF4 humanized antibody and its uses

Abstract

The present invention relates to an anti-TM4SF4 (TransMembrane 4 Superfamily Member 4) humanized antibody and its uses. The anti-TM4SF4 humanized antibody of the present invention can specifically bind to TM4SF4 with high affinity, but exhibits low immunogenicity in humans, and can therefore be useful for detecting TM4SF4 or targeting cancer cells and cancer stem cells that overexpress TM4SF4.

Description

[Technical field]

[0001] The present invention relates to TM4SF4 (TransMembrane 4 Superfamily The present invention relates to a humanized antibody capable of specifically binding to Member 4) with high affinity and exhibiting low immunogenicity in humans, and a composition for preventing or treating cancer comprising the same. [Background technology]

[0002] TM4SF4 (TransMembrane 4 Superfamily Member 4) is a type of tetraspanin protein. Other proteins in this category, TM4SF1 and TM4SF5, are upregulated in expression in many tumors and have been reported to be involved in epithelial-mesenchymal transition and cell migration, and many cancer cell-related studies are currently being conducted. TM4SF4 has been reported to be involved in cell death, differentiation, and cell penetration ability in cancer cells. Recently, the present researchers reported that TM4SF4 protein promotes the growth, self-renewal ability, and metastasis / invasion of cancer stem cells in human lung cancer cells, and proposed that TM4SF4 promotes the activation of IGF1Rβ / AKT / NFκB or JAK2 (or FAK) / STAT3, which are signaling systems important in cancer development, and thereby enhances the properties of cancer stem cells through promoted cytokine secretion, making tumors more malignant (Choi SI et al., Oncotarget. 2014; 5(20): 9823-9837, Choi SI et al., Oncotarget. 2017; 8(60): 101284-101297).

[0003] Antibodies are used as therapeutic agents due to their high binding specificity to target antigens and their stability in the human body. In particular, anti-cancer antibodies have been improved to humanized antibodies, single-chain antibodies, double antibodies, drug-fusion antibodies, etc. based on the development of antibody engineering technology, and are being used with significantly improved efficacy in cancer treatment. However, due to the diversity of cancer characteristics and the induction of treatment resistance due to the expression of new antigens, it has been pointed out that there are limitations to the types of antigens currently used for targeting cancer cells, and research is ongoing to discover new cancer-specific antigens and derive antibodies against them.

[0004] In particular, in the case of cancer that shows resistance to targeted drugs and radiation therapy used in existing cancer treatments and recurs, it has been reported that the characteristics of cancer stem cells play an important role, and it is becoming increasingly important to discover antigens that can be used to target cancer stem cells and secure specific antibodies.

[0005] Meanwhile, in order to develop a monoclonal antibody that specifically binds to a specific antigen, a method is mainly used in which an antigen is injected into a non-human animal and the antibody produced by the animal's immune system is used. However, the antibody produced by this method is not a human protein, since it is an antibody derived from an animal other than human, and therefore may have problems with immunogenicity when administered to the human body. That is, when an antibody derived from a species other than human is administered to the human body, it may induce the generation of HAMA (Human Anti-Mouse Antibody), and the therapeutic efficacy of the antibody derived from the heterologous species may be reduced.

[0006] Therefore, a method has been proposed to solve the above-mentioned problems by obtaining the sequence that plays an important role in binding to an antigen from the amino acid sequence constituting the antibody derived from the non-human species and replacing the remaining portion with the sequence of a human antibody. However, the chimeric antibody or humanized antibody produced by this method is a fusion of two different protein regions, and therefore cannot function as an antibody or has poor binding ability to an antigen. Therefore, it is still difficult to develop a humanized antibody that does not induce HAMA in the human body and has high specificity and affinity for the antigen. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a novel humanized antibody or antigen-binding fragment thereof that can specifically bind with high affinity to TM4SF4 (TransMembrane 4 Superfamily Member 4) protein, which is overexpressed on the surface of cancer cells, while exhibiting low immunogenicity when administered to the human body.

[0008] Another object of the present invention is to provide a polynucleotide, expression vector, and host cell capable of encoding and expressing the humanized antibody or antigen-binding fragment thereof, and a method for producing the humanized antibody or antigen-binding fragment thereof. The present invention also provides a method for producing the antibody or antigen-binding fragment thereof, comprising the step of culturing the host cell.

[0009] It is also an object of the present invention to provide compositions, kits for use in detecting TM4SF4, and methods for detecting TM4SF4.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, a composition for inhibiting the growth of cancer stem cells, and a composition for supporting anti-cancer radiation therapy. [Means for solving the problem]

[0011] In order to achieve the above-mentioned object, there is provided a humanized antibody or an antigen-binding fragment thereof which specifically binds to TM4SF4 (TransMembrane 4 Superfamily Member 4), comprising: a heavy chain variable region comprising FR-H1 having the amino acid sequence of SEQ ID NO: 1, FR-H2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, FR-H3 having the amino acid sequence of SEQ ID NO: 4, and FR-H4 having the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising FR-L1 having the amino acid sequence of SEQ ID NO: 6, FR-L2 having the amino acid sequence of SEQ ID NO: 7, FR-L3 having the amino acid sequence of SEQ ID NO: 8, and FR-L4 having the amino acid sequence of SEQ ID NO: 9.

[0012] Other aspects of the invention provide a polynucleotide comprising a nucleotide sequence encoding the humanized antibody or antigen-binding fragment thereof, an expression vector comprising the polynucleotide, and a host cell comprising the expression vector.

[0013] Another aspect of the present invention provides a composition for detecting TM4SF4, which comprises the humanized antibody or an antigen-binding fragment thereof, and a kit for detecting TM4SF4, which comprises the composition for detecting TM4SF4.

[0014] Another aspect of the present invention provides a method for detecting TM4SF4, comprising the step of contacting a sample to be detected that is suspected of containing TM4SF4, the sample comprising the humanized antibody or antigen-binding fragment thereof.

[0015] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, a composition for inhibiting the growth of cancer stem cells, and a composition for supplementing anti-cancer radiation therapy, each comprising the humanized antibody or an antigen-binding fragment thereof. Effect of the Invention

[0016] The humanized antibodies of the present invention can specifically bind to TM4SF4 while not binding to substances such as BSA, and therefore can be used to detect TM4SF4 or to detect cells that overexpress TM4SF4. In particular, the humanized antibody of the present invention has a characteristic that it is similar to an antibody derived from a mouse, or has a significantly higher binding affinity than a chimeric antibody, and therefore has excellent effects.

[0017] In addition, the humanized antibody of the present invention has an advantage that it is unlikely to induce HAMA (Human Anti-Mouse Antibody) and has low immunogenicity when administered to the human body because the remaining sequences, excluding most of the CDR sequences, are derived from human antibodies or are partially modified from the human antibody sequences. Therefore, the humanized antibody of the present invention has an advantage that it can solve the problem of immune reactions that may occur when using antibodies derived from mice.

[0018] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic diagram illustrating a recombinant PCR process for producing a gene sequence encoding the heavy chain variable region designated Hz2B7-1.0 of the humanized antibody of the present invention. [Diagram 2] FIG. 1 shows the results of constructing the Hz2B7-1.0 heavy chain variable region gene, where A shows the results of agarose gel electrophoresis of T1, T2, and T3 sections, and B shows the results of electrophoresis of DNA containing the final Hz2B7-1.0 heavy chain variable region gene formed by linking T, T2, and T3. [Diagram 3] FIG. 1 shows the results of agarose gel electrophoresis of DNA containing a gene encoding the light chain variable region of the humanized antibody of the present invention, designated Hz2B7-0.1. [Figure 4] Based on the amino acid sequence of the humanized antibody Hz2B7-1.1 of the present invention and the epitope sequence of the antigen TM4SF4, the interaction between these was analyzed through docking simulation, and the main amino acid residues of the antibody that interact with the epitope and the binding free energy values ​​are shown. Among the antibody atoms shown, pink indicates carbon, blue indicates nitrogen, and red indicates oxygen. Carbon atoms of the epitope are shown in green. [Diagram 5] This figure shows a comparison of the amino acid sequences of the heavy chain (A) and light chain (B) of the original mouse antibody (2B7) and a similar human antibody (human-3QRG) with the amino acid sequences of four types of heavy chains and three types of light chains of the newly produced humanized antibody Hz2B7 during the process of producing the humanized antibody Hz2B7 of the present invention. The hyphen (-) indicates that the amino acid is the same as the original mouse antibody amino acid, and the boxes indicate the positions of amino acids discovered in the present invention to be important for epitope binding. [Figure 6] FIG. 1A shows the results of agarose gel electrophoresis of DNA containing the genes of the heavy chain variable region designated Hz2B7-2.0 (Hz2B7 HC V46A), the heavy chain variable region designated Hz2B7-3.0 (Hz2B7 HC W55Y), and the heavy chain variable region designated Hz2B7-4.0 (Hz2B7 HC W55S) of the humanized antibodies of the present invention; and FIG. 1B shows the results of agarose gel electrophoresis of DNA containing the genes of the light chain variable region designated Hz2B7-0.2 (Hz2B7 LC N31V) and the light chain variable region designated Hz2B7-0.3 (Hz2B7 LC N31F). [Figure 7] FIG. 1 shows the results of agarose gel electrophoresis of the heavy and light chain genes of a chimeric antibody used as a comparative example (A), the results of electrophoresis of a vector containing the heavy chain gene (B), and the results of electrophoresis of a vector containing the light chain gene (C). [Figure 8] FIG. 2 shows the structure of a vector containing the gene for the chimeric antibody. [Figure 9]Figure A shows the results of SDS-PAGE and Coomassie blue staining of the humanized and chimeric antibodies of the present invention, the mouse-derived 2B7 antibody, and a human antibody IgG, while Figure B shows the results of Western blotting of the antibodies, in which the secondary antibody used binds to the IgG gamma and kappa chains of human antibodies. [Figure 10] This figure shows the results of indirect ELISA using 10 types of humanized antibodies, a chimeric antibody (Chi2B7), and human IgG (Isotype hIgG) of the present invention. A is a graph showing the binding affinity of all antibodies to the TM4SF4-BSA antigen, B is the binding affinity of Hz2B7-1.1, Hz2B7-1.2, and Hz2B7-1.3, which show higher binding affinity among the humanized antibodies, and C is the binding affinity of all antibodies to BSA. [Figure 11] This figure shows a comparative analysis of the antibody affinity of five types of anti-TM4SF4 humanized antibodies for the TM4SF4 protein epitope using surface plasmon resonance (SPR) analysis. First, a biotin-bound TM4SF4 peptide was attached to a sensor chip, and the five types of humanized antibodies were allowed to flow, while the antibody association rate (Ka), dissociation rate (Kd), and equilibrium dissociation constant (KD, Kd / Ka) were calculated. [Figure 12] This figure shows the results of FACS analysis using a flow cytometer on lung cancer, human primary hepatocytes, and hepatoma cell lines using the humanized antibodies (Hz2B7-1.1, Hz2B7-1.2, Hz2B7-1.3) of the present invention, the chimeric antibody (Chi2B7), and human IgG. A is a graph comparing the binding ability of the antibodies to lung cancer cell lines A549 cells and Calu-3 cell lines, and B is a graph confirming the binding ability of the Hz2B7-1.1 and Hz2B7-1.2 antibodies to human primary hepatocytes (hPH) and hepatoma cell lines Huh-7, SNU-387, and SNU-449 cell lines. [Figure 13]CHO-DG44 cells were transformed by introducing a gene encoding the humanized antibody of the present invention, clones having G418 resistance were selected, and the antibody production amount of each clone was compared by measuring the OD value via ELISA. 1 μg of human IgG was used as a positive control. [Figure 14] The antibody production amounts were compared by sandwich ELISA for the Hz2B7-1.1-4H12 clone and the Hz2B7-1.2-4A12 clone, in which the antibody gene was amplified using MTX. FIG. 1A shows a comparison of the antibody production amounts of the Hz2B7-1.1-4H12 clone not treated with MTX and the clone treated with 0.08 μM MTX, and FIG. 1B shows a comparison of the antibody production amounts of the Hz2B7-1.2-4A12 clone. [Figure 15] FIG. 13 shows a comparison of antibody production amounts (μg / 10 6 cells / 24 hr) of the Hz2B7-1.1-4H12 clone and the Hz2B7-1.2-4A12 clone, in which antibody genes were amplified by treatment with 0.08 μM MTX, as measured by sandwich ELISA. [Figure 16] 1 is a graph measuring and comparing the stability of mouse and humanized antibodies in human serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention will be described in detail below.

[0021] 1. Humanized antibodies and antigen-binding fragments thereof that specifically bind to TM4SF4 One aspect of the present invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds to TM4SF4 and exhibits low immunogenicity in the human body.

[0022] The term "antibody" in the present invention refers to an immunoglobulin molecule that is immunologically reactive by specifically binding to an epitope of an antigen. The antibody may include a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure (full-length antibody), a functional fragment having at least an antigen-binding function (antigen-binding fragment), and a recombinant antibody. The antibody of interest may be a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from a population of substantially identical antibodies, and such a monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be linked to a heavy chain by a disulfide bond. The antibody may comprise heavy chain (HC) and light chain (LC) polypeptides, and the heavy and light chains may comprise a variable region and a constant region.

[0023] The constant region is a site that mediates the binding of the antibody to various types of cells of the immune system (e.g., T-cells), host tissues including components of the complement system, etc. The constant region has the same function regardless of the type of antigen, if the antibody is of the same type derived from the same species, and the amino acid sequence constituting the constant region is the same or highly similar for each antibody. The constant region can be classified into heavy chain constant region (which can be abbreviated as CH) and light chain constant region (which can be abbreviated as CL). The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and / or epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and / or alpha 2 (α2) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types. IgG includes subtypes IgG1, IgG2, IgG3, and IgG4.

[0024] The variable region is an antibody site having specificity for an antigen, and can be classified into a heavy chain variable region (which can be abbreviated as VH) and a light chain variable region (which can be abbreviated as VL). The variable region may include three CDRs (complementary-determining regions) and four FRs (framework regions). The CDRs may be cyclic sites involved in antigen recognition, and the specificity for the antigen may be determined by the amino acid sequence of the CDRs. The CDRs may be referred to as CDR1, CDR2, and CDR3 depending on the order, and may be referred to as CDR-H1, CDR-H2, and CDR-H3 for the heavy chain variable region and CDR-L1, CDR-L2, and CDR-L3 for the light chain variable region depending on which polypeptide of the heavy chain and light chain the CDR belongs to. Similarly, FRs may be designated as FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy chain variable region, and as FR-L1, FR-L2, FR-L3, and FR-L4 for the light chain variable region. The CDRs and FRs may be arranged in the following order in each variable region, from the N-terminus (amino-terminus) to the C-terminus (carboxy-terminus): FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, and FR-H4 for the heavy chain variable region, and FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 for the light chain variable region.

[0025] The term "antigen-binding fragment" in the present invention refers to any fragment of the humanized antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment may be referred to interchangeably with the terms "fragment", "antibody fragment", etc., and may include Fab, Fab', F(ab') 2 , Fv, etc., but are not limited thereto.

[0026] The Fab has a structure including a light chain variable region, a heavy chain constant region, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. The Fab' is different from the Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab') 2 is generated by disulfide bond formation between cysteine ​​residues in the hinge region of Fab'. The Fv refers to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the light ... The heavy chain variable region and the light chain variable region are linked by a covalent bond via a carboxyl group or directly at the C-terminus, so that they can form a dimer-like structure such as a double-chain Fv. The antigen-binding fragment can be produced by using a proteolytic enzyme (e.g., whole antibody can be digested with papain to obtain Fab, or by digestion with pepsin to obtain F(ab')). 2 The present invention can be produced, but is not limited to, by the use of recombinant DNA technology, which can be obtained by the use of recombinant DNA technology (e.g., by using recombinant DNA technology, fragments can be obtained), or by recombinant DNA technology.

[0027] The term "humanized antibody" in the present invention refers to an antibody that exhibits reduced immunogenicity in humans or is non-immunogenic. The humanized antibody may be produced, for example, by combining CDRs (complementarity determining regions) derived from an individual other than human (non-human species) with a constant region derived from a human antibody and FRs (framework regions) of a variable region derived from a human antibody. The humanized antibody may be produced by grafting the CDRs of a non-human antibody between the FR sequences of a human antibody through a CDR-grafting method.

[0028] In the present invention, the term "FR (framework regions)" refers to the portion of the variable region of an immunoglobulin molecule other than the complementarity determining region. There are four framework regions in each of the light chain and heavy chain (framework region 1, framework region 2, framework region 3, and framework region 4).

[0029] In the present invention, the term "human antibody" refers to an antibody in which both the light chain and the heavy chain are derived from humans. Depending on the difference in the constant region of the heavy chain, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having a γ heavy chain, IgM having a μ heavy chain, IgA (including IgA1 and IgA2) having an α heavy chain, IgD having a δ heavy chain, or IgE having an ε heavy chain. In principle, the light chain includes one or more of a κ chain and a λ chain.

[0030] The term "chimeric antibody" as used herein means an antibody whose variable regions originate from an individual other than human (non-human species) and whose constant regions originate from a species different from said individual, e.g., human.

[0031] The humanized antibody and chimeric antibody will be described in more detail. When comparing mouse-derived antibodies with human antibodies, the order of sequence similarity to human antibodies is mouse antibody - chimeric antibody - humanized antibody - human antibody. Therefore, compared with mouse antibodies and chimeric antibodies, humanized antibodies have a higher similarity to human antibodies, and therefore have the characteristic of low immunogenicity when administered to the human body.

[0032] The term "epitope" as used herein means a specific site on an antigen that can be specifically recognized and bound by an immunoglobulin, an antibody, or an antigen-binding fragment thereof. The epitope may be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0033] The humanized antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region including FR-H1 having the amino acid sequence of SEQ ID NO: 1, FR-H2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, FR-H3 having the amino acid sequence of SEQ ID NO: 4, and FR-H4 having the amino acid sequence of SEQ ID NO: 5, and a light chain variable region including FR-L1 having the amino acid sequence of SEQ ID NO: 6, FR-L2 having the amino acid sequence of SEQ ID NO: 7, FR-L3 having the amino acid sequence of SEQ ID NO: 8, and FR-L4 having the amino acid sequence of SEQ ID NO: 9, and specifically binds to TM4SF4 (TransMembrane 4 Superfamily Member 4).

[0034] The FR-H1 having the amino acid sequence of SEQ ID NO: 1 may be the FR1 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", "Hz2B7-3.0", or "Hz2B7-4.0". The FR-H2 having the amino acid sequence of SEQ ID NO: 2 may be the FR2 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-3.0", or "Hz2B7-4.0", and the FR-H2 having the amino acid sequence of SEQ ID NO: 3 may be the FR2 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-2.0". The FR-H3 having the amino acid sequence of SEQ ID NO: 4 may be the FR3 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", "Hz2B7-3.0", or "Hz2B7-4.0". The FR-H4 having the amino acid sequence of SEQ ID NO:5 may be the FR4 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", "Hz2B7-3.0" or "Hz2B7-4.0".

[0035] The FR-L1 having the amino acid sequence of SEQ ID NO: 6 may be the FR1 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.1", "Hz2B7-0.2", or "Hz2B7-0.3". The FR-L2 having the amino acid sequence of SEQ ID NO: 7 may be the FR2 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.1", "Hz2B7-0.2", or "Hz2B7-0.3". The FR-L3 having the amino acid sequence of SEQ ID NO: 8 may be the FR3 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.1", "Hz2B7-0.2", or "Hz2B7-0.3". The FR-L4 having the amino acid sequence of SEQ ID NO: 9 may be the FR4 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.1", "Hz2B7-0.2", or "Hz2B7-0.3".

[0036] The TM4SF4 is a type of tetraspanin protein, and is known to be involved in cell death, differentiation, and cell penetration in cancer cells. In addition, the TM4SF4 is known to promote the growth and metastasis of cancer stem cells, and may enhance the characteristics of cancer stem cells to further increase the malignancy of tumors. The TM4SF4 protein may be a membrane protein present in the cell membrane, and a portion of TM4SF4 may be exposed to the outside of the cell. The exposed portion may include two loop structures, and a portion of the amino acid sequence of the exposed portion of the TM4SF4 may be an epitope that the humanized antibody or antigen-binding fragment thereof of the present invention can specifically recognize and bind to. The epitope of TM4SF4 may include, for example, the amino acid sequence of "TWGYPFHDGDYLNDE" (in the order from the N-terminus to the C-terminus).

[0037] The heavy chain variable region may further comprise at least one CDR selected from the group consisting of CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 77, and CDR-H3 having the amino acid sequence of SEQ ID NO: 14. In particular, the CDR-H2 may have the amino acid sequence of SEQ ID NO: 78, specifically, any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0038] The light chain variable region may further comprise at least one CDR selected from the group consisting of CDR-L1 having the amino acid sequence of SEQ ID NO: 79, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. In particular, the CDR-L1 may have any one amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the CDR-L3 may have the amino acid sequence of SEQ ID NO: 19.

[0039] The CDR-H1 having the amino acid sequence of SEQ ID NO: 10 may be the CDR1 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", "Hz2B7-3.0" or "Hz2B7-4.0". The CDR-H2 having the amino acid sequence of SEQ ID NO: 11 may be the CDR2 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-1.0" or "Hz2B7-2.0", the CDR-H2 having the amino acid sequence of SEQ ID NO: 12 may be the CDR2 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-3.0", and the CDR-H2 having the amino acid sequence of SEQ ID NO: 13 may be the CDR2 sequence of the heavy chain variable region referred to in the present invention as "Hz2B7-4.0". The CDR-H3 having the amino acid sequence of SEQ ID NO: 14 may be a CDR3 sequence of a heavy chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", "Hz2B7-3.0", or "Hz2B7-4.0".

[0040] The CDR-L1 having the amino acid sequence of SEQ ID NO: 15 may be the CDR1 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.1", the CDR-L1 having the amino acid sequence of SEQ ID NO: 16 may be the CDR1 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.2", and the CDR-L1 having the amino acid sequence of SEQ ID NO: 17 may be the CDR1 sequence of the light chain variable region referred to in the present invention as "Hz2B7-0.3". The CDR-L2 having the amino acid sequence of SEQ ID NO: 18 may be the CDR2 sequence of the light chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", or "Hz2B7-0.3". The CDR-L3 having the amino acid sequence of SEQ ID NO: 19 may be the CDR3 sequence of the light chain variable region referred to in the present invention as "Hz2B7-1.0", "Hz2B7-2.0", or "Hz2B7-0.3".

[0041] The heavy chain variable region may have any one of the amino acid sequences selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.

[0042] The light chain variable region may have any one of the amino acid sequences selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.

[0043] The heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 may be the sequence of a heavy chain variable region referred to in the present invention as "Hz2B7-1.0", the heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 may be the sequence of a heavy chain variable region referred to in the present invention as "Hz2B7-2.0", the heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 may be the sequence of a heavy chain variable region referred to in the present invention as "Hz2B7-3.0", and the heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 may be the sequence of a heavy chain variable region referred to in the present invention as "Hz2B7-4.0".

[0044] The light chain variable region having the amino acid sequence of SEQ ID NO: 24 may be the sequence of a light chain variable region referred to in the present invention as "Hz2B7-0.1", the light chain variable region having the amino acid sequence of SEQ ID NO: 25 may be the sequence of a light chain variable region referred to in the present invention as "Hz2B7-0.2", and the light chain variable region having the amino acid sequence of SEQ ID NO: 26 may be the sequence of a light chain variable region referred to in the present invention as "Hz2B7-0.3".

[0045] The humanized antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain constant region and / or a light chain constant region of an antibody derived from a human, and the heavy chain constant region and / or the light chain constant region of the antibody derived from a human may be used without limitation as long as it does not inhibit the specific binding property of the humanized antibody or antigen-binding fragment thereof to TM4SF4. For example, the heavy chain constant region may be a heavy chain constant region having the amino acid sequence of SEQ ID NO:27, and the light chain constant region may be a light chain constant region having the amino acid sequence of SEQ ID NO:28.

[0046] The amino acid sequences described above may include variants having different sequences due to deletion, insertion, substitution, or a combination thereof of amino acid residues, within a range that does not affect the structure, function, activity, etc. of a polypeptide containing the amino acid sequences. The amino acid sequences may also include amino acids that have undergone typical modifications known in the art, and the amino acid modifications may be, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. The humanized antibodies or antigen-binding fragments thereof of the present invention include not only those that contain the amino acid sequences described above, but also those that have substantially the same amino acid sequences as the amino acid sequences described above or variants thereof. The term "having substantially the same amino acid sequence" may include, but is not limited to, an amino acid sequence having a homology of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more to the amino acid sequence described above.

[0047] The humanized antibody or antigen-binding fragment thereof of the present invention has an equilibrium dissociation constant (KD) of 3.0×10 -8 M or less, for example, 2.9×10 -8 M or less, 2.8×10 -8 M or less, 2.7×10 -8 M or less, 2.6×10 -8 M or less or 2.5 x 10 -8 M or less, specifically, 2.4×10 -8 It may be M or less.

[0048] According to a specific embodiment of the present invention, the binding ability of the humanized antibody of the present invention to TM4SF4 was measured and compared through indirect ELISA. As a result, it was confirmed that the humanized antibody of the present invention exhibited significantly higher binding ability than a chimeric antibody (an antibody combining the variable region of mouse-derived ECL-2B7 antibody and the constant region of a human antibody) (FIG. 10, A and B). In particular, among humanized antibodies with various combinations of light chain variable region and heavy chain variable region, the binding ability of the humanized antibody called Hz2B7-1.2 was shown to be the best (FIG. 10 and FIG. 11). In addition, since the binding ability of the humanized antibody of the present invention was not shown in the indirect ELISA experiment performed on BSA, it was confirmed that the antibody specifically binds only to TM4SF4 (FIG. 10).

[0049] According to another specific embodiment of the present invention, the results of a FACS experiment using the humanized antibody of the present invention confirmed that the humanized antibody of the present invention has excellent binding ability to Calu-3 and A549 cells, which are lung cancer cell lines that overexpress TM4SF4 on the cell surface (Figure 12). When compared with the results of the FACS experiment using the chimeric antibody, the humanized antibody of the present invention was measured to have a higher binding ability to lung cancer cells, and it was confirmed that the effect of the humanized antibody called Hz2B7-1.2 was the most excellent, as in the indirect ELISA experiment (Figures 10 to 12).

[0050] 2. Technology for expressing TM4SF4-specific humanized antibodies Yet another aspect of the invention provides polynucleotides, expression vectors, host cells and production methods that can be used to express and produce the humanized antibody or antigen-binding fragment thereof.

[0051] The explanations regarding the humanized antibody, its antigen-binding fragment, TM4SF4, etc. are the same as those explained in "1. Humanized antibody that specifically binds to TM4SF4 and its antigen-binding fragment," so to avoid repetition, we will omit the explanations below and only explain the contents related to polynucleotides, expression vectors, and host cells.

[0052] The term "polynucleotide" as used herein includes DNA and RNA molecules, and nucleotides, which are the basic building blocks of polynucleotides, do not exist in nature. In addition to nucleotides having the same structure, analogues having modified sugar or base moieties may also be included.

[0053] The polynucleotide of the present invention comprises a nucleotide sequence encoding a humanized antibody or antigen-binding fragment thereof.

[0054] "Encoding the humanized antibody or antigen-binding fragment thereof" means that the polynucleotide encodes genetic information that enables a protein having the amino acid sequence of the humanized antibody or antigen-binding fragment thereof of the present invention to be synthesized through normal protein expression processes such as transcription, translation, etc. In this regard, the scope of the present invention may include not only a protein having the exact same amino acid sequence as the humanized antibody or antigen-binding fragment thereof, but also a polynucleotide that encodes a protein having substantially the same amino acid sequence as the protein, or a protein having the same and / or similar activity as the protein, as described above.

[0055] The polynucleotide may include a base sequence optimized according to the type of organism into which it is to be introduced and expressed, and the expression system, such as transcription and translation, of the organism.

[0056] Specifically, the polynucleotide may include at least one base sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0057] The base sequence of SEQ ID NO:29 may code for the amino acid sequence of SEQ ID NO:1, the base sequence of SEQ ID NO:30 may code for the amino acid sequence of SEQ ID NO:2, the base sequence of SEQ ID NO:31 may code for the amino acid sequence of SEQ ID NO:3, the base sequence of SEQ ID NO:32 may code for the amino acid sequence of SEQ ID NO:4, the base sequence of SEQ ID NO:33 may code for the amino acid sequence of SEQ ID NO:5, the base sequence of SEQ ID NO:34 may code for the amino acid sequence of SEQ ID NO:6, the base sequence of SEQ ID NO:35 may code for the amino acid sequence of SEQ ID NO:7, the base sequence of SEQ ID NO:36 may code for the amino acid sequence of SEQ ID NO:8, the base sequence of SEQ ID NO:37 may code for the amino acid sequence of SEQ ID NO:9, the base sequence of SEQ ID NO:38 may code for the amino acid sequence of SEQ ID NO:10, and the base sequence of SEQ ID NO:39 may code for the amino acid sequence of SEQ ID NO:11. The base sequence of SEQ ID NO: 40 may code for the amino acid sequence of SEQ ID NO: 12, the base sequence of SEQ ID NO: 41 may code for the amino acid sequence of SEQ ID NO: 13, the base sequence of SEQ ID NO: 42 may code for the amino acid sequence of SEQ ID NO: 14, the base sequence of SEQ ID NO: 43 may code for the amino acid sequence of SEQ ID NO: 15, the base sequence of SEQ ID NO: 44 may code for the amino acid sequence of SEQ ID NO: 16, the base sequence of SEQ ID NO: 45 may code for the amino acid sequence of SEQ ID NO: 17, the base sequence of SEQ ID NO: 46 may code for the amino acid sequence of SEQ ID NO: 18, the base sequence of SEQ ID NO: 47 may code for the amino acid sequence of SEQ ID NO: 19, the base sequence of SEQ ID NO: 48 may code for the amino acid sequence of SEQ ID NO: 20, the base sequence of SEQ ID NO: 49 may code for the amino acid sequence of SEQ ID NO: 21, and the base sequence of SEQ ID NO: 50 may code for the amino acid sequence of SEQ ID NO: 22. the base sequence of SEQ ID NO:51 may encode the amino acid sequence of SEQ ID NO:23, the base sequence of SEQ ID NO:52 may encode the amino acid sequence of SEQ ID NO:24, the base sequence of SEQ ID NO:53 may encode the amino acid sequence of SEQ ID NO:25, the base sequence of SEQ ID NO:54 may encode the amino acid sequence of SEQ ID NO:26, the base sequence of SEQ ID NO:55 may encode the amino acid sequence of SEQ ID NO:27, and the base sequence of SEQ ID NO:56 may encode the amino acid sequence of SEQ ID NO:28.

[0058] The polynucleotide of the present invention may comprise a nucleotide sequence substantially identical to the listed nucleotide sequence. The substantially identical nucleotide sequence may be, for example, a nucleotide sequence having a homology of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more to the listed nucleotide sequence, including cases where the same amino acids can be synthesized when transcribed and translated, but is not limited thereto.

[0059] The expression vector of the present invention comprises the above-mentioned polynucleotide.

[0060] The term "expression vector" as used herein means a means for expressing a specific gene in a host cell, and specifically includes, but is not limited to, plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0061] The expression vector may further include regulatory sequences such as a promoter, a terminator, etc., in addition to the nucleotide sequence encoding the humanized antibody or antigen-binding fragment thereof, and the nucleotide sequence encoding the humanized antibody or antigen-binding fragment thereof may be operatively linked to a promoter. The term "operably linked" refers to a functional link between a regulatory sequence (e.g., a promoter, a signal sequence, an array of transcriptional regulator binding sites, etc.) and another nucleotide sequence, such that the regulatory sequence can regulate the transcription and / or translation of the other nucleotide sequence.

[0062] The expression vector system of the present invention may be constructed by a variety of methods known in the art.

[0063] The expression vectors may be constructed for use in prokaryotic or eukaryotic hosts.

[0064] For example, when the expression vector is used as a prokaryotic cell host, it generally contains a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, a promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C, J. Bacteriol, (1984) 158:1018-1024), and the left-handed promoter of phage λ (pLλ promoter, Herskowitz, I and Hage n, D, Ann Rev Genet, (1980) 14: 399-445) may be used as a regulatory site. When Bacillus bacteria are used as the host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl Environ Microbiol (1998) 64: 3932-3938; Mol Gen Genet (1999) 64: 3932-3938) may be used as a regulatory site. 996) 250:734-741) or any promoter capable of expression in Bacillus may be used as the regulatory site. The expression vector may be produced by manipulating a plasmid (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or a virus (e.g., SV40, etc.) frequently used in the art.

[0065] When the expression vector is intended for use in a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, and may generally have a polyadenylation sequence as a transcription termination sequence. The expression vector may have a CMV promoter.

[0066] The expression vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Examples of sequences to be fused include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA). In addition, since the protein expressed by the expression vector of the present invention is a humanized antibody or an antigen-binding fragment thereof, when this characteristic is taken into consideration, the expressed protein can be easily purified through a protein A column or the like even without additional sequences for purification.

[0067] The expression vector may contain antibiotic resistance genes commonly used in the art as selection markers, for example, resistance genes to ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0068] The expression vector may be a vector system in which the light chain and the heavy chain are simultaneously expressed in one vector, or a system in which the light chain and the heavy chain are each expressed in a separate vector. In the latter case, the two vectors may be, for example, co-transformed. The vectors encoding the light and heavy chains can be introduced into host cells via co-transformation or targeted transformation. Co-transformation is a method in which vector DNAs encoding the light and heavy chains are simultaneously introduced into host cells, and then cells expressing both the light and heavy chains are selected. Targeted transformation is a method in which cells transformed with a vector containing the light chain (or heavy chain) are selected, and the selected cells are transformed again with a vector containing the heavy chain (or light chain), and finally cells expressing both the light and heavy chains are selected.

[0069] The host cell of the present invention comprises the expression vector.

[0070] The host cell may be any host cell known in the art, so long as it is capable of stably and continuously cloning and expressing the expression vector of the present invention. For example, Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis may be used. The host cell may be a prokaryotic host cell, such as, but not limited to, a strain of Bacillus, such as Bacillus thuringiensis, Streptomyces, Pseudomonas, e.g., Pseudomonas putida, Proteus mirabilis, or Staphylococcus, e.g., Staphylococcus carnosus.

[0071] When the host cell is a eukaryotic host cell, the host cell is of the genus Aspergillus The host cells may be, but are not limited to, fungi such as Streptococcus cerevisiae, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals. The host cells may be COS7 cells (monkey kidney cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, or 293 cells.

[0072] Transformation and / or transfection of the host cell may be carried out by any method for introducing nucleic acid into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique according to the host cell, as is known in the art. Specifically, electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitate, calcium chloride (CaCl 2 ) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine and desiccation / repression-mediated transformation methods, etc., but are not limited to these.

[0073] A method for producing the humanized antibody or antigen-binding fragment thereof of the present invention comprises culturing the host cell.

[0074] The method of producing the humanized antibody or antigen-binding fragment thereof may further comprise expressing the humanized antibody or antigen-binding fragment thereof in the host cell.

[0075] The host cells may be cultured using appropriate media and culture conditions known in the art. Those skilled in the art can easily adjust the culture process according to the selected strain. Cell culture is classified into suspension culture and adherent culture according to the cell growth method, and into batch, fed-batch, and continuous culture methods according to the culture method. The medium used for culture should appropriately meet the requirements of a specific strain.

[0076] In animal cell culture, the medium contains a variety of carbon sources, nitrogen sources and trace element components. Examples of usable carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these carbon sources may be used alone or in combination.

[0077] The nitrogen sources include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL) and soybean meal, and inorganic sources such as iodine, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. It may contain a nitrogen source, which may be used alone or in combination.

[0078] The medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts as phosphorus sources, as well as metal salts such as magnesium sulfate or ferrous sulfate, and may also contain amino acids, vitamins, and appropriate precursors.

[0079] In the culturing step, the pH of the culture can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. Also, during the culturing, the generation of bubbles can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Also, to maintain the culture in an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture can usually be 20°C to 45°C, or 25°C to 40°C.

[0080] The production method may further include a step of recovering the humanized antibody or antigen-binding fragment thereof expressed in the host cell. The humanized antibody or antigen-binding fragment thereof obtained by culturing the transformed host cell may be used in an unpurified state, or may be further purified to a high purity using various conventional methods, such as dialysis, salt precipitation, chromatography, etc. When chromatography is used, the type and order of columns may be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc., depending on the characteristics of the antibody, the culture method, etc.

[0081] 3. Use of the humanized antibody of the present invention for detecting TM4SF4 In another aspect, the present invention provides a use of the humanized antibody or antigen-binding fragment thereof for detecting TM4SF4. Specifically, the present invention provides a composition for detecting TM4SF4, a kit for detecting TM4SF4, and a method for detecting TM4SF4.

[0082] The explanations regarding the humanized antibody, its antigen-binding fragment, TM4SF4, etc. are the same as those explained in "1. Humanized antibody that specifically binds to TM4SF4 and its antigen-binding fragment," so they will be omitted to avoid repetition.

[0083] The composition for detecting TM4SF4 of the present invention comprises the humanized antibody or an antigen-binding fragment thereof, and the kit for detecting TM4SF4 of the present invention comprises the composition for detecting TM4SF4.

[0084] Furthermore, a method for detecting TM4SF4 of the present invention includes a step of contacting the humanized antibody or its antigen-binding fragment with a detection target sample suspected to contain TM4SF4.

[0085] The TM4SF4 detection composition and a kit containing the same can effectively detect TM4SF4 by contacting the humanized antibody or its antigen-binding fragment of the present invention that specifically binds to TM4SF4 with a sample to be detected to form an antigen-antibody complex.

[0086] The antigen-antibody complex means a combination of TM4SF4 and an antibody that recognizes it, for identifying tumor or cancer cells expressing TM4SF4 in a sample. The TM4SF4 detection composition, the kit containing the same, or the method for quantifying the TM4SF4 antigen using the humanized antibody or the antigen-binding fragment thereof may be performed by confirming the formation of an antigen-antibody complex. The formation of the antigen-antibody complex may be confirmed by enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, immunoimmunology, immunoassay ... The enzyme immunoassay (ELISA) may be performed by, but is not limited to, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, immunodiffusion assay, complement fixation assay, protein chip, etc. The enzyme immunoassay (ELISA) includes various ELISA methods such as direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid support, direct sandwich ELISA using a labeled or other antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, and indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting with an antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support.

[0087] Labels that enable the formation of antigen-antibody complexes to be measured qualitatively or quantitatively include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes. Enzymes include, but are not limited to, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, and β-lactamase.

[0088] 4. Use of the humanized antibody of the present invention for preventing or treating cancer, inhibiting the growth of cancer stem cells, and supplementing anti-cancer radiation therapy Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, a composition for inhibiting the growth of cancer stem cells, and a composition for supplementing anti-cancer radiation therapy, each comprising the humanized antibody or an antigen-binding fragment thereof.

[0089] The explanations regarding the humanized antibody, its antigen-binding fragment, TM4SF4, etc. are the same as those explained in "1. Humanized antibody that specifically binds to TM4SF4 and its antigen-binding fragment," so they will be omitted to avoid repetition.

[0090] However, the humanized antibody or antigen-binding fragment thereof of the present invention may include a CDR sequence that is identical to the CDR sequence of the 2B7 antibody or a CDR sequence that is a modified version of a portion of the CDR sequence. For the characteristics and effects of the 2B7 antibody, please refer to Korean Patent Application No. 2020-0168467.

[0091] The pharmaceutical composition for preventing or treating cancer of the present invention comprises the humanized antibody or an antigen-binding fragment thereof.

[0092] The humanized antibody or antigen-binding fragment thereof can bind to TM4SF4 with high affinity, and since TM4SF4 is known to be overexpressed on the surface of cancer cells, the humanized antibody or antigen-binding fragment thereof may be used to target cancer cells.

[0093] The compositions, in which the humanized antibody or antigen-binding fragment thereof is used alone or in combination with a conventional pharma- ceutically acceptable carrier, may be used in the treatment, prevention and diagnosis of hyperproliferative diseases such as cancer.

[0094] The cancer is specifically lung cancer, gastric cancer, colon cancer, rectal cancer, triple negative breast cancer, glioma, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, endometrial cancer, salivary gland cancer, or The cancer in the present invention may be, but is not limited to, thyroid cancer, more specifically, lung cancer, breast cancer, liver cancer, kidney cancer, stomach cancer, pancreatic cancer, or brain cancer. In particular, the cancer in the present invention may be, but is not limited to, cancer caused by overexpression, amplification, mutation, or activation of TM4SF4. That is, the composition comprising the humanized antibody or binding fragment thereof of the present invention has a growth inhibitory effect on all carcinomas regardless of abnormal expression or mutation of TM4SF4, so that the medical use of the present invention is not limited by the expression mode or the presence or absence of mutation of TM4SF4.

[0095] The composition may be in the form of a pharmaceutical composition, a quasi-drug composition, or a health food composition.

[0096] The cancer prevention or treatment composition of the present invention may further include a pharma- ceutically acceptable carrier. The term "pharma-ceutically acceptable" means that the compound does not have toxicity beyond the range that is acceptable for the application (prescription) target, even if it does not suppress the activity of the active ingredient, and the "carrier" is defined as a compound that facilitates the addition of the compound to cells or tissues.

[0097] The pharmaceutical composition of the present invention may be administered alone or in admixture with a convenient carrier, etc., and the dosage form of such administration may be a single dose or multiple dose dosage form. The pharmaceutical composition may be a solid formulation or a liquid formulation. The solid formulation may include, but is not limited to, powder, granules, tablets, capsules, suppositories, etc. The solid formulation may include, but is not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. The liquid formulation may include, but is not limited to, solutions such as water and propylene glycol solutions, suspensions, emulsions, etc., and may be prepared by adding suitable colorants, flavoring agents, stabilizers, viscosity agents, etc. For example, a powder may be prepared by simply mixing the tri-hydroxy derivative of a polyunsaturated fatty acid, which is the active ingredient of the present invention, with a suitable pharma- ceutical acceptable carrier, such as lactose, starch, microcrystalline cellulose, etc. Granules can be prepared by mixing the tri-hydroxy derivative of polyunsaturated fatty acid of the present invention, a suitable pharma- ceutically acceptable carrier, and a suitable pharma- ceutically acceptable binder such as polyvinylpyrrolidone, hydroxypropylcellulose, etc., followed by a wet granulation method using a solvent such as water, ethanol, isopropanol, etc., or a dry granulation method using compression force. Tablets can be prepared by mixing the granules with a suitable pharma- ceutically acceptable lubricant such as magnesium stearate, followed by tableting using a tablet press.

[0098] The pharmaceutical composition may be administered, depending on the disease to be treated and the condition of the individual, as an oral agent, an injection (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), an inhalant, a nasal agent, a vaginal agent, a rectal agent, a sublingual agent, a transdermal agent, an external agent, or the like, but is not limited thereto. Depending on the administration route, the composition may be formulated into a suitable dosage unit dosage form containing commonly used non-toxic pharma- ceutically acceptable carriers, additives, and vehicles.

[0099] The pharmaceutical composition may be administered at about 0.0001 mg / kg to about 10 g / kg daily, with a daily dosage of about 0.001 mg / kg to about 1 g / kg. However, the dosage may vary depending on the degree of purification of the mixture, the condition of the patient (age, sex, weight, etc.), the severity of the condition being treated, etc. If necessary, the total daily dosage may be administered in several portions throughout the day for convenience.

[0100] The composition for inhibiting cancer stem cell growth of the present invention comprises the humanized antibody or an antigen-binding fragment thereof.

[0101] The cancer stem cell (CSC) refers to an undifferentiated cell that has the ability to differentiate into various cancer cells. Cancer stem cells exist in about 1-2% of malignant tumor tissue and have the self-renewal ability and pluripotency, which are characteristics of normal stem cells. However, due to abnormalities in the autoregulatory function, cell division activation leads to an increase in cell numbers, and the cells differentiate into malignant tumor cells. Due to these characteristics of cancer stem cells, while general cancer cells are eliminated through anti-cancer treatment, cancer stem cells survive, and it is known that some of the surviving cancer stem cells cause cancer recurrence and metastasis.

[0102] Specifically, the cancer stem cells of the present invention may be cancer cells in which aldehyde dehydrogenase 1 (ALDH1) protein, which is one of the markers for cancer stem cells, is overexpressed or the protein activity is positive.

[0103] The humanized antibody or antigen-binding fragment thereof of the present invention can selectively inhibit cancer stem cells, and in particular can achieve excellent anti-cancer effects by killing cancer cell populations that contain cancer stem cells that are highly resistant to anti-cancer treatment. The humanized antibody or antigen-binding fragment thereof can inhibit the growth of cancer stem cells by reducing the self-renewal, invasion, and migration capabilities of cancer stem cells.

[0104] The antibody or antigen-binding fragment thereof can be used to prevent or treat cancers with cancer stem cell characteristics, but is not limited thereto. Cancers with cancer stem cell characteristics are resistant to existing anti-cancer treatments and have poor prognosis, so a treatment different from the existing anti-cancer treatments must be applied. For example, even if a patient has the same cancer, if the cancer has a high ratio of cancer stem cells, the patient will not be able to obtain a cancer treatment effect with existing anti-cancer treatments such as administration of anti-cancer drugs or radiation therapy. Therefore, even if the cancer is the same type, if the ratio of cancer stem cells is high in the cells at the cancer lesion site, it is very important to apply a new treatment different from the existing anti-cancer treatment.

[0105] The cancer having the cancer stem cell characteristics may be a cancer having a high ratio of cancer stem cells in the cell group constituting the cancer. Considering that the ratio of cancer stem cells in general cancer cells is about 1% or more and less than 5%, for example, a cancer having a ratio of cancer stem cells of 5% or more, 10% or more, 30% or more, 50% or more, or 70% or more in the cell group constituting the cancer may be defined as having cancer stem cell characteristics, and as described above, it may be characterized as being resistant to existing anti-cancer treatments and having a poor prognosis of anti-cancer treatments. Specifically, the cancer having the cancer stem cell characteristics in the present invention may be a cancer that overexpresses ALDH1. The cancer that overexpresses ALDH1 may be a cancer in which the ratio of cancer stem cells that express ALDH1 or have positive activity is relatively higher than that of general cancers.

[0106] Specifically, the cancer that overexpresses ALDH1 may be any one or more selected from the group consisting of lung cancer, breast cancer, liver cancer, kidney cancer, gastric cancer, pancreatic cancer and brain cancer, but is not limited thereto.

[0107] The prevention or treatment of cancer may be to prevent or treat cancer chemoresistance, cancer recurrence, or cancer metastasis during or after cancer treatment by reducing the regenerative ability, growth ability, invasion ability, or migration ability of cancer stem cells.

[0108] The composition for use as an adjuvant in anti-cancer radiation therapy of the present invention comprises the humanized antibody or an antigen-binding fragment thereof.

[0109] The composition comprises the humanized antibody or antigen-binding fragment thereof as an active ingredient for increasing the radiosensitivity of cancer-related cells.

[0110] The cancer-associated cells are cells that constitute a cancer, and may have characteristics that, compared to normal cells, are not uniform in shape, grow indefinitely, and have weak cohesion with surrounding cells. Specifically, the cancer-associated cells may be cancer cells or cancer stem cells, and in particular may be cancer stem cells.

[0111] The cancer stem cells may be undifferentiated cells capable of differentiating into various cancer cells, specifically, cancer cells expressing ALDH1 or showing positive activity. The cancer stem cells of the present invention may be characterized in that their cell proliferation is not inhibited by irradiation, their self-renewal ability is not reduced, and their migration and invasion abilities are not inhibited.

[0112] Furthermore, the cancer-related cells may be those that have low sensitivity to radiation, that is, those that are highly resistant to radiation therapy, or those that are substantially insensitive to radiation and therefore cannot be treated with anti-cancer therapy by radiation exposure.

[0113] The anti-cancer treatment may involve suppressing the proliferation of cancer-related cells, suppressing metastasis and infiltration, and inducing cell death through radiation, surgery, chemotherapy, etc. The anti-cancer treatment of the present invention may involve administering the humanized antibody or antigen-binding fragment thereof in combination with radiation. When the humanized antibody or antigen-binding fragment is administered in combination with radiation in this manner, the antibody or antigen-binding fragment increases the radiosensitivity of cancer-related cells, maximizing the anti-cancer therapeutic effect of radiation, and further preventing cancer recurrence and metastasis.

[0114] The present invention will now be described in detail with reference to examples.

[0115] However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0116] [Examples and Comparative Examples]

[0117] [Example 1] Design of a humanized antibody that specifically binds to TM4SF4 and preparation of its expression vector A humanized antibody capable of binding specifically to the TM4SF4 protein with high affinity was produced. To this end, the amino acid sequence of the complementarity determining regions (CDRs) of the variable region of the antibody site for forming a specific bond with TM4SF4 was determined by using the amino acid sequence of the constant region of a human antibody and the amino acid sequence of the framework regions (FRs) of the variable region, resulting in a novel humanized antibody that has specific binding ability to TM4SF4 but exhibits low immunogenicity when administered to the human body.

[0118] Specifically, the CDR sequences of the humanized antibody of the present invention were those of mouse-derived antibodies capable of specifically binding to TM4SF4, as follows: CDR-H1 (SEQ ID NO: 10), CDR-H2 (SEQ ID NO: 11), and CDR-H3 (SEQ ID NO: 14) of the heavy chain variable region, and CDR-L1 (SEQ ID NO: 15), CDR-L2 (SEQ ID NO: 18), and CDR-L3 (SEQ ID NO: 19) of the light chain variable region. In addition, the FR sequences were those of the heavy chain variable region, FR-H1 (SEQ ID NO: 1), FR-H2 (SEQ ID NO: 2), FR-H3 (SEQ ID NO: 4), and FR-H4 (SEQ ID NO: 5), and FR-L1 (SEQ ID NO: 6), FR-L2 (SEQ ID NO: 7), FR-L3 (SEQ ID NO: 8), and FR-L4 (SEQ ID NO: 9). The CDR sequences were those of the ECL-2B7 antibody, which is a mouse-derived TM4SF4 antibody, and the FR sequences were those of the 3QRG antibody, which is a human antibody. The 3QRG antibody was selected from a group of human antibody candidates similar to the heavy and light chain amino acid sequences of the ECL-2B7 antibody using the BLASTP analysis program of the National Center for Biotechnology Information (NCBI). The CDR sequence was grafted to the FR sequence by a CDR-grafting method, and the 3QRG antibody was synthesized. The humanized antibody of the present invention was produced. First, a signal peptide sequence was added to the front of the CDR sequence of the heavy chain variable region in the CDR sequence (SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 14) to convert the amino acid sequence of the CDR sequence into a base sequence encoding the CDR sequence using a reverse translation program (https: / / www.bioinformatics.org / sms2 / rev_trans.html). Then, a gene encoding the heavy chain variable region of the humanized antibody of the present invention was synthesized by linking the FR sequence (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5) using recombinant PCR. The gene, which is about 450 bp in length, was divided into three sections, and as shown in FIG. 1, fragments of the heavy chain variable region gene corresponding to T1 (164 bp) were synthesized using the ECL-2B7-SH-5' and ECL-2B7-SH-3' primers, T2 (167 bp) was synthesized using the ECL-2B7-HC-1-5' and ECL-2B7-HC-1-3' primers, and T3 (157 bp) was synthesized using the ECL-2B7-HC-2-5' and ECL-2B7-HC-2-3' primers. Then, the T1 and T2 fragments were ligated via recombinant PCR to synthesize a fragment of 310 bp in size, and the fragment in which T1 and T2 were ligated was again ligated to the T3 fragment using primers Ch57-HC-5' and ECL-2B7-WH-3' to synthesize a heavy chain variable region (Hz2B7-1.0) gene of the humanized antibody of the present invention of about 450 bp having EcoRI and ApaI restriction enzyme cleavage sites at both ends. The heavy chain variable region was constructed so that it was arranged in the order of FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, and FR-H4, and this was confirmed by agarose gel (Figure 2). The primer sequences used in this process are shown in Table 1 below.The gene for the light chain variable region (Hz2B7-0.1) of the humanized antibody of the present invention was synthesized by a gene synthesis company (Bionics, Korea) in the order of FR-L1 (SEQ ID NO: 6), CDR-L1 (SEQ ID NO: 15), FR-L2 (SEQ ID NO: 7), CDR-L2 (SEQ ID NO: 18), FR-L3 (SEQ ID NO: 8), CDR-L3 (SEQ ID NO: 19), and FR-L4 (SEQ ID NO: 9). The gene for the light chain variable region (Hz2B7-0.1) of about 450 bp in length was synthesized and confirmed by agarose gel (Figure 3). Then, the heavy chain variable region gene was cloned into the EcoRI-ApaI position of the pdCMV-dhfr vector using T4 ligase (NEB, USA), and the light chain variable region gene was cloned into the HindIII-BsiWI position of the pdCMV-dhfr vector using T4 ligase (Figure 8), thereby preparing the vector pdCMV-dhfrC-Hz2B7-1.1 encoding the humanized antibody of the present invention (Hz2B7-1.1).

[0119] [Table 1]

[0120] [Example 2] Design of mutant antibodies of the humanized antibody of the present invention and preparation of expression vectors

[0121] [2-1] Analysis of interactions between the humanized antibody of the present invention and epitopes and molecular modeling In order to prepare a mutant antibody by modifying a part of the amino acid sequence of the humanized antibody of the present invention designed in Example 1, the interaction between the humanized antibody and the target epitope of TM4SF4 was first confirmed. Generally, the binding strength of an antigen-antibody can be qualitatively predicted through docking simulation. Since the X-ray structure of the humanized antibody in Example 1 is not known, the structure was predicted through homology modeling, and the humanized antibody in Example 1 was then subjected to the following steps: The binding strength between the antibody and the epitope was confirmed. To this end, the mouse chimeric antibody X836, which has a high similarity in amino acid sequence to the humanized antibody of Example 1, was used as a template for homology modeling using BLAST with the MODELLER program version 8.2. Of the 235 amino acids of the X836 antibody, 176 amino acids matched with the humanized antibody of Example 1, indicating an amino acid sequence similarity of about 75%, and based on this, the three-dimensional structure of the humanized antibody of Example 1 could be predicted (FIG. 4).

[0122] Since the epitope of the antigen, TM4SF4 protein, is a short polypeptide chain, it was treated as a single organic compound and a docking simulation was performed on the CDR region of the humanized antibody of Example 1 to calculate the binding free energy and binding mode. Specifically, the calculation was performed using a 15-mer oligopeptide consisting of the amino acid sequence Thr-Trp-Gly-Tyr-Pro-Phe-His-Asp-Gly-Asp-Tyr-Leu-Asn-Asp-Glu from amino acid 126 to 140 of the TM4SF4 protein as a simplified model for the entire antigen. The binding free energy of the epitope for the humanized antibody was calculated to be -11.6 kcal / mol, and the K iThe calculated value is about 3 nM. This means that a very strong antigen-antibody bond is formed between the humanized antibody of the present invention and the epitope of TM4SF4. A modified version of AutoDock version 4.2.6 developed by Scripps Research Institute, USA, was used as the docking program, and some terms of the protein-ligand binding free energy function were improved to improve accuracy. Specifically, the parameters included in the electrostatic interaction term, hydrogen bond energy term, and ligand hydration energy term were more accurately optimized and used in the docking simulation. In the electrostatic interaction part between the antibody and the epitope, the dielectric constant value of the antibody molecule was directly calculated and used, and in the hydrogen bond energy term, the accuracy of the calculation was improved by expressing it as a product of Morse functions to reflect the interdependence of the angle and distance. In the ligand hydration energy part, the number of parameters was increased to 69, and the accuracy was improved by optimizing it through a genetic algorithm. The weighting factors for van der Waals bonds, hydrogen bonds, electrostatic interactions, entropy, and hydration energy terms were 0.1485, 0.0656, 0.1146, 0.3113, and 0.1711, respectively, which were used as given in the existing AutoDock program.

[0123] From the results of the complex structure analysis of the humanized antibody and epitope of Example 1 obtained by the docking simulation, it was possible to identify the amino acid sites that play an important role in binding. Among the amino acids of the humanized antibody Hz2B7-1.1, 5 amino acids, including arginine (Arg, R) at position 99 of the light chain, asparagine (Asn, N) at position 58 of the heavy chain, tyrosine (Tyr, Y) at position 60 of the heavy chain, glycine (Gly, G) at position 102 of the heavy chain, and serine (Ser, S) at position 103 of the heavy chain, belong to the CDR region and are predicted to play a key role in the function of the antibody by forming hydrogen bonds with the epitope (Figures 4 and 5). The van der Waals bond was shown to be relatively weak, and among the hydrophobic amino acids, only tyrosine at position 38 of the light chain and tyrosine at position 98 of the light chain were observed at the interface of the antibody-epitope complex (Figures 4 and 5).

[0124] Amino acids that directly interact with the epitope through hydrogen bonds or van der Waals bonds play an important role in the function of the antibody, and therefore must be maintained. In order to increase the binding strength to the epitope, it is necessary to replace amino acids that have weaker binding strength or are located at a greater distance. For example, when asparagine (Asn, N) at position 31, which corresponds to CDR1 of the light chain variable region of the humanized antibody Hz2B7-1.1 and is the portion to which the phenylalanine and tryptophan residues of the epitope bind in Figures 4 and 5, is replaced with a more hydrophobic aromatic amino acid, it is predicted that the binding strength will increase as the van der Waals bonds between the epitope and the antibody are strengthened. In addition, when asparagine (Asn, N) at position 31 corresponds to CDR2 of the heavy chain variable region of the humanized antibody Hz2B7-1.1, which is the portion to which the phenylalanine and tryptophan residues of the epitope bind in Figures 4 and 5, the binding strength will increase. Tryptophan (Trp, W) at position 55 of the epitope is adjacent to the aspartic acid of the epitope, and therefore, when substituted with an amino acid capable of forming stronger hydrogen bonds, it is predicted that the binding strength will increase. Therefore, positions 31, which corresponds to CDR1 of the light chain variable region, and 55, which corresponds to CDR2 of the heavy chain variable region, are important positions capable of increasing affinity with the epitope (FIGS. 4 and 5). In addition, three amino acids, namely threonine (Thr, T) at position 100, which corresponds to CDR3 of the light chain variable region, tryptophan at position 54, which corresponds to CDR2 of the heavy chain variable region, and asparagine at position 56, which corresponds to CDR2 of the heavy chain variable region, are also located relatively close to the epitope, and therefore, when these are substituted with amino acids with different chemical properties, it is predicted that new hydrogen bonds or van der Waals bonds with the epitope will be induced, thereby increasing the efficacy of the antibody. Therefore, positions 100 corresponding to CDR3 of the light chain variable region, and positions 54 and 56 corresponding to CDR2 of the heavy chain variable region are also important positions that can increase affinity with the epitope (Figures 4 and 5).

[0125] [2-2] Design of mutant humanized antibody amino acid sequence and vector construction Based on the docking simulation analysis of Example 2-1, alanine (Ala, A) at position 46 of the heavy chain variable region of the humanized antibody of Example 1 was replaced with valine (Val, V), and the heavy chain variable region Hz2B7-2.0(HC A46V) was designed by changing FR-H2 of the humanized antibody sequence of Example 1 to the amino acid sequence of SEQ ID NO: 3 (FIG. 5).

[0126] In addition, tryptophan (Trp, W) at position 55 in the heavy chain variable region of the humanized antibody of Example 1 was replaced with serine (Ser, S) or tyrosine (Tyr, Y), respectively, to design heavy chain variable regions Hz2B7-3.0 (HC W55S) and Hz2B7-4.0 (HC W55Y) with the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, in the humanized antibody sequence of Example 1 ( FIG. 5 ). For this purpose, primers h2B7-HC-W55S-5' and h2B7-HC-W55S-3', and primers h2B7-HC-W55Y-5' and h2B7-HC-W55Y-3' were synthesized, and recombinant PCR was performed using primers h2B7-HC-SLIC-3' and h2B7-HC-SLIC-3' for in-fusion cloning (Figure 6A). The sequence of the synthesized gene was analyzed using a kit (EZ-Fusion TM The mutant humanized antibodies were then cloned into the pdCMV-dhfr-h2B7 vector cleaved with EcoR1 and ApaI using a HT Cloning Kit (Enzymomics, Korea) (FIG. 8). After confirming the mutations by sequencing, pdCMV-dhfr-Hz2B7-3.0 and pdCMV-dhfr-Hz2B7-4.0 capable of expressing the mutant humanized antibodies were prepared.

[0127] In addition, by replacing asparagine (Asn, N) at position 31 of the light chain variable region of the humanized antibody of Example 1 with phenylalanine (Phe, F) or valine (Val, V), respectively, the light chain variable region Hz2B7-0.2 (LC N31F) and the light chain variable region Hz2B7-0.3 (LC N31V) were designed with the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and these were confirmed by agarose gel (Figure 5). For this purpose, we synthesized the primers h2B7-LC-N31F-5' and h2B7-LC-N31F-3', and the primers h2B7-LC-N31V-5' and h2B7-LC-N31V-3', and performed recombinant PCR using the primers h2B7-LC-SLIC-3' and h2B7-LC-SLIC-3' for infusion cloning (Figure 6B). The sequence of the synthesized gene was analyzed using a kit (EZ-Fusion TM The mutant humanized antibody was expressed by the pdCMV-dhfr-h2B7-0.2 and pdCMV-dhfr-h2B7-1.1 vectors, which were digested with HindIII and BsiWI, using a HT Cloning Kit (Enzymomics, Korea) (Figure 8). After confirming the mutations by sequence analysis, the mutant humanized antibody was expressed by the pdCMV-dhfr-h2B7-0.2 and pdCMV-dhfr-h2B7-1.1 vectors, which were digested with HindIII and BsiWI, respectively, (Figure 8). dCMV-dhfr-Hz2B7-0.3 was produced.

[0128] The primer sequences used in this process are shown in Table 2 below.

[0129] [Table 2]

[0130] [Comparative Example] Design of chimeric antibody targeting TM4SF4 and preparation of expression vector The humanized antibodies of Examples 1 and 2 were prepared by using mouse antibody CDR sequences for the antibody heavy / light chain variable regions, and human antibody sequences for the antibody constant region and FR sequences for the antibody variable region. Therefore, in order to compare with the humanized antibody of the present invention, a chimeric antibody was prepared in which the entire heavy / light chain variable regions were derived from a mouse antibody and the constant region was derived from a human antibody. In other words, the chimeric antibody can be considered to be an antibody intermediate between a mouse antibody and a humanized antibody in terms of similarity to a human antibody.

[0131] First, PCR was performed on the heavy and light chain genes of the mouse-derived ECL-2B7 antibody, and DNA of about 400 bp and 390 bp was isolated using a gel extraction kit (FAVORGEN, Taiwan). Then, in order to synthesize the signal sequence of the heavy chain gene, 5'-signal-EcoR1 (5'-GAC GAA TTC ACT CTA ACC ATG PCR was performed using the 5'-signal-EcoR1 primer and the ECL-2B7-SH-3' (CTT CAC CTC GGA GTG GAC ACC TGT AGT TA-3') primer (Figure 7A). In addition, to amplify the heavy chain variable region, PCR was performed using the 2B7-SH-5' (5'-GTC CAC TCC GAG GTG AAG CTG GAG GAG TC) primer and the ECL-2B7-HC-Chi-3' (TTG GGC CCT TGG TGG AGG CTG CAG AGA CAG TGA CCA G-3') primer with the heavy chain variable region DNA as a template (Figure 7A). Then, recombinant PCR was performed using the 5'-signal-EcoR1 primer and the ECL-2B7-HC-Chi-3' primer to link the heavy chain signal sequence and the heavy chain variable region gene. Through this, a gene in which the heavy chain variable region and the heavy chain signal sequence were linked was obtained, which was then run on a 1% agarose gel to confirm the identity of the gene. TMA DNA corresponding to about 450 bp was isolated using a PCR Purification Kit (Farvorgen, Taiwan) (FIG. 7A). The heavy chain variable region gene to which the signal sequence was linked was treated with EcoRI and ApaI, and then the FavorPrep GEL TMThe heavy chain variable region gene was isolated using a PCR Purification Kit (FIG. 7B). The heavy chain variable region gene thus obtained was cloned into the EcoRI and ApaI sites of the pdCMV-dhfr vector containing the heavy chain constant region (IgG1) gene of a human antibody using T4 DNA ligase (NEB, USA) (FIG. 8). Similarly, for the light chain gene, PCR was performed using the pdCMV-dhfr-ch57 vector as a template with the ch57-LC-whole 5' (5'-CTG CAA AGC TTC GGC ACG AGC A) and ECL-2B7-SL-3' (CAC AAT ATC TCC TTC AAC ACC AGA CAA CC-3') primers to synthesize the signal sequence, and PCR was performed using the ECL-2B7-SL-5' (5'-GTT GAA GGA GAT ATT GTG ATG ACC CAG TCT) and ECL-2B7-LC-chi-3' (CCA CCG TAC GTT TGA TTT CCA GCT T-3') primers to amplify the light chain variable region gene (Figure 7A). In order to link the signal sequence and the light chain variable region, recombinant PCR was performed using ch57-LC-whole 5' primer and ECL-2B7-LC-chi-3' primer, and the product was then developed on a 1% agarose gel and isolated in the same manner as the heavy chain variable region gene (FIG. 7A). The light chain variable region gene was isolated by treating with HindIII and BsiwI, and then cloned into the HindIII and BisWI sites of the pdCMV-dhfr-chi2B7-HC vector (FIG. 7C) into which the heavy chain gene previously prepared, including the light chain constant region (Ck) gene of a human antibody, was inserted, to prepare a vector capable of expressing the chimeric antibody (pdCMV-dhfr-chi2B7, FIG. 8). The vector was transformed into E. coli DH5α using the RbCl2 method, and E. coli with a size of about 450 bp was selected. This E. coli was cultured overnight in 5 ml of LB medium containing 50 μg / ml ampicillin, and then the plasmid DNA was isolated using a kit (DNA-spin TMPlasmid DNA was isolated using a Plasmid DNA purification kit (INTRON, Korea) and the nucleotide sequence was confirmed (Bionics, Korea). As a result, it was confirmed that the nucleotide sequence of the cDNA isolated from the transformant was identical to that of the variable region gene of the ECL-2B7 antibody and was correctly linked to the heavy and light chain constant region genes of the human antibody.

[0132] [Experimental Example 1] Expression and purification of humanized and chimeric antibodies Antibody proteins were expressed and purified from the humanized antibody vectors of the present invention prepared in Examples 1 and 2 and the chimeric antibody vector of the comparative example.

[0133] First, to express and purify the chimeric antibody ("Chi2B7"), a 150 mm Using a culture dish of 1 x 10 7HEK293T cells (human embryonic kidney cell line) were cultured in 20 ml of DMEM medium (Biowest, France). 50 μg of the pdCMV-dhfr-chi2B7 expression vector and 75 μl of polyethyleneimine (1 mg / ml) were mixed with the cultured cells, and then mixed with 500 μl of transfection optimization medium and evenly sprinkled on the cell culture medium. The next day, the supernatant was collected and filled with new medium to collect the chimeric antibody expressed from the cell line, and the supernatant was placed in a column packed with beads (Protein G agarose beads, Amicogen, Korea) to allow the antibody to bind to the beads. The beads were then washed with PBS (pH 8.0), and the antibody was eluted from the beads using 10 ml of 0.1 M glycine (pH 2.8) and 1 M Tris-HCl (pH 9.0) to purify the Chi2B7 chimeric antibody, which was confirmed by SDS-PAGE and Coomassie blue staining (Figure 9A). In addition, whether the purified chimeric antibody had the constant region of a human antibody was detected by Western blotting using goat antibodies (goat-α-hIgG-gamma chain-HRP, Invitrogen, USA, and goat-α-hIgG-kappa chain-HRP, Bethyl, USA) as secondary antibodies, and it was confirmed that the chimeric antibody contained the constant region of a human antibody (Figure 9B).

[0134] In addition, the humanized antibodies of Examples 1 and 2 were expressed and purified. Through Examples 1 and 2, four types of heavy chain variable regions and three types of light chain variable regions of the humanized antibodies of the present invention were produced, and various types of humanized antibodies can be produced by combining these. Thus, the expression vectors of Examples 1 and 2 were introduced into HEK293T cells, and the humanized antibodies were expressed in the same manner as the chimeric antibodies, and then separated and purified using Protein G beads and a column.

[0135] In order to confirm the purity of the purified chimeric antibody, the humanized antibody of the present invention, and the mouse-derived 2B7 antibody, 10% SDS-PAGE was performed. Six representative types (Hz2B7-1.1, Hz2B7-1.2, Hz2B7-1.3, Hz2B7-2.1, Hz2B7-3.2, Hz2B7-4.3) out of the total 10 humanized antibodies were analyzed, and 1 μg of each antibody was used for SDS-PAGE, Coomassie blue staining, and Western blotting analysis (FIG. 9). As a result of staining using a solution for Coomassie blue staining (PageBlue staining solution, Thermo Scientific, USA), the heavy and light chains of each antibody were confirmed at positions of about 55 kDa and 25 kDa (FIG. 9A). This means that both the chimeric antibody and the humanized antibody are IgG type antibodies and have a partial sequence of a human antibody. For Western blotting, goat antibodies (goat-α-hIgG-gamma chain-HRP, Invitrogen, USA, and goat-α-hIgG-kappa chain-HRP, Bethyl, USA) were used as secondary antibodies, and bands were detected using an ECL kit (Western Bright ECL kit, Advance, USA). As a result, since the ECL-2B7 antibody is a mouse antibody, it was not detected by Western blotting, whereas the heavy and light chains of the other antibodies (chimeric and humanized antibodies) were detected as in the SDS-PAGE results, and it was confirmed that they had human antibody sequences, confirming the purity of the purified antibodies (Figure 9B).

[0136] [Experimental Example 2] Comparison of TM4SF4 antigen-binding ability of humanized antibodies by ELISA Using the humanized and chimeric antibodies of the present invention, indirect ELISA (indirect ELISA) The binding ability to TM4SF4 was confirmed via ELISA. Specifically, the humanized antibodies were produced by combining the four heavy chain variable region variants and the three light chain variable region variants designed in Examples 1 and 2, and named Hz2B7-1.1 and Hz2B7-1 Ten types of forms, Hz2B7-1.2, Hz2B7-1.3, Hz2B7-2.1, Hz2B7-3.1, Hz2B7-3.2, Hz2B7-3.3, Hz2B7-4.1, Hz2B7-4.2 and Hz2B7-4.3, were prepared. For this purpose, 100 μl of TM4SF4-BSA (1 μg / ml) or BSA solution (1 μg / ml) dissolved in a coating buffer solution (100 mM carbonate / bicarbonate coating buffer, pH 9.6) was added to the bottom of each well of a 96-well plate and allowed to adsorb for 16 hours or more at 4°C. The coated antigen was then washed three times with wash buffer (0.05% PBS-T, Tween-20), and 200 μl of blocking buffer (5% skim milk in wash buffer) was added to the bottom of each well where the antigen was not adsorbed, and reacted at 4°C for more than 16 hours, and then washed three times again with wash buffer. The humanized antibodies and chimeric antibodies of the 10 combinations purified in Experimental Example 1 were then diluted to various concentrations using blocking buffer, and 100 μl of each was added to each well according to concentration and reacted at room temperature for 2 hours. After washing three times again, 1 mg / ml of an antibody (α-human IgG-Kappa chain-HRP (Bethyl, USA) was diluted 10,000-fold with blocking buffer, and 100 μl was added to each well and reacted at room temperature for 1 hour. After the reaction was completed, the plate was washed three times with washing buffer and then diluted with substrate solution (OPD solution, 30% H 2 O 2 100 μl of OPD stock (40 mg / ml, adjusted to 10 ml with phosphate citrate buffer) was added to each well and incubated at room temperature. 2 SO 4 The reaction was terminated by adding 50 μl Finally, to measure the activity of the solid-bound enzyme, the absorbance was measured at 490 nm using an ELISA reader (Figure 10).

[0137] As a result, none of the 10 humanized antibodies of the present invention bound to BSA, but bound to the TM4SF4-BSA antigen with high specificity (Figure 10). Therefore, it was confirmed that the humanized antibodies of the present invention not only have the ability to bind to TM4SF4, but also have the property of specifically binding to it. In addition, when the affinity to TM4SF4-BSA was compared, all of the 10 humanized antibodies showed significantly higher affinity for TM4SF4 than the chimeric antibody Chi2B7. Furthermore, each of the 10 humanized antibodies showed a different binding affinity, and among them, the Hz2B7-1.2 humanized antibody showed a significantly increased binding affinity compared to Hz2B7-1.1, confirming that it had the best binding ability to TM4SF4 (Figures 10A and B).

[0138] [Experimental Example 3] Comparison of the sensitivity of anti-TM4SF4 humanized monoclonal antibodies by SPR analysis Surface plasmon resonance (SPR) analysis was performed to compare the binding ability of the constructed anti-TM4SF4 humanized monoclonal antibodies to the human TM4SF4 peptide (hTM4SF4 aa 126-140) antigen.

[0139] There are five types of humanized antibodies as Analyte. As shown in Table 3, one mutant type (HzE2B7-1.1), two light chain mutant types (HzE2B7-1.2 (LC N31F), HzE2B7-1.3 (LC N31V)), and two heavy / light chain mutant types (HzE2B7-4.3 (HC W55Y, LC N31V), HzE2B7-3.2 (HC W55S, LC N31F)) were analyzed. A synthetic peptide (Biotin- GSAGGSS TWGYPFHDGDYLNDE, GSAGGSS: the space sequence between biotin and TWGYPFHDGDYLNDE, which is TM4SF4 aa 126-140) was used as a ligand.

[0140] [Table 3]

[0141] First, a streptavidin-conjugated sensor chip (Series S Sensor Chip SA, Cytiva) was inserted into a Biacore T200 (Cytiva) and activation buffer (1M NaCl, 50mM NaOH) was poured for 30 seconds, followed by HBS-EP buffer (Cytiva) to activate and stabilize the sensor chip surface. A ligand (Biotin-hTM4SF4 aa 126-140) was poured into the stable sensor chip at a concentration of 1nM to 128nM to immobilize the ligand on the sensor chip surface, and a regeneration buffer (Regeneration buffer, 20mM NaOH) was poured to construct a streptavidin-conjugated sensor chip with a stably bound biotin-peptide ligand. Analyte antibodies for affinity analysis were prepared by diluting 512 nmole / L to a concentration of 16 nmol / L by serial dilution. Each concentration of analyte antibodies was passed through the constructed ligand-bound streptavidin-bound sensor chip at a speed of 30 ul / min for 480 seconds to induce binding, and then HBS-EP buffer solution was passed through at the same time and speed to obtain the degree to which the analyte antibodies dissociated from the ligand peptide in the form of a response unit. The association rate (K) was calculated using Biacore T200 evaluation software (Cytiva) by excluding the response value of the non-ligand-bound sensor chip obtained in the same manner. a ) and dissociation rate (K d ) and the equilibrium dissociation constant (KD, K d / K a) values ​​were calculated. As the calculation standard, a mass transfer fitting model for the interaction between the ligand peptide and the analyte antibody was analyzed using 1:1 binding. Based on the equilibrium dissociation constants (KD) shown in Figure 11, the binding ability of the anti-TM4SF4 humanized monoclonal antibodies to the human TM4SF4-peptide (hTM4SF4 aa 126-140) antigen was 6.03 x 10 for the HzE2B7-1.2 (LC N31F) antibody. -9 The lowest in M ​​is HzE2B7-1.1 at 2.442 × 10 -8 M,HzE2B7-1.3(LC N31V) is 3.542×10 -8 M, HzE2B7-4.3 (HC W55Y LC N31V) is 8.165×10 -8 M, HzE2B7-3.2 (HC W55S LC N31F) is 1.119 × 10 -7 M (Table 3). It is preferable that the equilibrium dissociation constant (KD) value of the humanized antibody is low, and the value of a typical commercially licensed humanized antibody is usually 10 -8 M or less, and the lower limit is not particularly limited. Therefore, the KD of the first designed humanized antibody (HzE2B7-1.1) is already a useful 2.442 × 10 -8 The HzE2B7-1.2 (LC N31F) antibody, which has a light chain with a 31st asparagine (N) substituted with a phenylalanine (F) based on information obtained through a docking model, has an antigen binding affinity that is approximately four times higher than that of the originally designed HzE2B7-1.1, with an equilibrium dissociation constant (KD) value of 6.03 × 10 -9 This shows that the It can be seen that the antibody has higher value.

[0142] [Experimental Example 4] Comparison of cancer cell binding ability of humanized antibodies by FACS Through Experimental Example 2, it was confirmed that the humanized antibody of the present invention has high affinity and specifically binds to TM4SF4, so the humanized antibody of the present invention was treated with cancer cells that are characterized by overexpressing TM4SF4 to confirm whether it could bind.

[0143] Specifically, lung cancer cell lines Calu-3 and A549 were cultured in DMEM medium (Biowest, France) with 10% fetal bovine serum (WelGene). Liver cancer cell lines Huh7, SNU-387, and SNU-449 were cultured in RPMI-1640 medium (Biowest) with 10% fetal bovine serum. After that, the cells that had grown to about 80% were treated with an enzyme (TrypLE TM Express Enzyme, Gibco TM The cells were then washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde (PFA) for 15 minutes at 4°C. After washing twice with PBA (0.1% BSA in PBS), 5x105 cells were added to each of the antibodies in PBA containing mouse 2B7 antibody (10μg / ml), chimeric antibody Chi2B7 (10μg / ml), humanized antibodies Hz2B7-1.1 (10μg / ml), Hz2B7-1.2 (10μg / ml), and Hz2B7-1.3 (10μg / ml), and mouse and human isotype control IgG1 antibody (10μg / ml), and reacted at 4°C for 1 hour. After the reaction, the cells were washed twice with PBA. The group containing mouse-derived 2B7 antibody and mouse isotype IgG (2 μg / ml, Invitrogen) was conjugated with FITC fluorescence, and the α-mouse antibody specific to mouse IgG was detected. IgG-FITC (2 μg / ml) antibody was added. FITC fluorescent was conjugated to the group containing human isotype IgG, chimeric antibody, and humanized antibody, and α-Human IgG-FITC (2 μg / ml, Invitrogen) antibody specific to human IgG was added and reacted for 30 minutes at 4° C. After the reaction, the cells were washed with PBA and suspended in 500 μl of PBA, and the specificity of the antibody to the antigen was confirmed using a flow cytometer (FACS Calibur, BD, USA).

[0144] As a result, it was confirmed that, like the mouse-derived 2B7 antibody, the humanized antibody of the present invention and the chimeric antibody of the comparative example specifically bind to Calu-3 and A549 lung cancer cells expressing TM4SF4 on the surface. In addition, it was confirmed that they did not bind to the surface of human primary hepatocytes (hPH), but also specifically bind to liver cancer cell lines Huh7, SNU-387 and SNU-449 liver cancer cells. Among them, the analysis of the humanized antibody Hz2B7-1.2 of the present invention confirmed that it had a significantly high binding ability, especially to the A549 cell line (Figure 12).

[0145] [Experimental Example 5] Preparation of a cell line capable of producing the humanized antibody of the present invention with high productivity To prepare a cell line capable of producing the humanized antibody of the present invention, we used the DG44 cell line, a CHO (Chinese ovary hamster cell) cell line in which the gene encoding dihydrofolate reductase is deleted. The DG44 cells were cultured in IMDM medium (Welgene, Korea) supplemented with HT (hypoxanthine-thymidine, Sigma, Germany) and Dialyed FBS (Gibco, USA). The cells were detached with trypsin-EDTA (Welgene, Korea) and then plated at 1×10 6 The expression vectors encoding the humanized antibodies Hz2B7-1.1 and Hz2B7-1.2 (LC N31F) of the present invention were prepared. For transfection, pdCMV-dhfr-hz2B7-1.1 and pdCMV-dhfr-hz2B7-1.2, which were linearized by treating with 15ug of PvuI restriction enzyme (Enzynomics, Korea), were mixed thoroughly with TOM medium (transfection optimized medium) to prepare the transfection medium, and 15μl of lipofectamine 2000 (Invitrogen, USA) was reacted with the TOM medium, and then mixed with the medium containing the vector and reacted for 20 minutes. The reacted solution was then dropped into the DG44 cells to perform transformation.

[0146] The cells transfected with the gene encoding the humanized antibody of the present invention were screened for neomycin resistance in a medium containing 100 μg / ml G418, and the supernatant of the cells with colonies formed was obtained. The humanized antibody of the present invention contained in the supernatant was used to confirm antibody production by sandwich ELISA. Five clones with high OD values ​​and excellent antibody expression levels were selected from each transformant, and the antibody gene of the present invention was amplified by treating with MTX (methotrexate). The antibody gene was amplified for one week by treating with MTX at a concentration of 0.02 μM, and the antibody production amount was measured again by ELISA, and five clones with increased production were selected again, and the gene was amplified again by increasing MTX to a concentration of 0.08 μM.

[0147] As a result, a total of five clones, 3A12, H12, 4A12, 4B12 and 4H12, were selected as cell lines that highly produce hz2B7-1.1 humanized antibody. Then, 4A9, 4A12, 4B9, 4C7 and 4H12 clones were selected as cell lines that highly produce hz2B7-1.2 antibody (LC N31F), and a total of seven clones were selected, including 7A8 and 6G10 clones obtained through further transformation (FIG. 13). Cell stacks of the 12 clones were made and cultured in IMDM medium supplemented with 10% dialyzed FBS. After the cells had grown to about 50%, MTX was treated at a concentration of 0.08 μM every two days to amplify the antibody genes. As a result of culturing with MTX treatment for approximately 30 days, a cell line resistant to MTX at a concentration of 0.08 μM was obtained, and the 4H12 clone was selected as a cell line highly expressing the Hz2B7-1.1 antibody, and the 4A12 clone was selected as a cell line highly expressing the Hz2B7-1.2 antibody.

[0148] To select single clones from the clones that survived in the 0.08 μM MTX solution, 3 × 10 4 After about 10 days, stacks were made using each colony formed and stored, and the remaining cells were examined for an increase in antibody production using sandwich ELISA. Specifically, 100 μl of α-human IgG-Fc specific antibody (2 μg / ml) dissolved in a coating buffer (100 mM carbonate / bicarbonate coating buffer, pH 9.6) was added to the bottom of each well of a 96-well plate and allowed to adsorb for 16 hours or more at 4°C. After washing and blocking, the 4H12 clone (Hz2B7-1.1-4H12), 4A12 clone (Hz2B7-1.2-4A12), and each single clone with amplified antibody genes selected at a concentration of 0.08 μM MTX were incubated at 37°C in CO 2After culturing in an incubator for 24 hours, the culture medium was collected. The culture medium and human IgG isotype control antibody were prepared by diluting them to various concentrations, and 100 μl of each was added to each well for reaction at room temperature for 2 hours. Then, α-human IgG-Kappa chain-HRP (1 mg / ml, Bethyl, USA) was diluted 10,000-fold using blocking buffer, and 100 μl was added to each well for reaction at room temperature for 1 hour. After the reaction was completed, the substrate OPD solution (30% H 2 O 2 100 μl of OPD stock (40 mg / ml, adjusted to 10 ml with phosphate citrate buffer) was added to each well and incubated at room temperature for 10 minutes. 2 SO 4 The reaction was terminated by adding 50 μl.

[0149] As a result of ELISA, clones that showed higher absorbance than the clones before amplification by MTX were identified among the clones Hz2B7-1.1-4H12-0.08 and Hz2B7-1.2-4A12-0.08 in which the antibody gene was amplified by MTX. The antibody concentration was calculated through quantitative analysis of antibody production (FIGS. 14 and 15). As a result, in the case of the Hz2B7-1.1-4H12 clone, the antibody production of cells before the antibody gene was amplified by MTX was about 2.8 μg / 10 6 The antibody production of clone Hz2B7-1.1-4H12-0.08-#1, in which the antibody gene was amplified by MTX, increased by approximately 3.8 times to 10.8 μg / 10 6 In the case of the Hz2B7-1.2-4A12 clone, the antibody production before amplification was 2.5 μg / 10 6 cell / 24hr, but this clone was treated with MTX at a concentration of 0.08μM In the case of the Hz2B7-1.2-4A12-0.08 clone amplified to 100 μg / 10, clone #9 was approximately 14.2 μg / 10 6 cell / 24hr, #20 clone was 7.9μg / 10 6The antibody production amount per cell / 24 hr was shown to be approximately 5.7-fold and 3.1-fold higher than that of clones in which the gene was not amplified, indicating that antibody productivity had increased by up to 5.7-fold (Figure 15).

[0150] Next, a serum stability experiment was performed to measure the stability of antibodies in blood. Mouse antibody 2B7 purified from a mouse hybridoma cell line and humanized antibodies Hz2B7-1.1 and Hz2B7-1.2 purified from a CHO cell line were cultured in 60% human serum (Sigma-Aldrich) at 37°C for up to 4 days, and their antigen binding ability was measured at 0, 3, 24, 48, 72, and 96 hours using indirect ELISA. Specifically, 0.5μg / ml of TM4SF4-BSA antigen was adsorbed using a coating buffer solution (100mM carbonate / bicarbonate coating buffer, pH 9.6) at 4°C for more than 16 hours, followed by washing and blocking. Then, the antibody cultured in human serum was used as the primary antibody, and the ELISA process was performed as before to confirm whether the antigen binding ability was maintained over time. After measuring absorbance, the antigen-binding ability at 0 hours was assumed to be 100%. As a result, it was confirmed that mouse antibody 2B7 had the same antigen-binding ability in human serum for up to 96 hours (FIG. 16A). In addition, it was confirmed that humanized antibodies Hz2B7-1.1 and Hz2B7-1.2, which have altered antibody sequences and structures, were not decomposed and maintained a certain level of antigen-binding ability in human serum for up to about 4 days (FIGS. 16B and C).

[0151] Therefore, by specifically amplifying the antibody gene using MTX in CHO-DG44 cells transformed with the humanized antibody gene of the present invention, the cell lines Hz2B7-1.1-4H12-0.08-#1 and Hz2B7-1.2-4A12-0.08-#9 producing the antibody of the present invention could be established.

[0152] Although the present invention has been described in detail above only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention, and it is natural that such modifications and variations fall within the scope of the claims.

Claims

1. a heavy chain variable region comprising FR-H1 having the amino acid sequence of SEQ ID NO:1, FR-H2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3, FR-H3 having the amino acid sequence of SEQ ID NO:4, and FR-H4 having the amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising FR-L1 having the amino acid sequence of SEQ ID NO:6, FR-L2 having the amino acid sequence of SEQ ID NO:7, FR-L3 having the amino acid sequence of SEQ ID NO:8, and FR-L4 having the amino acid sequence of SEQ ID NO:9; Including, A humanized antibody or an antigen-binding fragment thereof that specifically binds to TM4SF4 (TransMembrane 4 Superfamily Member 4).

2. The heavy chain variable region further comprises at least one CDR selected from the group consisting of CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 77, and CDR-H3 having the amino acid sequence of SEQ ID NO: 14; The humanized antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region further comprises at least one CDR selected from the group consisting of CDR-L1 having the amino acid sequence of SEQ ID NO: 79, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO:

80.

3. The CDR-H2 has any one amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13; The CDR-L1 has any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof according to claim 2, wherein the CDR-L3 has the amino acid sequence of SEQ ID NO:

19.

4. the heavy chain variable region further comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 10, a CDR-H2 having the amino acid sequence of SEQ ID NO: 11, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 14; The humanized antibody or antigen-binding fragment thereof of claim 3, wherein the light chain variable region further comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 16, CDR-L2 having the amino acid sequence of SEQ ID NO: 18, and CDR-L3 having the amino acid sequence of SEQ ID NO:

19.

5. the heavy chain variable region has any one amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; The humanized antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region has any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO:

26.

6. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising at least one selected from the group consisting of a heavy chain constant region having the amino acid sequence of SEQ ID NO: 27 and a light chain constant region having the amino acid sequence of SEQ ID NO:

28.

7. The antigen-binding fragments include Fab, F(ab'), F(ab') ２ 6. The antibody or antigen-binding fragment thereof according to claim 1 , which is any one selected from the group consisting of Fv and Fc.

8. The antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (KD) of 2.4 x 10 －８ M or below The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

9. A polynucleotide comprising a base sequence encoding the humanized antibody or antigen-binding fragment thereof described in any one of claims 1 to 5.

10. An expression vector comprising the polynucleotide of claim 9.

11. A host cell comprising the expression vector of claim 10.

12. A method for producing a humanized antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 10.

13. A composition for detecting TM4SF4, comprising a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

14. A kit for detecting TM4SF4, comprising the composition for detecting TM4SF4 according to claim 13.

15. A method for detecting TM4SF4, comprising the step of contacting a humanized antibody or its antigen-binding fragment described in any one of claims 1 to 5 with a sample to be detected that is suspected to contain TM4SF4.

16. A pharmaceutical composition for preventing or treating cancer, comprising a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

17. The pharmaceutical composition for preventing or treating cancer according to claim 16, wherein the prevention or treatment of cancer is to prevent or treat at least one selected from the group consisting of cancer chemo-resistance during cancer treatment, cancer chemo-resistance after cancer treatment, cancer recurrence, and cancer metastasis.

18. The pharmaceutical composition for preventing or treating cancer according to claim 16, wherein the cancer is at least one selected from the group consisting of lung cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colorectal cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, multiple myeloma, acute myeloid leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibrous tumor and brain cancer.

19. A composition for inhibiting the growth of cancer stem cells, comprising a humanized antibody or its antigen-binding fragment described in any one of claims 1 to 5.

20. A composition for use in radiation-induced anti-cancer treatment, comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

21. The composition for supporting anti-cancer radiation treatment according to claim 20, wherein the humanized antibody or antigen-binding fragment thereof enhances the sensitivity of cancer cells, including cancer stem cells, to radiation.

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