Anti-TM4SF4 humanized antibodies and uses thereof

Abstract

The present invention relates to: a humanized antibody capable of binding specifically to tetratransmembrane protein superfamily member 4 (TM4SF4) with high affinity and exhibiting low immunogenicity in humans; and a composition for preventing and treating cancer or assisting immune anti-cancer therapy, said composition comprising the humanized antibody.

Description

Technical Field

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

[0002] [Cross-reference to Related Applications]

[0003] This application claims priority to Korean Patent Application No. 10-2022-0177553, filed on December 16, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Tetraspanin superfamily member 4 (TM4SF4) is a tetraspanin protein. It has been reported that other proteins in this category, such as TM4SF1 and TM4SF5, are upregulated in many types of tumors and are involved in epithelial-mesenchymal transition and cell migration, and many studies related to cancer cells have been conducted. In some reports, it has been reported that TM4SF4 is involved in apoptosis, differentiation, and cell invasion ability in cancer cells. Recently, the research team of the present invention reported that the TM4SF4 protein promotes the growth, self-renewal ability, and metastasis / invasion of cancer stem cells in human lung cancer cells, and showed that TM4SF4 promotes the activation of important signal transduction systems IGF1Rβ / AKT / NFκB or JAK2 (or FAK) / STAT3 in cancer development, and enhances cancer stem cell characteristics through promoted cytokine secretion, thereby making tumors more malignant (Choi SI et al., Oncotarget. 2014; 5(20):9823-9837, Choi SI et al., Oncotarget. 2017; 8(60):101284-101297).

[0005] Antibodies are used as therapeutic agents due to their high binding specificity to target antigens and stability in the human body. In particular, based on the development of antibody engineering technology, antibodies with anti-cancer functions have been improved into humanized antibodies, single-chain antibodies, bispecific antibodies, and drug conjugate antibodies, and have been used to greatly enhance the efficacy of cancer treatment. However, due to the diversity of cancer characteristics and the induction of treatment resistance by the expression of neoantigens, the existing types of antigens used to target cancer cells have been pointed out to be limited, and continuous research has been carried out to find novel cancer-specific antigens and obtain antibodies against such antigens.

[0006] In particular, in the case of cancers that are resistant to targeted drugs or radiotherapy used in existing cancer treatments and have recurred, the properties of cancer stem cells have been reported to play an important role, and it has become increasingly important to identify antigens that can be used to target cancer stem cells and obtain specific antibodies.

[0007] Meanwhile, to develop monoclonal antibodies that specifically bind to a particular antigen, a method has been mainly used in which the antigen is injected into an animal other than a human, and the antibodies produced using the immune system of that animal are utilized. However, the antibodies produced by the above method are not human proteins because they are derived from animals other than humans, and thus, when administered to humans, immunogenicity problems may arise. That is, when an antibody derived from a species other than a human is administered to a human body, it can induce the production of human anti-mouse antibodies (HAMA), and the therapeutic efficacy of the heterologous antibody can be reduced.

[0008] Therefore, a method for solving the above problems has been proposed, which involves obtaining the sequences that play an important role in antigen binding from the amino acid sequences constituting antibodies derived from species other than humans, and replacing the remaining parts with human antibody sequences. However, there is a possibility that the chimeric antibodies or humanized antibodies produced by this method may not function as antibodies, or their binding affinity for antigens may be reduced because they are a fusion of two different protein regions. Therefore, it remains difficult to develop a humanized antibody that exhibits high specificity and affinity for antigens without inducing the production of HAMA in the human body.

[0009] In addition, a method is needed to maximize the effect of anti-cancer immunotherapy by inhibiting the expression of TM4SF4 in cancer cells and increasing the anti-cancer immune activity that has already emerged. Therefore, the inventors of the present invention have attempted to develop an antibody that assists anti-cancer immunotherapy by inhibiting the activity of the TM4SF4 protein in cancer cells. Summary of the Invention

[0010] Technical Problem

[0011] An object of the present invention is to provide a novel humanized antibody or its antigen-binding fragment that can specifically bind with high affinity to the tetraspanin superfamily member 4 (TM4SF4) protein overexpressed on the surface of cancer cells and can exhibit low immunogenicity when administered to a human body.

[0012] Another object of the present invention is to provide a polynucleotide, expression vector, host cell that encodes a humanized antibody or its antigen-binding fragment and is capable of expressing it, and a method for producing a humanized antibody or its antigen-binding fragment. Yet another object of the present invention is to provide a method for producing the antibody or its antigen-binding fragment, the method comprising culturing a host cell.

[0013] Another object of the present invention is to provide a composition and a kit for detecting TM4SF4, and a method for detecting TM4SF4.

[0014] 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 assisting radiotherapy for cancer treatment.

[0015] Another object of the present invention is to provide a composition for assisting anti-cancer immunotherapy.

[0016] Technical solution

[0017] One aspect of the present invention provides a humanized antibody or an antigen-binding fragment thereof, which comprises: a heavy chain variable region, the 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, FR-H3 having the amino acid sequence of SEQ ID NO:3, and FR-H4 having the amino acid sequence of SEQ ID NO:4; and a light chain variable region, the light chain variable region comprising FR-L1 having the amino acid sequence of SEQ ID NO:5, FR-L2 having the amino acid sequence of SEQ ID NO:5, FR-L3 having the amino acid sequence of SEQ ID NO:7, and FR-L4 having the amino acid sequence of SEQ ID NO:8, wherein the humanized antibody or an antigen-binding fragment thereof specifically binds to tetraspanin superfamily member 4 (TM4SF4).

[0018] Furthermore, in the humanized antibody or an antigen-binding fragment thereof, 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:9, CDR-H2 having the amino acid sequence of SEQ ID NO:10, and CDR-H3 having the amino acid sequence of SEQ ID NO:11, and 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:12 or SEQ ID NO:13, CDR-L2 having the amino acid sequence of SEQ ID NO:14, and CDR-L3 having the amino acid sequence of SEQ ID NO:15.

[0019] In another aspect of the present invention, in the humanized antibody or an antigen-binding fragment thereof, the humanized antibody or an antigen-binding fragment thereof has an amino acid sequence in which the 31st amino acid asparagine in CDR1 of the light chain variable region is substituted with phenylalanine.

[0020] In yet another aspect of the present invention, there is provided a polynucleotide having a base sequence encoding two types of humanized antibodies or antigen-binding fragments, an expression vector comprising the polynucleotide, and a host cell comprising the expression vector.

[0021] Another aspect of the present invention provides a composition for detecting TM4SF4, the composition comprising the humanized antibody or antigen-binding fragment, and a kit for detecting TM4SF4, the kit comprising the composition for detecting TM4SF4.

[0022] Another aspect of the present invention provides a method for detecting TM4SF4, the method comprising contacting the humanized antibody or its antigen-binding fragment with a sample to be tested that is expected to contain TM4SF4.

[0023] 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 assisting radiotherapy for anti-cancer treatment, the composition comprising the humanized antibody or its antigen-binding fragment.

[0024] Beneficial effects

[0025] The humanized antibody of the present invention can be effectively used to detect TM4SF4 or target cancer cells or cancer stem cells overexpressing TM4SF4 because it can specifically bind to TM4SF4 without binding to substances such as BSA. In particular, the humanized antibody of the present invention exhibits excellent technical effects because it has a significantly higher binding affinity compared to antibodies derived from mice or chimeric antibodies.

[0026] In addition, since the humanized antibody of the present invention uses an amino acid sequence derived from a human antibody or is obtained by modifying a partial amino acid sequence other than the CDR sequence, the humanized antibody of the present invention is less likely to induce human anti-mouse antibodies (HAMA) when administered to humans, and thus has the effect of low immunogenicity. Therefore, the humanized antibody of the present invention has the advantage of being able to solve the immune response problems that may occur when using antibodies derived from mice.

[0027] In addition, the humanized antibody of the present invention can reduce the growth, migration and invasion of cancer cells as well as radioresistance, and can reduce the radiation resistance of cancer cells, thereby preventing or treating cancer, inhibiting the growth of cancer stem cells, and assisting radiotherapy for anti-cancer treatment.

[0028] In addition, the humanized antibody of the present invention has excellent effects on anti-cancer immunity by inhibiting the expression of PD-L1 and enhancing antibody-dependent cytotoxicity.

[0029] However, the effects of the present invention are not limited to the above effects, and other effects not mentioned can be obviously understood by those skilled in the art from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shows the results of a colony formation assay for confirming changes in cell growth by treating the lung adenocarcinoma cell line A549 and pancreatic cancer cell line MIAPaCa-2 expressing TM4SF4 and the H1299 cell line not expressing TM4SF4 with a humanized anti-TM4SF4 antibody.

[0031] Figure 2 Shows the results of confirming changes in radioresistance by colony formation assay when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0032] Figure 3 Shows the results of confirming changes in cell migration by wound healing when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0033] Figure 4 Shows the results of confirming changes in cell migration and invasion by transwell when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0034] Figure 5 Shows the results of confirming changes in the stem cell characteristics of cancer cells by cell sphere formation when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0035] Figure 6 Shows the results of confirming that the EMT phenomenon of cells is significantly inhibited by Western blot when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0036] Figure 7 Shows the results of confirming that the EMT phenomenon of cells is significantly inhibited by immunofluorescence when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0037] Figure 8 Shows the results of confirming the expression levels of intracellular ALDH1 and CD44 when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and MIAPaCa-2 cells (which are pancreatic cancer cell lines).

[0038] Figure 9 Shows the results of confirming the antibody-dependent cell cytotoxicity (ADCC) function of the humanized antibody of the present invention in the lung adenocarcinoma cell line A549 and the MIAPaCa-2 cell (which is a pancreatic cancer cell line).

[0039] Figure 10 Shows the results of confirming the protein and gene expression changes of programmed death ligand 1 (PD-L1) and B7H4 (which is a ligand of an immune checkpoint receptor in cancer cells) when the humanized antibody of the present invention is used to treat the lung adenocarcinoma cell line A549 and the MIAPaCa-2 cell (which is a pancreatic cancer cell line). Detailed Description of the Invention

[0040] Hereinafter, the present invention will be described in detail.

[0041] 1. Humanized antibody specifically binding to TM4SF4 or antigen-binding fragment thereof

[0042] One aspect of the present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds to TM4SF4 and exhibits low immunogenicity in humans.

[0043] The term "antibody" in the present invention refers to an immunoglobulin molecule that has an immune response by specifically binding to an antigenic epitope. Antibodies can include all monoclonal antibodies, polyclonal antibodies, antibodies with a full-length chain structure (full-length antibodies), functional fragments with at least antigen-binding function (antigen-binding fragments), and recombinant antibodies. Specifically, the antibody of the present invention can be a monoclonal antibody or its antigen-binding fragment. A monoclonal antibody refers to an antibody molecule composed of a single molecule obtained from a substantially identical group of antibodies, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. A full-length antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain can be linked to the heavy chain by a disulfide bond. An antibody can contain heavy chain (HC) and light chain (LC) polypeptides, and the heavy chain and the light chain can include a variable region and a constant region.

[0044] The constant region is the site that mediates the binding of antibodies to various cells of the immune system (such as T cells, etc.), host tissues containing components of the complement system, etc. Regardless of the type of antigen, as long as it is the same type of antibody derived from the same species, the constant region has the same function, and the amino acid sequence constituting the constant region is the same or highly similar for each antibody. The constant region can be divided into the heavy-chain constant region (which can be abbreviated as CH) and the light-chain constant region (which can be abbreviated as CL). The heavy-chain constant region has types of γ (gamma), μ (mu), α (alpha), δ (delta), and / or ε (epsilon), and has γ1 (gamma 1), γ2 (gamma 2), γ3 (gamma 3), γ4 (gamma 4), α1 (alpha 1), and / or α2 (alpha 2) as subclasses. The light-chain constant region has types of κ (kappa) and λ (lambda). IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0045] The variable region is the antibody site that is specific to the antigen and can be divided into the heavy-chain variable region (which can be abbreviated as VH) and the light-chain variable region (which can be abbreviated as VL). The variable region can include three complementarity-determining regions (CDRs) and four framework regions (FRs). The CDRs can be loop sites involved in antigen recognition, and the specificity of the antigen can be determined according to the amino acid sequences of the CDRs.

[0046] Depending on whether the CDR is a heavy-chain polypeptide or a light-chain polypeptide, the CDRs can be called CDR1, CDR2, and CDR3 in order, and can be called CDR-H1, CDR-H2, and CDR-H3 for the heavy-chain variable region, and can be called CDR-L1, CDR-L2, and CDR-L3 for the light-chain variable region.

[0047] The FR refers to the part of the variable region of the immunoglobulin molecule other than the complementarity-determining regions. Depending on whether the FR is a heavy-chain polypeptide or a light-chain polypeptide, the FRs can be called FR1, FR2, FR3, and FR4 in order, and can be called FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy-chain variable region, and can be called FR-L1, FR-L2, FR-L3, and FR-L4 for the light-chain variable region.

[0048] Similarly, for the heavy chain variable region, the FRs can be referred to as FR-H1, FR-H2, FR-H3, and FR-H4, and for the light chain variable region, the FRs can be referred to as FR-L1, FR-L2, FR-L3, and FR-L4. In addition, the CDRs and FRs can be arranged in each variable region in the following order. The order is from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus): for the heavy chain variable region, FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, and FR-H4; for the light chain variable region, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4.

[0049] The term "antigen-binding fragment" in the present invention refers to any fragment of the humanized antibody of the present invention that has the antigen-binding function of an antibody. The antigen-binding fragment can be used interchangeably with terms such as "fragment" and "antibody fragment", and the antigen-binding fragment can be Fab, Fab', F(ab')2, Fv, etc., but is not limited thereto.

[0050] Fab has a structure that includes the light chain and heavy chain variable regions, the light chain constant region, and the first heavy chain constant region (CH1 domain), and has one antigen-binding site. Fab' is different from Fab in that Fab' has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. When the cysteine residues in the hinge region of Fab' form a disulfide bond, F(ab')2 is produced. Fv refers to the smallest antibody fragment that only has the heavy chain variable region and the light chain variable region. The double-chain Fv can be formed by non-covalently linking the heavy chain variable region and the light chain variable region, and the single-chain Fv can generally be formed by covalently linking the heavy chain variable region and the light chain variable region via a peptide linker, or directly linking the heavy chain variable region and the light chain variable region at the C-terminus, thereby forming a structure such as a dimer, such as a double-chain Fv. The antigen-binding fragment can be prepared by using a protease (for example, Fab can be obtained by limited cleavage of the whole antibody with papain, and the F(ab')2 fragment can be obtained by cleavage of the whole antibody with pepsin), or by genetic recombination techniques, but is not limited thereto.

[0051] The term "humanized antibody" in the present invention refers to an antibody that exhibits reduced immunogenicity or non-immunogenicity in humans. A humanized antibody can be produced by combining, for example, the CDRs derived from a non-human entity (non-human species) with the constant region derived from a human antibody and the FRs in the variable region derived from a human antibody. A humanized antibody can be produced by transplanting the CDRs of a non-human species antibody between the FR sequences of a human antibody by the CDR transplantation method.

[0052] A humanized antibody refers to an antibody in which both the heavy-chain variable region and the light-chain variable region are derived from humans. According to the differences in the heavy-chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) with a γ-chain heavy chain, IgM with a μ-chain heavy chain, IgA (including IgA1 and IgA2) with an α-chain heavy chain, IgD with a δ-chain heavy chain, or IgE with an ε-chain heavy chain. Additionally, in principle, the light chain includes at least one of the κ-chain and the λ-chain.

[0053] Compared with murine antibodies or chimeric antibodies, humanized antibodies are characterized by a higher similarity to human antibodies and thus have lower immunogenicity when administered to humans.

[0054] The term "epitope" in the present invention refers to a specific site on an antigen that can be specifically recognized and bound by an immunoglobulin, an antibody, or an antigen-binding fragment thereof. An epitope can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed through the tertiary folding of a protein.

[0055] TM4SF4 is a four-transmembrane protein and is a protein known to be involved in apoptosis, differentiation, and cell invasion ability in cancer cells. In addition, TM4SF4 is known to promote the growth and metastasis of cancer stem cells and can enhance the characteristics of cancer stem cells and make tumors more malignant. The TM4SF4 protein can be a membrane protein present in the cell membrane, and a part of TM4SF4 can be exposed to the outside of the cell. The exposed part can have two loop structures, and some amino acid sequences of the extracellular exposed part of TM4SF4 can be epitopes that can be specifically recognized and bound by the humanized antibody or its antigen-binding fragment of the present invention. The epitope of TM4SF4 can have, for example, an amino acid sequence of "TWGYPFHDGDYLNDE" (in the order from the N-terminus to the C-terminus, SEQ ID NO:2).

[0056] Epitope regions can be, for example, GGCARCLGGTLIPLAFFGFLANILLFFPGG (SEQ ID NO: 23) at positions 4 - 33 from the N-terminus of the reference TM4SF4 antigen (SEQ ID NO: 1), LGSGVLMIFPALVFL (SEQ ID NO: 24) at positions 53 - 67, NNDCCGCCGN (SEQ ID NO: 25) at positions 71 - 80, STIFAVVGFLGAGYSFIISAI (SEQ ID NO: 26) at positions 92 - 112, KGPKCLM (SEQ ID NO: 27) at positions 116 - 122, WGYPFHD (SEQ ID NO: 28) at positions 127 - 133, and CREPLNVVPWNLTLFSILLVVGGIQMVLCAIQVVNGLLGTLCGDCQCCGCCGG (SEQ ID NO: 29) at positions 146 - 198. More specifically, it can be TWGYPFHDGDYLNDE (SEQ ID NO: 2) at positions 126 - 140 starting from the N-terminus of the reference TM4SF4 antigen (SEQ ID NO: 1).

[0057] The humanized antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises FR-H1 with the amino acid sequence of SEQ ID NO: 1, FR-H2 with the amino acid sequence of SEQ ID NO: 2, FR-H3 with the amino acid sequence of SEQ ID NO: 3, and FR-H4 with the amino acid sequence of SEQ ID NO: 4. The light chain variable region comprises FR-L1 with the amino acid sequence of SEQ ID NO: 5, FR-L2 with the amino acid sequence of SEQ ID NO: 6, FR-L3 with the amino acid sequence of SEQ ID NO: 7, and FR-L4 with the amino acid sequence of SEQ ID NO: 8, and specifically binds to transmembrane 4 superfamily member 4 (TM4SF4).

[0058] The FR-H1 having the amino acid sequence of SEQ ID NO:1 can be the FR1 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The FR-H2 having the amino acid sequence of SEQ ID NO:2 can be the FR2 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention, and the FR-H3 having the amino acid sequence of SEQ ID NO:3 can be the FR3 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The FR-H4 having the amino acid sequence of SEQ ID NO:4 can be the FR4 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention.

[0059] The FR-L1 having the amino acid sequence of SEQ ID NO:5 can be the FR1 sequence of the light chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The FR-L2 having the amino acid sequence of SEQ ID NO:6 can be the FR2 sequence of the light chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The FR-L3 having the amino acid sequence of SEQ ID NO:7 can be the FR3 sequence of the light chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The FR-L4 having the amino acid sequence of SEQ ID NO:8 can be the FR4 sequence of the light chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention.

[0060] 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:9, CDR-H2 having the amino acid sequence of SEQ ID NO:10, and CDR-H3 having the amino acid sequence of SEQ ID NO:11, and preferably comprises CDR-H1, CDR-H2, and CDR-H3.

[0061] 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:12 or SEQ ID NO:13, CDR-L2 having the amino acid sequence of SEQ ID NO:14, and CDR-L3 having the amino acid sequence of SEQ ID NO:15, and preferably comprises CDR-H1, CDR-H2, and CDR-H3.

[0062] The CDR-H1 having the amino acid sequence of SEQ ID NO:9 can be the CDR1 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The CDR-H2 having the amino acid sequence of SEQ ID NO:10 can be the CDR2 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention, and the CDR-H3 having the amino acid sequence of SEQ ID NO:11 can be the CDR3 sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention.

[0063] The CDR-L1 having the amino acid sequence of SEQ ID NO:12 can be the CDR1 sequence of the light chain variable region referred to as "Hz2B7-1.1" in the present invention, the CDR-L1 having the amino acid sequence of SEQ ID NO:13 can be the CDR1 sequence of the light chain variable region referred to as "Hz2B7-1.2" in the present invention, and the CDR-L2 having the amino acid sequence of SEQ ID NO:14 can be the CDR2 sequence of the light chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention. The CDR-L3 having the amino acid sequence of SEQ ID NO:15 can be the CDR3 sequence of the light chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention.

[0064] The heavy chain variable region can have the amino acid sequence of SEQ ID NO:16.

[0065] The light chain variable region can have the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18.

[0066] The heavy chain variable region having the amino acid sequence of SEQ ID NO:16 can be the sequence of the heavy chain variable region referred to as "Hz2B7-1.1" or "Hz2B7-1.2" in the present invention.

[0067] The light chain variable region having the amino acid sequence of SEQ ID NO:17 can be the sequence of the light chain variable region referred to as "Hz2B7-1.1" in the present invention, and the light chain variable region having the amino acid sequence of SEQ ID NO:18 can be the sequence of the light chain variable region referred to as "Hz2B7-1.2" in the present invention. The humanized antibody or its antigen-binding fragment of the present invention may comprise the heavy chain constant region and / or the 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 can be used without limitation as long as the humanized antibody or its antigen-binding fragment does not inhibit the property of specifically binding to TM4SF4.

[0068] The above amino acid sequence may include variants with different sequences due to deletions, insertions, substitutions of amino acid residues, or combinations thereof, as long as the scope does not affect the structure, function, activity, etc. of the polypeptide containing the amino acid sequence. In addition, the amino acid sequence may contain amino acids with common modifications known in the art, and examples of amino acid modifications include phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation.

[0069] In another aspect of the present invention, the humanized antibody or its antigen-binding fragment may have an amino acid sequence in which the 31st amino acid asparagine in CDR1 of the light chain variable region is substituted with phenylalanine.

[0070] The humanized antibody or its antigen-binding fragment of the present invention not only has the above amino acid sequence, but also has an amino acid sequence that is substantially the same as it or a variant thereof. The meaning of having a substantially the same amino acid sequence is that the amino acid sequence has a homology of 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 99.5% or higher with the above amino acid sequence, but is not limited thereto.

[0071] 2. Technology for expressing TM4SF4-specific humanized antibody

[0072] Another aspect of the present invention provides a polynucleotide, an expression vector, a host cell, and a production method that can be used to express and produce a humanized antibody or its antigen-binding fragment.

[0073] Since the descriptions of the humanized antibody, its antigen-binding fragment, TM4SF4, etc. are the same as those described in "1. Humanized antibody or its antigen-binding fragment that specifically binds to TM4SF4", to avoid repeated description, the description is omitted here, and only the content related to the polynucleotide, expression vector, and host cell will be described below.

[0074] The term "polynucleotide" in the present invention includes DNA and RNA molecules, and the nucleotide, which is the basic structural unit of the polynucleotide, may include not only naturally occurring nucleotides but also analogs in which the sugar or base moiety is modified.

[0075] The polynucleotide of the present invention has a base sequence encoding a humanized antibody or its antigen-binding fragment.

[0076] Encoding a humanized antibody or an antigen-binding fragment thereof refers to genetic information that encodes a protein capable of synthesizing an amino acid sequence of the humanized antibody or an antigen-binding fragment thereof in the normal protein expression process of a polynucleotide (such as transcription or translation). In this case, the scope of the present invention may even include such polynucleotides: which not only encode a protein having an amino acid sequence identical to the amino acid sequence of the humanized antibody or an antigen-binding fragment thereof, but also encode a protein having an amino acid sequence substantially identical to the amino acid sequence of the above protein, or encode a protein having the same and / or similar activity as the protein.

[0077] The polynucleotide may have an optimized base sequence according to the type of organism into which the polynucleotide is to be introduced and expressed and the expression system (such as transcription or translation) of the organism.

[0078] Specifically, the polynucleotide may have the base sequence of SEQ ID NO:19 or SEQ ID NO:20.

[0079] The base sequence of SEQ ID NO:19 may encode the amino acid sequence of "Hz2B7-1.1", and the base sequence of SEQ ID NO:20 may encode the amino acid sequence of "Hz2B7-1.2".

[0080] The polynucleotide of the present invention may have a base sequence substantially identical to the base sequences listed above. Substantially identical base sequences include, for example, cases where the same amino acids can be synthesized when transcribed and translated, and may be base sequences having 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 99.5% or higher homology to the above base sequences, but are not limited thereto.

[0081] The expression vector of the present invention contains a polynucleotide.

[0082] The term "expression vector" in the present invention refers to a means for expressing a specific gene in a host cell. Specifically, the expression vector includes plasmid vectors; cosmid vectors; and viral vectors, such as phage vectors, adenovirus vectors, retroviral vectors or adeno-associated virus vectors, but are not limited thereto.

[0083] In addition to the base sequence encoding a humanized antibody or an antigen-binding fragment thereof, the expression vector may further have regulatory sequences, such as a promoter or a terminator, and the base sequence encoding a humanized antibody or an antigen-binding fragment thereof may be operably linked to the promoter. Operable linkage refers to a functional linkage between a regulatory sequence (such as a promoter, a signal sequence, an array of transcriptional regulatory factor binding sites, etc.) and another base sequence, whereby the regulatory sequence can regulate the transcription and / or translation of the other base sequence.

[0084] The expression vector system of the present invention can be constructed by various methods known in the art.

[0085] The expression vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0086] For example, when the expression vector uses a prokaryotic cell as a host, the expression vector generally contains a strong promoter capable of promoting transcription (such as the tac promoter, the lac promoter, the lacUV5 promoter, the lpp promoter, the pLλ promoter, the pRλ promoter, the rac5 promoter, the amp promoter, the recA promoter, the SP6 promoter, the trp promoter, the T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When Escherichia coli (E. coli) (such as HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the Escherichia coli tryptophan biosynthesis pathway (Yanofsky, C, J Bacteriol, (1984) 158: 1018-1024) and the left promoter of bacteriophage λ (pLλ promoter; Herskowitz, I and Hagen, D, Ann Rev Genet, (1980) 14: 399-445) can be used as regulatory sites. When a Bacillus bacterium is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl Environ Microbiol (1998) 64: 3932-3938; Mol Gen Genet (1996) 250: 734-741) or any promoter that can be expressed in Bacillus bacteria can be used as a regulatory site. The expression vector can be produced by manipulating commonly used plasmids in the art (such as pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), bacteriophages (such as λgt4·λB, λ-Charon, λΔz1, M13, etc.) or viruses (such as SV40, etc.).

[0087] In the case where a eukaryotic cell is used as a host for the expression vector, a promoter derived from the mammalian cell genome (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, or 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, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and the expression vector can generally have a polyadenylation sequence as a transcription termination sequence. The expression vector may contain the CMV promoter.

[0088] In addition, the expression vector can be fused with other sequences to facilitate the purification of the antibody expressed therefrom. Examples of the 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, considering its characteristics, the expressed protein can be easily purified by a protein A column or the like without an additional purification sequence.

[0089] The expression vector contains an antibiotic resistance gene commonly used in the art as a selection marker, and may contain, for example, resistance genes against ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0090] The expression vector can be a vector system in which the light chain and the heavy chain are expressed simultaneously in one vector, or a system in which the light chain and the heavy chain are expressed separately in different vectors. In the latter case, the two vectors can be introduced into the host cell by, for example, co-transformation or targeted transformation. Co-transformation is a method of simultaneously introducing each vector DNA encoding the light chain and the heavy chain into the host cell, and then selecting cells that express both the light chain and the heavy chain. Targeted transformation is a method of selecting cells transformed with a vector containing the light chain (or heavy chain), transforming the selected cells again with a vector containing the heavy chain (or light chain), and finally selecting cells that express both the light chain and the heavy chain.

[0091] The host cell of the present invention contains the expression vector.

[0092] As the host cell, any host cell known in the art can be used as long as it can stably and continuously clone and express the expression vector of the present invention, and examples of host cells include, but are not limited to, prokaryotic host cells such as Escherichia coli, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas such as Pseudomonas putida, Proteus mirabilis, and Staphylococcus such as Staphylococcus carnosus.

[0093] When the host cell is a eukaryotic host cell, fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, and Neurospora crassa, other lower eukaryotes, higher eukaryotes such as insect-derived cells and cells derived from plants or mammals can be used. The host cell can 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, HuT78 cells or 293 cells, but are not limited thereto.

[0094] Transformation and / or transfection into the host cell can be carried out using any method for introducing nucleic acids into an organism, cell, tissue or organ, and can be carried out by selecting appropriate standard techniques according to the host cell, as known in the art. Specifically, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, polyethylene glycol (PEG), dextran sulfate, liposome transfection, desiccation / inhibition-mediated transformation methods, etc. can be carried out, but the present invention is not limited thereto.

[0095] A method for producing the humanized antibody or antigen-binding fragment thereof of the present invention includes culturing the host cell.

[0096] The method for producing the humanized antibody or antigen-binding fragment thereof may further include expressing the humanized antibody or antigen-binding fragment thereof in the host cell.

[0097] The host cells can be cultured according to appropriate culture media and culture conditions known in the art. Those skilled in the art can easily adjust and use the culture process according to the selected strain. According to the cell growth method, cell culture is divided into suspension culture and adherent culture, and according to the culture method, it is divided into batch culture, fed-batch culture, and continuous culture. The culture medium used should appropriately meet the requirements of the specific strain.

[0098] In animal cell culture, the culture medium contains various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used 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 can be used alone or in combination.

[0099] Examples of nitrogen sources include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and soybean powder, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and these nitrogen sources can be used alone or in combination.

[0100] The culture medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts as phosphorus sources. In addition, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, suitable precursors, etc. can be included.

[0101] In the culturing step, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. In addition, during the culture, foam generation can be inhibited by using an antifoaming agent such as polyethylene glycol fatty acid ester. In addition, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture can generally be a temperature of 20°C to 45°C or 25°C to 40°C.

[0102] The production method may further include recovering the humanized antibody or its antigen-binding fragment expressed in the host cell. The humanized antibody or its antigen-binding fragment obtained by culturing the transformed host cell can be used in an unpurified state, or can be further purified to high purity using various conventional methods such as dialysis, salt precipitation, and chromatography. When using chromatography, the type and order of the column can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc. according to the characteristics of the antibody, the culture method, etc.

[0103] 3. Use of the humanized antibody of the present invention for detecting TM4SF4

[0104] Another aspect of the present invention provides the use of a humanized antibody or an 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.

[0105] Since the descriptions of the humanized antibody, its antigen-binding fragment, TM4SF4, etc. are the same as those described in "1. Humanized antibody or antigen-binding fragment thereof that specifically binds to TM4SF4", the description is omitted to avoid repetition.

[0106] The composition for detecting TM4SF4 of the present invention comprises a humanized antibody or an antigen-binding fragment thereof, and the kit for detecting TM4SF4 of the present invention comprises the composition for detecting TM4SF4.

[0107] In addition, the method for detecting TM4SF4 of the present invention includes contacting a humanized antibody or an antigen-binding fragment thereof with a sample to be detected that is expected to contain TM4SF4.

[0108] The composition for detecting TM4SF4 and the kit containing the same can effectively detect TM4SF4 by contacting a humanized antibody or an antigen-binding fragment thereof that specifically binds to TM4SF4 with the sample to be detected to form an antigen-antibody complex.

[0109] The antigen-antibody complex refers to the combination of TM4SF4 and an antibody that recognizes TM4SF4 to identify tumors or cancer cells expressing TM4SF4 in a sample.

[0110] The method for quantifying the TM4SF4 antigen using the composition for detecting TM4SF4, the kit containing the same, or a humanized antibody or an antigen-binding fragment thereof can be carried out by confirming the formation of the antigen-antibody complex, and the confirmation of the formation of the antigen-antibody complex can be carried out by enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemical staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, immunodiffusion assay, complement fixation assay, protein chip, etc., but not limited thereto. Enzyme-linked immunosorbent assay (ELISA) includes various ELISA methods. For example, direct ELISA uses a labeled antibody that recognizes an antigen attached to a solid-phase support; indirect ELISA uses a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid-phase support; direct sandwich ELISA uses another labeled antibody that recognizes an antigen in an antigen-antibody complex attached to a solid-phase support; and indirect sandwich ELISA, in which it reacts with another labeled antibody that recognizes an antigen in an antigen-antibody complex attached to a solid-phase support, and then uses a labeled secondary antibody that recognizes the other labeled antibody.

[0111] Examples of markers capable of qualitatively or quantitatively measuring the formation of antigen - antibody complexes include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes. Examples of enzymes include, but are not limited to, β - glucuronidase, β - D - glucosidase, β - D - galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, and GDP - enzyme, RNase, glucose oxidase, and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, and β - lactamase.

[0112] 4. Use of the humanized antibody of the present invention for preventing or treating cancer, for inhibiting the growth of cancer stem cells and for assisting radiotherapy for cancer treatment 5. Use of the humanized antibody of the present invention for assisting anti-cancer immunotherapy

[0113] 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 assisting radiotherapy for cancer treatment, which comprise a humanized antibody or an antigen - binding fragment thereof.

[0114] Since the descriptions of the humanized antibody, its antigen - binding fragment, TM4SF4, etc. are the same as those described in "1. Humanized antibody or its antigen - binding fragment specifically binding to TM4SF4", in order to avoid repetitive description, the description is omitted.

[0115] However, the humanized antibody or its antigen - binding fragment of the present invention may have a CDR sequence identical to the CDR sequence of the ECL - 2B7 antibody or a CDR sequence with a modified part of the CDR sequence of the ECL - 2B7 antibody, and for the characteristics and functions of the ECL - 2B7 antibody, reference can be made to Korean Patent Publication No. 2021 - 0071856.

[0116] The pharmaceutical composition for preventing or treating cancer of the present invention comprises a humanized antibody or an antigen - binding fragment thereof.

[0117] The humanized antibody or its antigen - binding fragment can bind to TM4SF4 with high affinity, and TM4SF4 is known to be overexpressed on the surface of cancer cells. Therefore, the humanized antibody or its antigen - binding fragment can be used to target cancer cells.

[0118] The humanized antibody or its antigen - binding fragment in the composition can be used alone or in combination with a conventional pharmaceutically acceptable carrier for the treatment, prevention, and diagnosis of hyperproliferative diseases such as cancer.

[0119] Cancer can be, for example, lung cancer, gastric cancer, colon cancer, colorectal cancer, triple-negative breast cancer, glioblastoma, head and neck cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, endometrial cancer, salivary gland cancer or thyroid cancer. More specifically, it can be lung cancer, breast cancer, liver cancer, kidney cancer, gastric cancer, pancreatic cancer or brain cancer, and particularly, it can be non-small cell lung cancer or pancreatic cancer, but is not limited thereto.

[0120] In the present invention, the cancer can be a cancer caused by overexpression, amplification, mutation or activation of TM4SF4, and can particularly refer to a cancer with overexpression of TM4SF4.

[0121] The composition can be in the form of a pharmaceutical composition, a quasi-pharmaceutical composition or a health food composition.

[0122] The composition for preventing or treating cancer of the present invention may further comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the substance does not inhibit the activity of the active ingredient and does not have toxicity exceeding what is adaptable for the administration (prescription) target, and "carrier" is defined as a compound that helps the compound enter cells or tissues.

[0123] The pharmaceutical composition of the present invention can be administered alone or in combination with any convenient carrier, etc., and the formulation to be administered can be a single-dose or repeated dosage form. The pharmaceutical composition can be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid preparations can contain, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. Liquid preparations include water, solutions such as propylene glycol solutions, suspensions, emulsions, etc., but are not limited thereto, and can be prepared by adding appropriate coloring agents, flavoring agents, stabilizers, thickening agents, etc. For example, a powder can be prepared by simply mixing a trihydroxy derivative of a polyunsaturated fatty acid (which is the active ingredient of the present invention) with a suitable pharmaceutically acceptable carrier (such as lactose, starch or microcrystalline cellulose). Granules can be prepared as follows: mixing a trihydroxy derivative of a polyunsaturated fatty acid of the present invention, a suitable pharmaceutically acceptable carrier and a suitable pharmaceutically acceptable binder (such as polyvinylpyrrolidone or hydroxypropyl cellulose), and then using a wet granulation method (using a solvent such as water, ethanol or isopropanol), or a dry granulation method using compressive force. In addition, tablets can be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate, and then using a tablet machine to tablet the mixture.

[0124] The pharmaceutical composition can be administered in the form of oral preparations, injections (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection or implant), inhalants, nasal administrations, vaginal administrations, rectal administrations, sublingual administrations, transdermal administrations, topical administrations, etc., depending on the disease to be treated and the individual condition, but not limited thereto. According to the administration route, the pharmaceutical composition can be formulated into appropriate dosage unit preparations, containing common and non-toxic pharmaceutically acceptable carriers, additives and excipients.

[0125] The pharmaceutical composition can be administered at a daily dose of about 0.0001 mg / kg to about 10 g / kg, and can be administered at a daily dose of about 0.001 mg / kg to about 1 g / kg. However, the dose can vary according to the purity of the mixture, the condition of the patient (age, gender, weight, etc.) and the severity of the disease being treated. If necessary, for convenience, the total daily dose can be administered in divided doses several times a day.

[0126] The composition for inhibiting the growth of cancer stem cells of the present invention comprises a humanized antibody or an antigen-binding fragment thereof.

[0127] Cancer stem cells (CSCs) refer to undifferentiated cells that have the ability to differentiate into various cancer cells. Cancer stem cells exist in malignant tumor tissues in an amount of about 1% to 2%, and have the self-renewal ability and pluripotency of normal stem cells, but have abnormal self-regulatory functions, which leads to the activation of cell division, resulting in an increase in cell number and differentiation into malignant tumor cells. Due to these characteristics of cancer stem cells, general cancer cells are removed by anti-cancer treatment, but cancer stem cells survive, and it is known that cancer recurrence and metastasis are caused by some surviving cancer stem cells.

[0128] Specifically, the cancer stem cells of the present invention can be cancer cells that overexpress aldehyde dehydrogenase 1 (ALDH1) protein (which is one of the markers of cancer stem cells) or have positive protein activity.

[0129] The humanized antibody or an antigen-binding fragment thereof of the present invention can selectively inhibit cancer stem cells, and in particular, can obtain excellent anti-cancer effects by killing cancer cell populations including cancer stem cells that are highly resistant to anti-cancer treatment. The humanized antibody or an antigen-binding fragment thereof can inhibit the growth of cancer stem cells by reducing the self-renewal ability, invasion ability and migration ability of cancer stem cells.

[0130] In a specific embodiment of the present invention, the adenocarcinoma cell line A549 in lung cancer cells and the MIAPaCa-2 cells as a pancreatic cancer cell line were treated with the humanized antibody of the present invention, and then the expression level of ALDH1 as a marker of cancer stem cell characteristics was measured. As a result, it was confirmed that the humanized antibody of the present invention inhibits the stem cell transformation of cancer cells because the expression level of the ALDH1 protein was significantly reduced.

[0131] The antibody or its antigen-binding fragment can be used for the prevention or treatment of cancers having cancer stem cell characteristics, but is not limited thereto. Cancers having cancer stem cell characteristics are resistant to existing anticancer treatments and have a poor prognosis; therefore, a treatment different from existing anticancer treatments should be applied to them. For example, even among patients with the same type of cancer, in the case where the cancer has a high proportion of cancer stem cells, the patient will not obtain a cancer treatment effect from existing known cancer treatments (such as administration of anticancer drugs or radiotherapy). Therefore, even if it is the same type of cancer, it is very important to apply a novel treatment method different from existing anticancer treatments in the case where the proportion of cancer stem cells in the cells at the cancer lesion site is high.

[0132] The above-mentioned cancers having cancer stem cell characteristics can be cancers having a high proportion of cancer stem cells in the cell population constituting the cancer. Considering that the proportion of cancer stem cells in general cancer cells is about 1% or higher and less than 5%, for example, when the proportion of cancer stem cells in the cell population constituting the cancer is 5% or higher, 10% or higher, 30% or higher, 50% or higher, or 70% or higher, the cancer can be defined as having cancer stem cell characteristics, and as described above, the characteristics of this cancer can lie in resistance to existing anticancer treatments and a poor prognosis of anticancer treatments. Specifically, in the present invention, cancers having cancer stem cell characteristics can be cancers overexpressing ALDH1. Cancers overexpressing ALDH1 can be cancers in which the proportion of cancer stem cells expressing ALDH1 or having positive activity thereof is relatively higher than that of general cancers.

[0133] Specifically, cancers overexpressing ALDH1 can be at least one 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.

[0134] The prevention or treatment of cancer can aim to prevent or treat cancer chemoresistance, cancer recurrence, or cancer metastasis by reducing the regenerative ability, growth ability, invasive ability, or migratory ability of cancer stem cells during or after cancer treatment.

[0135] In a specific embodiment of the present invention, the humanized antibody of the present invention was used to treat the adenocarcinoma cell line A549 in lung cancer cells and the MIAPaCa-2 cells as a pancreatic cancer cell line, and then the migration and invasion of the cells were observed through a transwell. As a result, it was shown that the migration and invasion of the cancer cells were reduced by about 40%, confirming that the humanized antibody of the present invention plays an important role in inhibiting cancer cell metastasis.

[0136] The composition for assisting radiotherapy in the treatment of cancer of the present invention comprises a humanized antibody or an antigen-binding fragment thereof.

[0137] The composition comprises a humanized antibody or an antigen-binding fragment thereof as an active ingredient for reducing the radioresistance of cancer-related cells.

[0138] Cancer-related cells are cells that constitute cancer and may have characteristics such as an irregular shape, unlimited proliferation, and weak binding to surrounding cells compared to normal cells. Specifically, cancer-related cells can be cancer cells or cancer stem cells, and specifically, can be cancer stem cells.

[0139] Cancer stem cells can be undifferentiated cells having the ability to differentiate into various cancer cells, and specifically, can be cancer cells expressing ALDH1 or having positive activity against ALDH1. In the present invention, cancer stem cells may have the characteristics that cell proliferation is not inhibited by radiation exposure, self-renewal ability is not reduced, and migration and invasion abilities are not inhibited.

[0140] In addition, cancer-related cells can be highly radiation-tolerant, that is, highly resistant to radiotherapy, and can have a relatively high resistance to radiation, making it impossible to use radiotherapy for cancer treatment.

[0141] Anticancer can aim to inhibit the proliferation of cancer-related cells, inhibit metastasis and invasion, and induce apoptosis through radiation exposure, surgery, and chemotherapy.

[0142] In the present invention, in cancer treatment, a humanized antibody or an antigen-binding fragment thereof can be administered in combination with radiation exposure. Therefore, when a humanized antibody or an antigen-binding fragment is administered in combination with radiation exposure, the radiation resistance of cancer-related cells is inhibited by the antibody or antigen-binding fragment, so that the anticancer treatment effect can be maximized by radiation exposure, and the recurrence and metastasis of cancer can be further prevented.

[0143] In a specific embodiment of the present invention, the adenocarcinoma cell line A549 in lung cancer cells and the MIAPaCa-2 cells as a pancreatic cancer cell line were treated with the humanized antibody of the present invention, and then the radioresistance of cancer cells was measured by analyzing colony formation. As a result, when treated with the humanized antibody of the present invention, it was confirmed that the number of colonies decreased after radiation exposure, resulting in the inhibition of cell growth. Therefore, it was confirmed that the humanized antibody of the present invention has the effect of reducing the radioresistance of cancer cells.

[0144] Confirmation of cell growth according to treatment of lung cancer cells and pancreatic cancer cells with anti-TM4SF4 humanized antibody

[0145] Another aspect of the present invention provides a composition for assisting anti-cancer immunotherapy, which comprises a humanized antibody or an antigen-binding fragment thereof.

[0146] Since the descriptions of the humanized antibody, its antigen-binding fragment, TM4SF4, etc. are the same as those described in "1. Humanized antibody or its antigen-binding fragment specifically binding to TM4SF4", in order to avoid repeated descriptions, the description is omitted.

[0147] Similarly, since the descriptions of the composition, anti-cancer, etc. are the same as those described in "4. Use of the humanized antibody of the present invention for preventing or treating cancer, for inhibiting the growth of cancer stem cells and for assisting radio-anti-cancer treatment", in order to avoid repeated descriptions, the description is omitted.

[0148] Assisting anti-cancer immunotherapy means that it can promote or assist immune cells to eliminate cancer cells by enhancing the immune system of cancer patients and inhibiting the immune escape mechanism of cancer cells. Specifically, it can enhance immune activity by enhancing antibody-dependent cell cytotoxicity (ADCC), inhibiting the expression of programmed death ligand (PD-L1), or inhibiting the expression of B7H4.

[0149] ADCC refers to the process by which immune cells lyse target cells that bind to specific antibodies on their cell surfaces, and requires immune cells called natural killer (NK) cells to bind to the antibodies. When the humanized antibody or its antigen-binding fragment of the present invention is treated, the humanized antibody or its antigen-binding fragment binds to cancer cells, and due to the binding, the antibody-dependent cell cytotoxicity function of NK cells can be enhanced.

[0150] In a specific embodiment of the present invention, the humanized antibody of the present invention was treated with hepatocytes (as a control) and A549 cells of the adenocarcinoma cell line in lung cancer cells and MIAPaCa-2 cells of the pancreatic cancer cell line, and then the antibody-dependent cell cytotoxicity (ADCC) function was confirmed. As a result, it was confirmed that ADCC was not observed in the control, while obvious ADCC was observed in cancer cells.

[0151] PD-L1 refers to the ligand of the immune checkpoint receptor of cancer cells, which causes the immune escape mechanism of cancer cells against immune cells, thereby preventing immune cells from eliminating cancer cells. When treated with the humanized antibody or its antigen-binding fragment of the present invention, the expression of the PD-L1 gene or protein can be inhibited.

[0152] B7-H4 is a negative regulator of T cell function, and the ligation of T cells can inhibit its growth, cytokine secretion, and cytotoxicity. B7-H4 is known to be overexpressed in various solid tumors, and the expression of B7-H4 in tumors is known to be associated with poor prognosis. In particular, B7-H4 directly inhibits T cell activity, which leads to the immune escape mechanism of cancer cells against immune cells, thereby preventing immune cells from eliminating cancer cells. When treated with the humanized antibody or its antigen-binding fragment of the present invention, the expression of the B7-H4 gene or protein can be inhibited.

[0153] In a specific embodiment of the present invention, the A549 cells of the adenocarcinoma cell line and the MIAPaCa-2 cells of the pancreatic cancer cell line in lung cancer cells were treated with the humanized antibody of the present invention, and then the changes in the expression levels of the PD-L1 and B7-H4 genes and proteins were measured. As a result, it was confirmed that the expressions of the PD-L1 and B7-H4 genes and proteins were significantly reduced.

[0154] Therefore, the humanized antibody and its antigen-binding fragment of the present invention have the effects of enhancing the ADCC effect of immune cells and inhibiting the expressions of PD-L1 and B7-H4, thereby enhancing the immune system of cancer patients and promoting immune cells to eliminate cancer cells by inhibiting the immune escape mechanism of cancer cells.

[0155] Hereinafter, the present invention will be described in detail with reference to the examples.

[0156] However, the following examples are intended to specifically illustrate the present invention, and the present invention is not limited by the following examples.

[0157] [Example 1]

[0158] Figure 1

[0159] Confirm whether the humanized antibody of the present invention reduces the growth of cancer cells in the A549 of the adenocarcinoma cell line and the MIAPaCa-2 of the pancreatic cancer cell line in lung cancer cells.

[0160] First, a protease inhibitor mixture (Sigma-Aldrich, St Louis, UAS) was mixed with TX100 lysis buffer [20 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, and 0.5% Triton X-100], and cancer cells were treated with the mixed solution.

[0161] Protein concentration was measured by Western blotting using Bradford reagent (Bio-Rad, Hercules, CA, USA). For Western blotting assay, equal amounts of protein were separated on an 8 - 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a Hybond nitrocellulose membrane (Amersham Pharmacia, Pittsburgh, PA, USA). The membrane was blocked with a phosphate-buffered saline (PBS) buffer containing skim milk (10%) and Tween 20 (0.1%) at room temperature for 1 hour. In the membrane, TM4SF4 and β-actin were treated with antibodies in a cold box overnight. Thereafter, the membrane was washed with Tris-buffered saline solution, treated with a peroxidase-labeled secondary antibody (Abcam), and then proteins were visualized using the Westzol enhanced chemiluminescence detection kit (iNtRON Biotechnolgy).

[0162] The TM4SF4 protein in A549, H460, and THLE-2 cells was subjected to Western blotting using the same method as described above. As a result, as Figure 1 shown in Figure 1 A, it was confirmed that TM4SF4 was expressed in A549 cancer cells but not in H460 and THLE-2 cells (

[0163] A). 3 Two × 10 3 A549 cells expressing TM4SF4 and two × 10

[0164] MIAPaCa-2 cells were cultured in 60-mm cell culture dishes and treated with the anti-TM4SF4 humanized antibody (Hz-2B7-1.2) of the present invention at concentrations of 1 μg / ml, 5 μg / ml, and 10 μg / ml, respectively. Five days after the treatment, A549 and MIAPaCa-2 cells were stained with a 0.5% (w / v) crystal violet solution, and colonies were observed and counted. The colony survival rate was expressed as a percentage (%) relative to the control. An IgG antibody was used as the antibody for the treatment control group. Figure 1 B and Confirmation of radioresistance in lung cancer cells and pancreatic cancer cells C). Thus, it was confirmed that the membrane protein TM4SF4 plays an important role in the growth of adenocarcinoma cell lines and pancreatic cancer cell lines in non-small cell lung cancer, and when TM4SF4 binds to the anti-TM4SF4 humanized antibody, the growth ability of cells is significantly reduced.

[0165] [Example 2]

[0166] Figure 2

[0167] To confirm whether cell growth would reduce radioresistance when treating adenocarcinoma cell line A549 and pancreatic cancer cell line MIAPaCa-2 cells with the humanized antibody of the present invention, the cancer cell lines were treated with the humanized antibody (Hz-2B7-1.2) of the present invention, and then the colony staining experiment was carried out using the same method as described in Example 1. At this time, the treatment with the humanized antibody was carried out before radiation (3 Gy) exposure.

[0168] As a result, when treating A549 cells of the adenocarcinoma cell line in non-small cell lung cancer and MIAPaCa-2 cells of the pancreatic cancer cell line with the humanized antibody of the present invention, both the number of cultured colonies and the number of colonies cultured after radiation exposure decreased, confirming a reduction in cell growth ( Confirmation of cell migration by wound healing in lung cancer cells and pancreatic cancer cells ). Therefore, it can be confirmed that when inhibiting the function of TM4SF4 by treating A549 and MIAPaCa-2 cells expressing TM4SF4 with the humanized antibody of the present invention, the radioresistance is reduced.

[0169] [Example 3]

[0170] Figure 3

[0171] This experiment was carried out to determine whether the treatment with the humanized antibody of the present invention not only affects cell growth but also affects cell migration ability. A549 and MiaPaCa-2 cells were treated with the humanized antibody (Hz-2B7-1.2) of the present invention. After 72 hours, A549 and MiaPaCa-2 cells were transferred to a 35 mm culture dish, cultured until the confluence rate was 90% or higher, and washed thoroughly with PBS. The medium was replaced with RPMI 1640 medium containing 0.5% FBS, and the cells were cultured overnight at 5% CO2 and 37 °C to induce inhibition of the function of TM4SF4. The bottom of the culture dish was scratched to wound the cells by drawing two lines using a 200 μl pipette tip, the culture dish was slowly washed again with PBS and refilled with cell medium, and the cells were cultured and observed after 36 hours. The change in the spacing between the wounds was expressed as the relative wound area.

[0172] As a result, as Confirmation of cell migration and invasion by transwell in lung cancer cells and pancreatic cancer cells shown, it was confirmed that the migration of A549 and MiaPaCa-2 cells treated with the humanized antibody of the present invention was significantly reduced compared to the control. In the control group, due to increased cell migration for up to 24 hours, the relative wound area decreased to 50% or less. Therefore, it was confirmed that the continuous cell migration was inhibited by treating the adenocarcinoma cell line A549 and the pancreatic cancer cell line MiaPaCa-2 with the humanized antibody of the present invention.

[0173] [Example 4]

[0174] 4-1. Confirmation of reduced cell migration in A549 and MIAPaCa-2 cells treated with the humanized antibody of the present invention

[0175] Using a transwell plate, changes in cell migration and cell invasion ability were confirmed according to the treatment with the humanized antibody of the present invention.

[0176] Figure 4

[0177] To observe cell migration, A549 and MIAPaCa-2 cells were treated with the humanized antibody (Hz-2B7-1.2) of the present invention in the same manner as described in Example 3 to induce functional inhibition of TM4SF4. After 72 hours, A549 and MIAPaCa-2 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Hyclone) and antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin, Hyclone), and introduced into the wells of a transwell plate (Cellbiolabs) at a density of 2×10 5 cells / 300 μl. 500 μl of RPMI 1640 medium without fetal bovine serum was added to the lower chamber, and the cells were cultured at 5% CO2 and 37 °C for 24 hours. After the culture was completed, the cells that had migrated to the lower surface of the transwell plate were stained with CyQuant TM GR staining reagent, and the number of stained cells was observed.

[0178] As a result, as 4-2. Confirmation of reduced cell invasion in A549 and MIAPaCa-2 cells treated with the humanized antibody of the present invention shown, it was confirmed that the migration of cells treated with the humanized antibody of the present invention in the adenocarcinoma cell lines A549 and MIAPaCa-2 was reduced by approximately 40% compared to the control.

[0179] Figure 4

[0180] To measure cell invasion, A549 and MIAPaCa-2 cells were treated with the humanized antibody (Hz-2B7-1.2) of the present invention in the same manner as in Example 3 to induce functional inhibition of TM4SF4. After 72 hours, 5×10 5 A549 and MIAPaCa-2 cells were prepared, coated with 10 μl of Matrigel TM on the upper surface of the transwell plate and dried, and then the cells were cultured in RPMI 1640 medium without fetal bovine serum at room temperature for 30 minutes. Thereafter, an experiment was performed using the transwell plate in the same manner as the cell migration measurement method described in Example 4-1. After the culture was completed, the upper surface of the transwell plate was removed with a cotton swab to observe the invading cells, the cells were stained with CyQuant TM GR staining reagent, and the number of stained cells was observed.

[0181] As a result, as Confirmation of cell sphere formation in lung cancer cells and pancreatic cancer cells shown, cell invasion in the adenocarcinoma cell lines A549 and MIAPaCa-2 cells was reduced by approximately 40%. Therefore, it was confirmed that the humanized antibody of the present invention has the effect of inhibiting cancer cell metastasis by reducing cell migration and invasion.

[0182] [Example 5]

[0183] Figure 5

[0184] After treatment with the humanized antibody (Hz-2B7-1.2) of the present invention, sphere formation assays were performed on A549 and MiaPaCa-2 cells. During the sphere formation assay, A549 and MiaPaCa-2 cells were added to stem cell-permissive Dulbecco's Modified Eagle Medium (DMEM-F12; Invitrogen), which contained epidermal growth factor (FGF, 20 ng / ml), basic fibroblast growth factor (20 ng / ml), and B27 serum. The cells were seeded at a density of 1 cell / well or 2 cells / well in an ultra-low attachment 96-well plate (Corning, Inc., Corning, NY, USA) and incubated overnight in a humidified incubator at 37 °C and 5% CO2. Thereafter, the presence of single cells in each well was visually confirmed, and the spheres were quantified using an inverted phase contrast microscope after 9 to 12 days. At the same time, approximately 2×10 3 cells were seeded into a 6-well plate under the above culture conditions, cultured in a humidified incubator at 5% CO2, and the number of spheres formed was quantified.

[0185] As a result, as Confirmation of whether epithelial-mesenchymal transition (EMT) in lung cancer cells and pancreatic cancer cells is inhibited shown, it was confirmed that sphere formation was significantly inhibited when treated with the humanized antibody of the present invention compared to treatment with an IgG antibody as a control. Therefore, it was confirmed that the inhibition of TM4SF4 function by treatment with the humanized antibody of the present invention plays an important role in inhibiting cancer stem cell properties.

[0186] [Example 6]

[0187] Figure 6

[0188] It was investigated whether the humanized antibody of the present invention inhibits the EMT phenomenon. Specifically, when A549 and MIAPaCa-2 cells were treated with the humanized antibody (Hz-2B7-1.2) of the present invention, changes in the protein expression levels of EMT markers such as E-cadherin, N-cadherin, vimentin, Snail, and β-actin were investigated by Western blotting and immunofluorescence.

[0189] The results of Western blotting and immunofluorescence experiments confirmed that after treatment with the humanized antibody of the present invention, the expression of E-cadherin increased and the expressions of N-cadherin and vimentin decreased ( Figure 7 and Confirmation of ALDH1 and CD44 expression in lung cancer cells and pancreatic cancer cells ). Therefore, this means that the humanized antibody of the present invention effectively inhibits the EMT phenomenon and effectively controls cancer.

[0190] [Example 7]

[0191] Figure 8

[0192] To confirm whether the humanized antibody (Hz-2B7-1.2) of the present invention reduces the expressions of representative markers ALDH1 and CD44 of cancer cell stemness, the changes in the expression levels were studied by immunofluorescence assay.

[0193] In the same manner as in the above example, treatment with the TM4SF4 humanized antibody was performed to induce functional inhibition of TM4SF4 in A549 and MIAPaCa-2 cells. After 72 hours, after treatment with antibodies labeled with fluorescence factors of each antibody against ALDH1 and CD44, the expression levels were confirmed by fluorescence microscopy.

[0194] As a result, as Confirmation of antibody-dependent cell cytotoxicity (ADCC) function in lung cancer cells and pancreatic cancer cells shown, it was confirmed that when treated with the humanized antibody of the present invention, due to the inhibition of TM4SF4 function, the intracellular expression levels of ALDH1 and CD44 were significantly reduced. That is, it was confirmed that the humanized antibody of the present invention is involved in regulating the expressions of ALDH1 and CD44, which are intracellular cancer stemness regulators.

[0195] [Example 8]

[0196] Figure 9

[0197] The ADCC assay was performed using an ADCC reporter gene bioassay, V variant and core kit (Catalog No. G7010, Promega). A549 cells, MIAPaCa-2 cells, and hepatocyte THLE cells were used as target cells, respectively. The target cells were seeded into white 96-well plates and incubated at 37 °C for 20 to 24 hours before the assay. The next day, the medium was removed from each well and 25 μl of ADCC assay buffer was added. Serial dilutions of the humanized antibody (Hz-2B7-1.2) of the present invention were prepared in a V-bottom 96-well plate and incubated at room temperature. 25 μl of the diluted antibody was seeded into the white 96-well plate. As the ADCC bioassay effector cells (CPM), cryopreserved cells that could be thawed, proliferated, and stored long-term were used as effector cells, and 25 μl of effector cells was added to the target cells (effector:target ratio was 12:1), and incubated at 37 °C for 6 hours. After 6 hours, the assay plate was incubated at room temperature (20 to 22 °C) for 15 minutes. Thereafter, a luciferase assay was performed to visualize cytotoxicity. 75 μl of Bio-Glo TM luciferase assay reagent was suspended in each sample (well) and incubated at room temperature for 5 to 30 minutes. The luciferase activity was measured using ONE-Glo luciferase assay reagent (Promega) and GLOMAX TM 96 microplate photometer (Promega). The luciferase activity was normalized with anti-IgG.

[0198] As a result, as Confirmation of programmed death ligand 1 (PD-L1) and B7H4 expression in lung cancer cells and pancreatic cancer cells shown, antibody-dependent cytotoxicity was clearly observed in A549 and MIAPaCa-2 cells, while no antibody-dependent cytotoxicity was observed in hepatocyte THLE cells used as a control. Therefore, it was confirmed that the humanized antibody of the present invention did not act on normal hepatocytes, but specifically acted on cancer cells, thereby enhancing cytotoxicity.

[0199] [Example 9]

[0200] Figure 10

[0201] Adenocarcinoma cell line A549 cells and pancreatic cancer cell line MIAPaCa-2 cells were treated with the humanized antibody (Hz-2B7-1.2) of the present invention, and then the changes in the protein levels of PD-L1 and B7H4 (PD-L1 and B7H4 are ligands of immune checkpoint receptors for immune escape present in cancer cells) were confirmed by Western blotting in the same manner as in Example 7. In addition, the changes in the gene expression of PD-L1 and B7H4 were confirmed using RT-PCR method.

[0202] As a result, as ​As shown, it was confirmed that the expression of PD-L1 and B7H4 proteins and genes in A549 and MIAPaCa-2 cells was significantly reduced according to the treatment with the humanized antibody of the present invention. This means that the humanized antibody of the present invention functions as an immune anti-cancer antibody.

Claims

1. A humanized antibody or an antigen-binding fragment thereof, comprising: A heavy chain variable region, the 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, FR-H3 having the amino acid sequence of SEQ ID NO:3, and FR-H4 having the amino acid sequence of SEQ ID NO:4; and A light chain variable region, the light chain variable region comprising: FR-L1 having the amino acid sequence of SEQ ID NO:5, FR-L2 having the amino acid sequence of SEQ ID NO:6, FR-L3 having the amino acid sequence of SEQ ID NO:7, and FR-L4 having the amino acid sequence of SEQ ID NO:8, Among them, The humanized antibody or an antigen-binding fragment thereof specifically binds to transmembrane 4 superfamily member 4 (TM4SF4).

2. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein, 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:9, CDR-H2 having the amino acid sequence of SEQ ID NO:10, and CDR-H3 having the amino acid sequence of SEQ ID NO:11, and 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:12 or SEQ ID NO:13, CDR-L2 having the amino acid sequence of SEQ ID NO:14, and CDR-L3 having the amino acid sequence of SEQ ID NO:

15.

3. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein, The heavy chain variable region has the amino acid sequence of SEQ ID NO:16, and The light chain variable region has the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:

18.

4. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein, The humanized antibody or an antigen-binding fragment thereof has an amino acid sequence in which the 31st amino acid asparagine in CDR1 of the light chain variable region is substituted with phenylalanine.

5. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The antigen-binding fragment is any one selected from the group consisting of Fab, F(ab′), F(ab′)2, and Fv.

6. A polynucleotide comprising a base sequence encoding the humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

7. An expression vector, the expression vector comprising the polynucleotide according to claim 6.

8. A host cell, the host cell comprising the expression vector according to claim 7.

9. A method for producing a humanized antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 8.

10. A composition for detecting TM4SF4, the composition comprising the humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

11. A kit for detecting TM4SF4, the kit comprising the composition for detecting TM4SF4 according to claim 10.

12. A method for detecting TM4SF4, the method comprising contacting a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4 with a sample to be tested expected to contain TM4SF4.

13. A pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

14. The pharmaceutical composition according to claim 13, wherein, The prevention or treatment of the cancer is the prevention or treatment of at least one selected from the group consisting of cancer chemoresistance during cancer treatment, cancer chemoresistance after cancer treatment, cancer recurrence, and cancer metastasis.

15. The pharmaceutical composition according to claim 13, wherein, The cancer is at least one selected from the group consisting of lung cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, multiple myeloma, acute myelocytic leukemia, malignant lymphoma, thymic carcinoma, osteosarcoma, fibroid tumor, and brain cancer.

16. The pharmaceutical composition according to claim 13, wherein, The cancer is non-small cell lung cancer or pancreatic cancer.

17. A composition for inhibiting the growth of cancer stem cells, the composition comprising a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

18. A composition for assisting radiochemotherapy for cancer, the composition comprising a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

19. The composition according to claim 18, wherein, The humanized antibody or an antigen-binding fragment thereof enhances the sensitivity of cancer cells including cancer stem cells to radiation.

20. A composition for assisting cancer immunotherapy, the composition comprising a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

21. The composition according to claim 20, wherein, The humanized antibody or an antigen-binding fragment thereof enhances immune activity by inhibiting the expression of PD-L1 or B7H4.

22. The composition according to claim 20, wherein, The humanized antibody or an antigen-binding fragment thereof enhances antibody-dependent cellular cytotoxicity (ADCC) by reacting with natural killer cells (NK cells).

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