Use of claudin-3 as a target for the diagnosis and treatment of small cell lung cancer

Abstract

This invention relates to the use of claudin-3 as a target for the treatment of small cell lung cancer, and more particularly to a small cell lung cancer prophylactic or therapeutic composition containing an anti-claudin-3 specific antibody as an active ingredient with reduced side effects. Since the composition according to the present invention does not exhibit ADCC activity in normal cells compared to small cell lung cancer cells, it can be usefully used in the development of small cell lung cancer-specific therapeutic drugs with fewer side effects.

Description

[Technical Field]

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2023-0077110, filed on 15 June 2023, and Republic of Korea Patent Application No. 10-2024-0002208, filed on 5 January 2024, and the entire above specification is a reference to this application. The present invention relates to the use of claudin-3 as a target for the diagnosis and treatment of small cell lung cancer, and more particularly to a small cell lung cancer prophylactic or therapeutic composition comprising an anti-claudin-3 specific antibody as an active ingredient with reduced side effects. [Background technology]

[0002] Lung cancer is a major cause of cancer death worldwide, accounting for one-quarter of all cancer deaths, and the overall five-year survival rate for lung cancer patients remains extremely low, at only around 15%. Lung cancer is classified into two subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer, with small cell lung cancer accounting for approximately 13% of all lung cancers. Small cell lung cancer, in particular, has limited treatment options, frequently experiences rapid recurrence and metastasis after treatment, and is recognized as the most deadly type of lung cancer, with an overall five-year survival rate of only 7% for small cell lung cancer patients, indicating a very poor prognosis.

[0003] Treatment for small cell lung cancer typically involves platinum-based chemotherapy, etoposide, and checkpoint inhibitors (such as atezolizumab or durvalumab) as first-line treatments. More recently, lurbinectedin was approved by the US FDA in 2020 as a treatment for small cell lung cancer.

[0004] However, such treatment methods are also used to treat other cancers and cannot be considered specific to small cell lung cancer. In the case of lurbectedin, various side effects are known, including leukopenia, lymphopenia, fatigue, anemia, dyspnea, vomiting, and cough. Therefore, there is an urgent need to develop new therapeutic drugs that have fewer side effects and are specific to small cell lung cancer.

[0005] Furthermore, small cell lung cancer (SLC) is diagnosed through a medical history and physical examination, imaging tests such as chest X-ray, CT, PET, and MRI, cytological and pathological examinations via biopsy, and measurements of blood tumor markers such as NSE and ProGRP. While these diagnostic methods confirm the presence or absence of SLC, determine the stage, and establish an appropriate treatment plan, there is a lack of diagnostic methods for specific treatment targets for SLC patients. Therefore, the discovery of new diagnostic biomarkers for targeted therapy for SLC patients is urgently needed.

[0006] On the other hand, claudin proteins are important structural and functional components of epithelial tight junctions located between two adjacent cells. They regulate cell-cell permeability, maintain ion homeostasis, and support cell adhesion and polarity. Claudin proteins are 22 to 27 kDa transmembrane proteins that multimerize within or across the cell membrane to form a protective barrier. To date, 24 types of claudin proteins have been reported, and it has been confirmed that these claudin proteins have various expression patterns in various organs. In particular, because claudin proteins are located in tight junctions, they are not exposed to the outside and are maintained between the membranes in normal cells. However, in cancer cells, which lose cellular identity and overgrow during the tumorigenetic process, the tight junction is lost, and claudin proteins are exposed, acting as cancer markers. This has led to high interest in them as cancer-specific markers or targets for therapeutic drugs. [Overview of the project] [Problems that the invention aims to solve]

[0007] As a result of the inventors' efforts to develop a small cell lung cancer-specific therapeutic agent with fewer side effects, it was confirmed that when targeting the claudin-3 protein, the antibody-dependent cellular cytotoxicity (ADCC) activity is low against normal cells, but high only against small cell lung cancer cells. It was also confirmed that claudin-3 is significantly highly expressed in small cell lung cancer patients, and the present invention was completed by confirming its potential as a diagnostic biomarker for small cell lung cancer patients.

[0008] Therefore, an object of the present invention is to provide a composition for preventing or treating small cell lung cancer with reduced side effects, comprising an anti-claudin-3 specific antibody as an active ingredient.

[0009] Another object of the present invention is to provide a composition for preventing or treating small cell lung cancer with reduced side effects, consisting of an anti-claudin-3 specific antibody.

[0010] Still another object of the present invention is to provide a composition for preventing or treating small cell lung cancer with reduced side effects, consisting essentially of an anti-claudin-3 specific antibody.

[0011] Still another object of the present invention is to provide a method for providing information for diagnosing small cell lung cancer, comprising the step of measuring the protein level of claudin-3 from a biological sample isolated from a subject.

[0012] Still another object of the present invention is to provide the use of an anti-claudin-3 specific antibody for manufacturing a composition for treating small cell lung cancer with reduced side effects.

[0013] Still another object of the present invention is to provide a method for treating small cell lung cancer with reduced side effects, comprising administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to an individual who needs it.

[0014] Still another object of the present invention is to provide a method for treating small cell lung cancer, which comprises administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to a patient with small cell lung cancer who has been administered a chemotherapy drug.

Means for Solving the Problems

[0015] In order to achieve the above object, the present invention provides a composition for preventing or treating small cell lung cancer with reduced side effects, which contains an anti-claudin-3 specific antibody as an active ingredient.

[0016] In order to achieve another object of the present invention, the present invention provides a composition for preventing or treating small cell lung cancer with reduced side effects, which consists of an anti-claudin-3 specific antibody.

[0017] In order to achieve still another object of the present invention, the present invention provides a composition for preventing or treating small cell lung cancer with reduced side effects, which is essentially composed of an anti-claudin-3 specific antibody.

[0018] In order to achieve still another object of the present invention, the present invention provides a method for providing information for diagnosing small cell lung cancer, which includes measuring the level of claudin-3 protein from a sample of a patient suspected of having small cell lung cancer.

[0019] In order to achieve still another object of the present invention, the present invention provides the use of an anti-claudin-3 specific antibody for producing a composition for treating small cell lung cancer with reduced side effects.

[0020] In order to achieve still another object of the present invention, the present invention provides a method for treating small cell lung cancer with reduced side effects, which includes administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to an individual who needs it.

[0021] To achieve yet another objective of the present invention, the present invention provides a method for treating small cell lung cancer, comprising administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to a small cell lung cancer patient who has been administered a chemotherapy drug. [Effects of the Invention]

[0022] This invention relates to a composition for the prevention or treatment of small cell lung cancer with reduced side effects, comprising an anti-claudin-3 specific antibody as an active ingredient. When targeting claudin-3, the ADCC activity of the antibody is low in normal cells but remarkably high in cancer cells, making it useful for developing small cell lung cancer-specific therapeutic agents with fewer side effects. [Brief explanation of the drawing]

[0023] [Figure 1a] Figure 1a shows that claudin-3 mRNA is expressed at the highest rate in small cell lung cancer among various cancer types. [Figure 1b] Figure 1b shows that claudin-3 mRNA is expressed at the highest rate in ovarian cancer among various cancer types. [Figure 1c] Figure 1c shows that claudin-3 mRNA is expressed at the highest rate in non-small cell lung cancer among various cancer types. [Figure 2] Figure 2 shows the results of the analysis of the correlation between claudin-3 protein and mRNA levels. [Figure 3] Figure 3 shows the results of flow cytometry analysis of the expression rate of claudin-3 protein in small cell lung cancer cell lines. [Figure 4a] Figure 4a shows the results of immunochemical staining analysis of the expression rate of claudin-3 protein in claudin-3-positive small cell lung cancer cell lines. [Figure 4b]Figure 4b shows the results of immunochemical staining analysis of the expression rate of claudin-3 protein in claudin-3-positive small cell lung cancer cell lines. [Figure 4c] Figure 4c shows the results of immunochemical staining analysis of the expression rate of claudin-3 protein in claudin-3-positive small cell lung cancer cell lines. [Figure 4d] Figure 4d shows the results of immunochemical staining analysis of the expression rate of claudin-3 protein in claudin-3-positive small cell lung cancer cell lines. [Figure 5a] Figure 5a shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5b] Figure 5b shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5c] Figure 5c shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5d] Figure 5d shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5e] Figure 5e shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5f] Figure 5f shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5g]Figure 5g shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5h] Figure 5h shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 5i] Figure 5i shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in a claudin-3-positive small cell lung cancer cell line using the LDH method. [Figure 6a] Figure 6a shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in claudin-3-positive normal cell lines using the LDH method. [Figure 6b] Figure 6b shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in claudin-3-positive normal cell lines using the LDH method. [Figure 6c] Figure 6c shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in claudin-3-positive normal cell lines using the LDH method. [Figure 6d] Figure 6d shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in claudin-3-positive normal cell lines using the LDH method. [Figure 6e] Figure 6e shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in claudin-3-positive normal cell lines using the LDH method. [Figure 6f] Figure 6f shows the results of confirming the anticancer effect of a claudin-3-targeted monoclonal antibody in claudin-3-positive normal cell lines using the LDH method. [Figure 7]Figure 7 shows the results of comparing the expression levels of claudin-3 and β-actin when small cell lung cancer cell lines were treated with the chemotherapy drugs etoposide, cisplatin, and carboplatin, respectively. [Figure 8a] Figure 8a shows the results of confirming the anticancer effect of a claudin-3-targeted CD3 biantibody in a claudin-3-positive small cell lung cancer cell line using the WST method. [Figure 8b] Figure 8b shows the results of confirming the anticancer effect of a claudin-3-targeted CD3 biantibody in a claudin-3-positive small cell lung cancer cell line using the WST method. [Figure 8c] Figure 8c shows the results of confirming the anticancer effect of a claudin-3-targeted CD3 biantibody in a claudin-3-positive small cell lung cancer cell line using the WST method. [Modes for carrying out the invention]

[0024] The present invention will be described in detail below.

[0025] The present invention provides a composition for the prevention or treatment of small cell lung cancer, comprising an anti-claudin-3 specific antibody as an active ingredient, with reduced side effects.

[0026] In this specification, the term "comprising" is used synonymously with "including" or "characterized by," and does not exclude additional components or steps of the method that are not specifically mentioned in the compositions or methods according to the present invention. The term "consisting of" means excluding additional elements, steps, or components that are not separately described. The term "essentially consisting of" means that, within the scope of the composition or method, it may include substances or steps that do not substantially affect its basic properties along with the substances or steps described.

[0027] While continuing research on small cell lung cancer, the inventors confirmed that claudin-3 mRNA and protein are expressed at significantly high levels in small cell lung cancer cell lines, thereby confirming that claudin-3 protein is a diagnostic and therapeutic biomarker in small cell lung cancer. Using an antibody targeting claudin-3 protein, the inventors confirmed ADCC (Antibody-dependent cellular cytotoxicity) activity against small cell lung cancer. This confirmed effective anticancer activity, particularly the fact that while ADCC activity is not readily apparent in normal cells even when claudin-3 is expressed, ADCC activity is readily apparent in small cell lung cancer even at low levels of claudin-3 expression. Specifically, according to one embodiment of the present invention, when cancer cell lines expressing claudin-3 and normal cell lines were treated with a high dose of anti-claudin-3 antibody, 30% to 80% ADCC activity was observed in cancer cells, but normal cell lines showed ADCC activity of 7.9% or less, or no ADCC cytotoxicity was observed. Based on these points, it appears that the antibody does not exhibit cytotoxicity to normal tissue cells.

[0028] This fact suggests that claudin-3 has potential as a diagnostic biomarker in small cell lung cancer, and that antibodies specific to anti-claudin-3 protein may be used as a treatment for small cell lung cancer with fewer side effects. In other words, cancer cells are characterized by rapid proliferation and division, and most anticancer drugs are designed to act on rapidly proliferating cells. However, there are cells in the body that, despite being normal cells, exhibit characteristics of rapid proliferation, much like cancer cells. These include gastrointestinal mucosal cells, bone marrow cells, hair follicle cells, oral epithelial cells, and germ cells. These cells, despite being normal, are affected and destroyed by anticancer drugs, ultimately leading to side effects such as leukopenia, lymphopenia, fatigue, anemia, dyspnea, vomiting, stomatitis, cough, and / or reproductive dysfunction. Unlike such anticancer drugs, the composition containing the anti-claudin-3 specific antibody as an active ingredient according to the present invention has the advantage of significantly reducing side effects, as ADCC activity does not appear if any cell in the body expresses claudin-3, as long as the cell in question is a normal cell, and ADCC activity is only expressed against small cell lung cancer cells that express claudin-3.

[0029] Due to these advantages, the compositions according to the present invention can be administered in much larger quantities compared to the known dosages / dosages of existing anticancer drugs, thereby achieving a relatively higher anticancer effect compared to existing anticancer drugs. For example, among monoclonal antibody therapies known in the industry, if the drug dose containing the antibody against a target expressed in normal cells is 375 mg / m², then Rituximab is 375 mg / m². 2 (Non-Hodgkin lymphoma); trastuzuamb is 8 mg / kg (approximately 240 mg / m²). 2 ) After initial loading, 6 mg / kg (approximately 180 mg / m²) 2 (Breast cancer, stomach cancer); bevacizumab is 5 mg / kg (approximately 150 mg / m²). 2 ) or 10 mg / kg (approximately 300 mg / m²) 2 (Metastatic colorectal cancer), 15 mg / kg (approximately 450 mg / m²) 2)(Non-small cell lung cancer), 10 mg / kg (about 300 mg / m 2 )(Renal cell cancer); cetuximab is 400 mg / m 2 After the initial loading, 250 mg / m 2 (Metastatic colorectal cancer, head and neck squamous cell cancer). In the case of a monoclonal antibody drug targeting Claudin-18.2, the clinical phase II dosage is 800 mg / m for zolbetuximab 2 After the initial loading, 600 mg / m 2 ; 10 mg / kg (about 300 mg / m) for TST001 2 ; 800 mg / m for ASKB589 2 ; 20 mg / kg (about 800 mg / m) for BIL93 2 ) are respectively set to such levels. Considering this fact, the composition according to the present invention is 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , 1100 mg / m 2 , 1200 mg / m 2 Or, 500 mg / m 2 to 1200 mg / m 2 , 600 mg / m 2 to 1200 mg / m 2 , 700 mg / m 2 to 1200 mg / m 2 , 800 mg / m 2 to 1200 mg / m 2 , 900 mg / m 2 to 1200 mg / m 2 , 1000 mg / m 2 to 1200 mg / m 2 , 1100 mg / m 2 to 1200 mg / m 2While it can be administered in higher doses, it is not limited thereto. Due to the low side effect characteristics of the composition according to the present invention, ordinary technicians can appropriately select and administer the composition according to the present invention in doses higher than those of monoclonal antibody drugs targeting claudin that are commonly known in the industry, thereby obtaining superior anticancer effects compared to other monoclonal antibody drugs.

[0030] In this invention, the "claudin-3 (CLDN3)" protein is a protein belonging to the claudin family that is located at the site where tight junctions occur and plays a unique role in removing intercellular space during tight junctions. Tight junctions are rigid structures that connect adjacent cell membranes in the tissues of organisms such as animals. Claudin-3 regulates the intercellular permeability of small solutes such as ions, has a molecular weight of 20-34 kDa, and is known as the most important backbone protein in tight junctions, possessing two extracellular loops and four transmembrane domains. The above-mentioned claudin-3 protein is also known to function as a barrier-forming protein, such as in the blood-brain barrier and the intestinal barrier, and is involved in regulating cell proliferation, migration, differentiation, and polarity.

[0031] In this invention, the term "antibody" is also called immunoglobulin (Ig) and is a general term for proteins that selectively act on antigens and are involved in the body's immune response. Whole antibodies found in nature generally consist of two pairs of polypeptides, a light chain (LC) and a heavy chain (HC), which are composed of several domains, or a structure based on these two pairs of LC / HC. There are five types of heavy chains that make up mammalian antibodies, represented by the Greek letters α, β, γ, δ, and μ, and each type of heavy chain constitutes a different type of antibody, such as IgA, IgD, IgE, IgG, and IgM. There are two types of light chains that make up mammalian antibodies, represented by λ and κ.

[0032] The heavy and light chains of an antibody are structurally divided into variable and constant regions based on the variability of their amino acid sequences. The constant region of the heavy chain consists of three or four heavy chain constant regions, such as CH1, CH2, and CH3 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the type of antibody, while the light chain consists of one constant region called LC. The variable and constant regions of the heavy and light chains are arranged side by side and linked by a single covalent disulfide bond. The light chain and heavy chain, as well as the variable region of the light chain, bind specifically to the antigen. Since the whole antibody is composed of two pairs of heavy and light chains (HC / LC), one whole antibody molecule has bivalent monospecificity, binding to the same two antigens through its two variable regions. The variable region of an antibody that binds to an antigen is called the antigen-binding site, and the part of the antigen surface that is recognized by the antibody is called the antigenic determinant (epitope).

[0033] The variable region of an antibody containing the antigen-binding site is subdivided into a framework region (FR) with low sequence variability and a complementary determining region (CDR) with high sequence variability (hypervariable region). VH and VL each have two CDRs and four FRs, arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. Among the variable regions of an antibody, the CDR, which has the highest sequence variability, is the site that directly binds to the antigen and is most important for the antigen specificity of the antibody.

[0034] The antibody or antibody fragment according to the present invention is not limited in type as long as it has the above-described CDR, VH and VL, or light chain and heavy chain configuration. The antibody may be in the form of IgG, IgA, IgM, IgE, or IgD, and is particularly preferably an IgG antibody. The above IgG includes, but is not limited to, subtypes such as IgG1, IgG2, IgG3, and IgG4. It may also be a monoclonal antibody derived from a single B cell, or a polyclonal antibody derived from multiple B cells, but it is preferable to be a monoclonal antibody, which is a group of antibodies whose heavy chain and light chain amino acid sequences are substantially identical. Furthermore, the antibody or antibody fragment of the present invention may be conjugated with enzymes, fluorescent substances, radioactive substances, proteins, etc., but is not limited thereto.

[0035] The antibody of the present invention may be derived from any animal, including mammals such as humans and birds, and preferably from humans, or it may be a chimeric antibody containing a portion of the antibody derived from humans and a portion of the antibody derived from another species of animal.

[0036] In this invention, the antibody fragment refers to the fragment that maintains the antigen-specific binding affinity of the whole antibody, and may specifically be in the form of Fab, F(ab'), F(ab')2, Fv, scFv, diabody, or dsFv.

[0037] Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one invariant domain in both the heavy chain and light chain. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and has a form in which two Fabs are linked by a disulfide bond at a heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to a Fab separated by reducing the disulfide bond of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment consisting only of the variable regions of the heavy chain and light chain. scFv (single chain variable fragment) is a recombinant antibody fragment in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked by a flexible peptide linker. A diabody refers to a fragment in which the VH and VL of an scFv are linked by a very short linker and cannot bind to each other, while the VH and VL of other scFvs of the same form are linked to each other to form a dimer. A dsFv refers to a polypeptide in which one amino acid residue each of the VH and VL is replaced with a cysteine ​​residue, and these polypeptides are linked together via disulfide bonds between the cysteine ​​residues. The amino acid residue to be substituted for the cysteine ​​residue can be selected based on the prediction of the antibody's three-dimensional structure according to methods known to the industry, such as those by Reiter et al.

[0038] In one embodiment of the present invention, the antibody according to the present invention is the antibody disclosed in the applicant's prior registered patent No. 10-2340989. That is, the antibody or fragment thereof according to the present invention may be an antibody comprising an antibody heavy chain variable region (VH) comprising a heavy chain complementarity determining region (VH-CDR1) comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain complementarity determining region (VH-CDR2) comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain complementarity determining region (VH-CDR3) comprising the amino acid sequence shown in SEQ ID NO: 3, and an antibody light chain variable region (VL) comprising a light chain complementarity determining region (VL-CDR1) comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain complementarity determining region (VL-CDR2) comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain complementarity determining region (VL-CDR3) comprising the amino acid sequence shown in SEQ ID NO: 6.

[0039] However, as mentioned above, the characteristic of the present invention that claudin-3 protein is an effective target for small cell lung cancer is not exerted by any single specific antibody specific to claudin-3, but rather stems from the characteristics of the claudin-3 protein itself in small cell lung cancer. Therefore, the effect can be obtained equally well by using any antibody that is specific to the claudin-3 protein, not just antibodies with the specific complementarity-determining region sequence described above.

[0040] Furthermore, in some embodiments, the antibodies according to the present invention may be chemically modified (e.g., one or more moieties are bound to the antibody), glycosylated (e.g., low fucosylation, non-fucosylation, or increased sialylation), or modified or produced in cell lines and / or cell culture conditions to alter one or more functional properties of the antibody.

[0041] In some embodiments, the antibody according to the present invention includes an Fc region with modified glycosylation to increase ADCC activity. That is, in some embodiments, the Fc region of the antibody according to the present invention may not contain fucose sugars. The non-fucosylated antibody may, but is not limited to, be produced using a cell line expressing a heterologous enzyme that depletes the intracellular fucose pool, or using a host cell line lacking the endogenous α-1,6-fucosyltransferase (FUT8) gene.

[0042] In one embodiment of the present invention, ADCC activity was measured using anti-claudin-3 and anti-CD3 bispecific antibodies, and it was confirmed that all experimentally tested cell lines exhibited excellent anticancer activity. Therefore, the antibody according to the present invention may be a bispecific antibody. The term "bispecific antibody" above means an antibody in which a single antibody has two types of binding sites specific to different antigens. The antibody of the present invention may be a bispecific antibody that can specifically bind to CD3 in addition to claudin-3 as described above, but is not limited thereto. It can be applied without limitation to any target known to be applicable to small cell lung cancer other than CD3, such as DLL3, B7H3, PD-L1, IGFR, VEGFR, FGFR, TROP2, and SEZ6.

[0043] In one embodiment of the present invention, it was confirmed that the expression level of claudin-3 protein increased when small cell lung cancer cell lines were treated with a chemotherapeutic drug. This means that in the case of antibody-based drugs that target claudin-3 protein, such as the composition according to the present invention, the increased binding opportunity can lead to superior anticancer activity. Therefore, the composition for the treatment of small cell lung cancer according to the present invention is characterized by being administered to small cell lung cancer patients who have been given chemotherapy drugs.

[0044] In the present invention, the above-mentioned chemotherapy drugs can be applied without limitation as long as they are applicable to patients with small cell lung cancer, but preferably they are cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

[0045] In the present invention, the content of the above composition is not greatly limited depending on the intended use or appearance, and may be, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, and more preferably 1 to 30% by weight, based on the total weight of the composition. Furthermore, the pharmaceutical composition according to the present invention may further contain additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient.

[0046] Furthermore, the present invention provides a method for providing information for the diagnosis of small cell lung cancer, which includes the step of measuring the level of claudin-3 protein in a sample from a patient suspected of having small cell lung cancer.

[0047] In the present invention, the method for providing information for the diagnosis of small cell lung cancer may further include the step of comparing the expression level of claudin-3 protein or the gene encoding it in a sample isolated from a patient suspected of having small cell lung cancer after the above step with that of a healthy individual.

[0048] In the method according to the present invention, the biological sample may be derived from a specific tissue or organ of the patient, preferably from lung tissue. More specifically, the biological sample includes, but is not limited to, a bodily fluid sample, which includes blood, serum, plasma, lymph, breast milk, urine, feces, and saliva.

[0049] In the method according to the present invention, the claudin-3 expression level can be measured by one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, Western blotting, exosome, circulating tumor cells, and flow cytometry, but is not limited thereto. A person with ordinary skills can carry out the method according to the present invention using any method known in the art that can measure the expression level of claudin-3 from a biological sample without limitation.

[0050] The present invention also provides the use of an anti-claudin-3 specific antibody for producing a small cell lung cancer treatment composition with reduced side effects.

[0051] The "effective amount" in this invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, detecting, diagnosing, or suppressing or reducing small cell lung cancer. The "individual" may be an animal, preferably a mammal, and may include humans, or it may be a cell, tissue, organ, etc. derived from an animal.

[0052] The term "treatment" in this invention broadly refers to improving symptoms of small cell lung cancer, which may include curing, substantially preventing, or improving the condition of the disease, and may include, but is not limited to, alleviating, curing, or preventing one or most of the symptoms arising from the disease.

[0053] The present invention also provides a method for treating small cell lung cancer with reduced side effects, which involves administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to an individual in need of it.

[0054] Furthermore, the present invention provides a method for treating small cell lung cancer, comprising administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to a small cell lung cancer patient who has been administered a chemotherapy drug.

[0055] The above-mentioned "individual" may be a small cell lung cancer patient who has been administered chemotherapy drugs, and these chemotherapy drugs may be cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

[0056] Furthermore, the present invention provides a method for treating small cell lung cancer, comprising administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to a small cell lung cancer patient who has been administered a chemotherapy drug.

[0057] The present invention will be described in detail below. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0058] [Example 1] High expression analysis of claudin-3 mRNA in small cell lung cancer We compared the distribution of claudin-3 mRNA levels in cancer cells of different cancer types using the DepMab database. As a result, we were able to confirm the presence of a large number of cells with specifically high claudin-3 mRNA levels in small cell lung cancer (see Figures 1a to 1c).

[0059] [Example 2] Correlation analysis between claudin-3 protein and mRNA Considering that mRNA expression levels and protein expression levels of specific genes do not always coincide, we analyzed the correlation between claudin-3 protein and mRNA expression in 51 cell lines. To this end, mRNA expression rate information for 51 cancer cell lines was obtained from the DepMab database, and the expression rate of claudin-3 protein in the relevant cell lines was confirmed using flow cytometry according to the following method.

[0060] After culturing 51 different cell lines, the cells were harvested and divided into 2 x 10⁶ cells. 6 Cells / mL (100 μl) were treated with 10 μg / mL anti-claudin-3 antibody (an antibody discovered using a claudin-3 high-expression cell line) at 4°C for 45 minutes, and then washed three times with PBS / 1% FBS buffer. Subsequently, the cells were treated with 1:100 diluted anti-human IgG-FITC antibody (Jackson Immunoresearch Laboratories) at 4°C for 45 minutes, washed three times with PBS / 1% FBS buffer, and then analyzed via flow cytometry (BD FACSCalibur). The correlation between claudin-3 protein and mRNA expression rates was then evaluated using the Pearson correlation coefficient. As a result, r = 0.5816 was confirmed, confirming a positive correlation between claudin-3 protein and mRNA expression rates (see Figure 2).

[0061] [Example 3] Analysis of claudin-3 protein expression levels in small cell lung cancer The presence or absence of claudin-3 protein expression was evaluated using flow cytometry in 16 small cell lung cancer cell lines (Type A: NCI-H209, NCI-H69, NCI-H1688, NCI-H187, NCI-H146, NCI-H719, NCI-H889; Type A / N: SCLC-21H, COR-L279, DMS 53; Type N: COR-L24, NCI-H417; Type P: NCI-H526, COR-L311; Type Y: NCI-H2286, SW1271). The specific method for evaluating protein expression rates was the same as in Example 2 above.

[0062] As a result, as can be seen in Figure 3, claudin-3 positivity was confirmed in all 16 cell lines used in the analysis, and it was confirmed that claudin-3 was expressed at high levels in all subtypes of small cell lung cancer except for Type Y small cell lung cancer.

[0063] [Example 4] Analysis of claudin-3 protein expression levels in claudin-3-positive small cell lung cancer cell lines From the claudin-3-positive cell lines identified in Example 3 above, cell lines expressing high / medium / low levels of claudin-3 protein were selected, and the expression levels of claudin-3 protein were evaluated by immunochemical staining.

[0064] First, based on the results of Example 3 and Figure 3 above, the H209, H146, and H889 cell lines were selected as high, medium, and low claudin-3 protein expression cell lines, respectively. After culturing each cell line, the cells were harvested and FFPE blocks were prepared using each cell line, and the expression level of claudin-3 protein was confirmed by immunohistochemical staining.

[0065] As a result, as can be seen in Figures 4a to 4d, we confirmed that even in cells with relatively low claudin-3 protein expression as determined by flow cytometry, immunochemical staining showed moderate or higher expression levels of 2+ and 1+.

[0066] [Example 5] Confirmation of anticancer activity of a monoclonal antibody targeting claudin-3 in claudin-3-positive small cell lung cancer cell lines. In claudin-3-positive small cell lung cancer cell lines, anti-claudin-3 monoclonal antibody (aABN501, afucosylated) and the NK92-MI-CD16a cell line expressing human CD16a were used to confirm anticancer activity.

[0067] Detailed information on the ABN501 antibody used in this embodiment can be found in Registered Patent No. 10-2340989 of the Republic of Korea, and more specifically, the complementarity-determining region (CDR) sequence of the antibody is as follows.

[0068] [Table 1]

[0069] Each cell line is 1x10 6 After seeding cells / mL (50 μl) into a 96-well plate, treat with 10 μg / mL to 0.001 ng / mL of anti-claudin-3 antibody at room temperature for 30 minutes, then divide into 4x10⁻¹⁴⁻¹ 6 After treating NK92-MI-CD16a cells at a concentration of cells / mL (25 μl), they were cultured for 4 hours. Subsequently, the anti-cancer effect was confirmed by LDH (lactate dehydrogenase) measurement.

[0070] As a result, as can be seen in Figures 5a to 5i, effective anticancer activity was confirmed in all claudin-3-positive cell lines.

[0071] [Example 6] Confirmation of anticancer activity of a single-clonal antibody targeting claudin-3 in claudin-3-positive normal cell lines In normal cell lines expressing claudin-3, we confirmed the anti-cancer activity of a single-clonal anti-claudin-3 antibody (aABN501, afucosylated) and the NK92-MI-CD16a cell line expressing human CD16a.

[0072] Each cell line is 2 x 10 5 After seeding cells / mL (100 μl) into a 96-well plate, culture until 100% confluency is reached. After removing the culture medium, treat with 10 μg / mL to 0.001 ng / mL (final concentration) of anti-claudin-3 antibody (75 μl) at room temperature for 30 minutes, then divide into 4x10 cells. 6 After treating cells / mL (25 μl) of NK92-MI-CD16a cells, they were cultured for 4 hours. Toxicity was then confirmed by LDH (lactate dehydrogenase) assay.

[0073] As a result, as can be seen in Figures 6a to 6f, we confirmed that cell death was not induced in claudin 3-positive normal cells.

[0074] Comparing these results with those of Example 5 and Figures 5a to 5i, when treated with a high dose of 66.6 nM (10 μg / mL) antibody, cancer cell lines all showed ADCC activity of 30% to 80%, although there were some differences depending on the cell line type and claudin-3 expression rate. In contrast, normal cells showed low ADCC activity of 7.9% or less. These values ​​in normal cells are, on average, achieved at doses more than 100 times lower than those of small cell lung cancer cell lines with low claudin-3 expression rates.

[0075] [Example 7] Increase in claudin-3 expression rate by chemotherapy in small cell lung cancer NCI-H146 and NCI-H526 cells, which are small cell lung cancer cell lines, are 1x10 6 After seeding cells into a 100π culture dice, they were treated with etoposide (200, 400 nM) or cisplatin (5 μM) and carboplatin (100 μM), which are chemotherapy drugs for small cell lung cancer. Lysates were obtained after incubation for 48 hours and 24 hours, respectively. Subsequently, the expression rates of claudin-3 and β-actin were confirmed by Western blotting.

[0076] As a result, as can be seen in Figure 7, we confirmed that chemotherapy increased the expression level of claudin-3 protein in small cell lung cancer cell lines. This means that the opportunities for claudin-3 targets to bind to antibody-based drugs increase, which can contribute to the enhancement of anticancer activity.

[0077] [Example 8] Confirmation of anticancer activity of claudin-3-targeted CD3 biantibody in claudin-3-positive small cell lung cancer cell lines Anticancer activity was confirmed in claudin-3-positive small cell lung cancer cell lines using an anti-claudin-3 x CD3 biantibody and human PBMCs. Each cell line was subjected to 4 x 10⁶ tests. 5 After seeding cells / mL (50 μl) into a U-type 96-well plate, the cells were treated with 0.1 pM–1 μM anti-claudin-3 x CD3 biantibody in a 37°C, 5% CO2 incubator for 30 minutes. Subsequently, 4x10⁻¹⁴ cells were collected. 6 hPBMC cells (#LOT 238230218-C) were treated at a concentration of cells / mL (50 μl) and cultured for 72 hours. The anticancer effect was then confirmed using the WST (DoGENBio, EZ-Cytox) method.

[0078] As a result, as can be seen in Figures 8a to 8c, effective anticancer activity was confirmed in all claudin-3-positive cell lines. [Industrial applicability]

[0079] As described above, the present invention relates to a composition for the prevention or treatment of small cell lung cancer with reduced side effects, comprising an anti-claudin-3 specific antibody as an active ingredient. When claudin-3 is targeted, the ADCC activity of the antibody is low in normal cells but remarkably high in cancer cells, making it useful for the development of small cell lung cancer-specific therapeutic agents with fewer side effects.

Claims

1. A composition for the prevention or treatment of small cell lung cancer, containing an anti-claudin-3 specific antibody as an active ingredient, with reduced side effects.

2. The composition is characterized by not exhibiting cytotoxic activity against normal cell tissue, and is a composition for the prevention or treatment of small cell lung cancer with reduced side effects as described in claim 1.

3. The aforementioned side effect is one selected from the group consisting of leukopenia, lymphopenia, fatigue, anemia, dyspnea, vomiting, stomatitis, cough, and reproductive dysfunction, characterized in that the side effect of the composition for the prevention or treatment of small cell lung cancer described in claim 1 is reduced.

4. The aforementioned antibody is The antibody heavy chain variable region (VH) includes a heavy chain complementarity determination region (VH-CDR1) containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain complementarity determination region (VH-CDR2) containing the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain complementarity determination region (VH-CDR3) containing the amino acid sequence shown in SEQ ID NO: 3; and An antibody containing an antibody light chain variable region (VL) that includes a light chain complementarity-determining region (VL-CDR1) containing the amino acid sequence shown in SEQ ID NO: 4, a light chain complementarity-determining region (VL-CDR2) containing the amino acid sequence shown in SEQ ID NO: 5, and a light chain complementarity-determining region (VL-CDR3) containing the amino acid sequence shown in SEQ ID NO: 6; A composition for the prevention or treatment of small cell lung cancer, characterized in that the side effects described in claim 1 are reduced.

5. The antibody is afucosylated, characterized in that the side effects of the small cell lung cancer prevention or treatment composition described in claim 1 are reduced.

6. The composition for the prevention or treatment of small cell lung cancer, according to claim 1, characterized in that the antibody is a bispecific antibody against claudin-3 and anti-CD3, with reduced side effects.

7. The composition for the prevention or treatment of small cell lung cancer, as described in claim 1, is characterized by being administered to a small cell lung cancer patient who has been given a chemotherapy drug, and has reduced side effects.

8. The composition for the prevention or treatment of small cell lung cancer, according to claim 7, characterized in that the chemotherapy drug is cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof, with reduced side effects.

9. A method for providing information for the diagnosis of small cell lung cancer, comprising the step of measuring the level of claudin-3 protein in a sample from a patient suspected of having small cell lung cancer.

10. Use of anti-claudin-3 specific antibodies to manufacture compositions for the treatment of small cell lung cancer with reduced side effects.

11. The use of an anti-claudin-3 specific antibody according to claim 10, characterized in that the aforementioned side effect is one selected from the group consisting of leukopenia, lymphopenia, fatigue, anemia, dyspnea, vomiting, stomatitis, cough, and reproductive dysfunction.

12. The aforementioned antibody is The antibody heavy chain variable region (VH) includes a heavy chain complementarity determination region (VH-CDR1) containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain complementarity determination region (VH-CDR2) containing the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain complementarity determination region (VH-CDR3) containing the amino acid sequence shown in SEQ ID NO: 3; and An antibody containing an antibody light chain variable region (VL) that includes a light chain complementarity-determining region (VL-CDR1) containing the amino acid sequence shown in SEQ ID NO: 4, a light chain complementarity-determining region (VL-CDR2) containing the amino acid sequence shown in SEQ ID NO: 5, and a light chain complementarity-determining region (VL-CDR3) containing the amino acid sequence shown in SEQ ID NO: 6; The use of the anti-claudin-3 specific antibody according to claim 10, characterized in that it is such.

13. The use of the anti-claudin-3 specific antibody according to claim 10, characterized in that the antibody is afucosylated.

14. The use of an anti-claudin-3 specific antibody according to claim 10, characterized in that the antibody is a bispecific antibody for anti-claudin-3 and anti-CD3.

15. A method for treating small cell lung cancer with reduced side effects, comprising administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to an individual in need.

16. The method for treating small cell lung cancer with reduced side effects according to claim 15, characterized in that the individual is a small cell lung cancer patient who has been administered a chemotherapy drug.

17. The method for treating small cell lung cancer with reduced side effects, as described in claim 16, characterized in that the chemotherapy drug is cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

18. A method for treating small cell lung cancer, comprising administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to a small cell lung cancer patient who has been administered a chemotherapy drug.

19. The method for treating small cell lung cancer according to claim 18, characterized in that the chemotherapy drug is cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

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