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

Abstract

The present invention relates to a use of claudin-3 as a target for treatment of small cell lung cancer, and more specifically to a side effect-reduced composition for prevention or treatment of small cell lung cancer, comprising an anti-claudin-3-specific antibody as an active ingredient. The composition, according to the present invention, does not exhibit ADCC activity in normal cells compared to small cell lung cancer cells, and thus may be usefully employed in the development of a small cell lung cancer-specific therapeutic agent having few side effects.

Description

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

[0001] This application claims the benefit of Korean Patent Application No. 10-2023-0077110, filed June 15, 2023, and Korean Patent Application No. 10-2024-0002208, filed January 5, 2024, the entire contents of which are incorporated herein by reference. 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 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.

[0002]

[0003] Lung cancer is a major cause of cancer deaths worldwide, accounting for a quarter of all cancer deaths. However, the five-year survival rate for lung cancer patients remains extremely low, at only 15%. Lung cancer is classified into two subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with SCLC accounting for approximately 13% of all lung cancers. SCLC, in particular, is recognized as the most lethal type of lung cancer due to limited treatment options and a high incidence of rapid recurrence and metastasis after treatment. The prognosis for SCLC patients is extremely poor, with a five-year survival rate of only 7%.

[0004] Treatment for this type of small cell lung cancer primarily includes platinum-based chemotherapy, etoposide, and checkpoint inhibitors (atezolizumab or durvalumab). Recently, Lubinectedin was approved by the US FDA in 2020 as a treatment for small cell lung cancer.

[0005] However, these treatment methods are also used to treat other cancers and cannot be considered specific treatments for small cell lung cancer, and in the case of Lurbinectedin, several side effects such as leukopenia, lymphopenia, fatigue, anemia, dyspnea, vomiting, and cough are known. Therefore, there is an urgent need to develop a new treatment method that is specific to small cell lung cancer and has fewer side effects.

[0006] Furthermore, small cell lung cancer is diagnosed through medical history and physical examination, imaging tests such as chest X-ray, CT, PET, and MRI, cytological and pathological tests through biopsy, and tests to measure blood tumor markers such as NSE and ProGRP. These diagnostic methods are ways to confirm the presence of small cell lung cancer, determine the stage, and establish an appropriate treatment plan, but there is no diagnostic method regarding diagnostic markers for specific treatment for small cell lung cancer patients. Therefore, there is an urgent need to discover new diagnostic biomarkers for targeted treatment for each small cell lung cancer patient.

[0007]

[0008] Meanwhile, claudin proteins are important structural and functional components of epithelial tight junctions located between two adjacent cells, and are proteins that regulate cell-cell permeability, maintain ion homeostasis, and support cell adhesion and polarity. Claudin proteins are transmembrane proteins of 22 to 27 kDa that multimerize within or across the cell membrane to form a protective barrier. To date, 24 types of claudin proteins have been reported, and these claudin proteins have been confirmed to have diverse expression patterns in various organs. In particular, claudin proteins are proteins located at tight junctions, so in normal cells they are not exposed to the outside but are maintained between the membranes. However, in cancer cells that lose their cellular conformation and overgrow during the tumorigenic process, claudin proteins lose tight junctions, which exposes them and acts as cancer markers. Therefore, claudin proteins are receiving great attention as cancer-specific markers or targets for therapeutics.

[0009]

[0010] Accordingly, the inventors of the present invention have made great efforts to develop a small cell lung cancer-specific treatment with fewer side effects, and as a result, they confirmed that when targeting the claudin-3 protein, while ADCC (Antibody-dependent cellular cytotoxicity) activity is low for normal cells, it exhibits high ADCC activity only for small cell lung cancer cells, and confirmed that claudin-3 is significantly overexpressed in small cell lung cancer patients, confirming its potential as a diagnostic biomarker for small cell lung cancer patients, thereby completing the present invention.

[0011]

[0012] Accordingly, the purpose of the present invention is to provide a composition for preventing or treating small cell lung cancer with reduced side effects, which comprises an anti-claudin-3 specific antibody as an active ingredient.

[0013]

[0014] Another 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.

[0015]

[0016] Another object of the present invention is to provide 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.

[0017]

[0018] 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.

[0019]

[0020] Another object of the present invention is to provide a use of an anti-claudin-3 specific antibody for preparing a composition for treating small cell lung cancer with reduced side effects.

[0021]

[0022] Another object of the present invention is to provide a method for treating small cell lung cancer with reduced side effects, which comprises administering an effective amount of a composition containing anti-claudin-3 as an active ingredient to a subject in need thereof.

[0023]

[0024] 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 small cell lung cancer patient receiving a chemotherapy drug.

[0025]

[0026] In order to achieve the above purpose, the present invention provides 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.

[0027]

[0028] 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, comprising an anti-claudin-3 specific antibody.

[0029]

[0030] 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 is essentially composed of an anti-claudin-3 specific antibody.

[0031]

[0032] In order to achieve another object of the present invention, the present invention provides a method for providing information for diagnosing small cell lung cancer, comprising the step of measuring the level of claudin-3 protein from a sample of a patient suspected of having small cell lung cancer.

[0033]

[0034] In order to achieve another object of the present invention, the present invention provides the use of an anti-claudin-3 specific antibody for preparing a composition for treating small cell lung cancer with reduced side effects.

[0035]

[0036] In order to achieve another object of the present invention, the present invention provides a method for treating small cell lung cancer with reduced side effects, comprising administering an effective amount of a composition comprising anti-claudin-3 as an active ingredient to a subject in need thereof.

[0037]

[0038] In order to achieve another object of the present invention, the present invention provides 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 small cell lung cancer patient who has received a chemotherapy drug.

[0039]

[0040] The present invention is described in detail below.

[0041]

[0042] The present invention provides 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.

[0043]

[0044] In this specification, the term "comprising" is used with the same meaning as "including" or "characterized by", and does not exclude additional components or method steps, etc. that are not specifically mentioned in the composition or method according to the present invention. In addition, the term "consisting of" means excluding additional elements, steps, or components, etc. that are not separately described. The term "essentially consisting of" means that, in the scope of the composition or method, it can include materials or steps, etc. that do not substantially affect the basic characteristics thereof, in addition to the materials or steps described.

[0045]

[0046] While continuing research on small cell lung cancer, the inventors confirmed that claudin-3 mRNA and protein were expressed at significantly high levels in small cell lung cancer cell lines, and through this, they confirmed that claudin-3 protein is a diagnostic and therapeutic biomarker in small cell lung cancer. In addition, when an antibody targeting claudin-3 protein was used to confirm ADCC (antibody-dependent cellular cytotoxicity) activity against small cell lung cancer, it was confirmed that it had effective anticancer activity. In particular, it was confirmed that while ADCC activity was not well observed in normal cells even when claudin-3 was expressed, ADCC activity was well observed in small cell lung cancer even when claudin-3 was expressed at a low level. That is, according to one embodiment of the present invention, when a high dose of anti-claudin-3 antibody was treated against cancer cell lines and normal cell lines expressing claudin-3, ADCC activity of 30% to 80% was observed in cancer cells, but ADCC activity of 7.9% or less was observed in normal cell lines, or no ADCC cytotoxicity was observed. Accordingly, it is expected that the antibody will not exhibit cytotoxicity against normal tissue cells.

[0047] These findings suggest that claudin-3 has the potential to be a diagnostic biomarker in small cell lung cancer, and that antibodies specific for the anti-claudin-3 protein may have fewer side effects when used as a treatment for small cell lung cancer. Cancer cells are characterized by rapid proliferation and division, and most anticancer drugs are designed to act on these rapidly proliferating cells. However, there are normal cells in the body that have characteristics of rapid proliferation similar to cancer cells. These cells, such as gastrointestinal mucosal cells, bone marrow cells, hair follicle cells, oral epithelial cells, and germ cells, 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 these anticancer agents, the composition comprising the anti-claudin-3 specific antibody according to the present invention as an active ingredient has the advantage of significantly reducing side effects because it does not exhibit ADCC activity if the cell in question is a normal cell, regardless of which cell in the body expresses claudin-3, and exhibits ADCC activity only against small cell lung cancer cells expressing claudin-3.

[0048]

[0049] Due to these advantages, the composition according to the present invention can be administered in a much larger amount compared to the known dosage / administration regimen of existing anticancer agents, and thus, a relatively higher anticancer effect can be obtained compared to existing anticancer agents. For example, in the case of a monoclonal antibody therapeutic agent known in the art, an antibody for a target expressed in normal cells and a drug dosage including the antibody, Rituximab is administered at 375 mg / m 2 (non-Hodgkin's lymphoma); trastuzuamb was administered at a dose of 8 mg / kg (approximately 240 mg / m 2 ) after initial loading of 6 mg / kg (approximately 180 mg / m 2 )(breast cancer, stomach cancer); bevacizumab is 5 mg / kg (approximately 150 mg / m2 ) or 10 mg / kg (approximately 300 mg / m 2 )(metastatic colorectal cancer), 15 mg / kg (approximately 450 mg / m 2 )(non-small cell lung cancer), 10 mg / kg (approximately 300 mg / m 2 )(renal cell carcinoma); cetuximab is 400 mg / m 2 250 mg / m after initial loading 2 (metastatic colorectal cancer, head and neck squamous cell carcinoma) and, in the case of a monoclonal antibody drug targeting claudin-18.2, the phase 2 clinical dose is zolbetuximab 800 mg / m 2 After initial loading, 600 mg / m 2 ; TST001 was administered at 10 mg / kg (approximately 300 mg / m 2 ); ASKB589 was 800 mg / m 2 ; BIL93 was administered at 20 mg / kg (approximately 800 mg / m 2 ) are known to be set to about 500 mg / m. Considering this fact, the composition according to the present invention 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 Up to 1200 mg / m 2 , 600 mg / m 2 Up to 1200 mg / m 2 , 700 mg / m 2 Up to 1200 mg / m 2 , 800 mg / m 2 Up to 1200 mg / m 2 , 900 mg / m 2 Up to 1200 mg / m 2 , 1000 mg / m 2 Up to 1200 mg / m 2, 1100 mg / m 2 Up to 1200 mg / m 2 Although it can be administered in a higher dose, it is not limited thereto, and due to the characteristics of the composition according to the present invention having fewer side effects, a person skilled in the art can appropriately select and administer the composition according to the present invention in a dose greater than the dose of a monoclonal antibody drug targeting claudin commonly known in the art, thereby obtaining a superior anticancer effect than other monoclonal antibody drugs.

[0050]

[0051] In the present invention, the 'claudin-3 (CLDN3)' protein is a protein belonging to the claudin family, and is a protein that exists in the part where tight junctions occur and plays a unique role in eliminating the space between cells in tight junctions. Tight junctions are rigid structures that connect adjacent cell membranes in the tissues of organisms such as animals. Claudin-3 regulates the permeability between cells of small solutes such as ions, has a molecular weight of 20-34 kDa, and is known to be the most important backbone protein in tight junctions with two extracellular loops and four transmembrane structural domains. The claudin-3 protein functions as a barrier-forming protein such as the blood-brain barrier and the intestinal barrier, and is also known to be involved in regulating cell proliferation, migration, differentiation, and polarity.

[0052]

[0053] In the present invention, the term 'antibody', also called immunoglobulin (Ig), is a general term for proteins that selectively act on antigens and participate in biological immunity. Whole antibodies found in nature are generally composed of two pairs of light chains (LC) and heavy chains (HC), which are polypeptides composed of multiple domains, or have a structure of two pairs of LC / HC as their basic units. There are five types of heavy chains that constitute mammalian antibodies, indicated by the Greek letters α, β, γ, δ, and μ, and depending on the type of heavy chain, they constitute different types of antibodies, such as IgA, IgD, IgE, IgG, and IgM. There are two types of light chains that constitute mammalian antibodies, indicated by λ and κ.

[0054] The heavy and light chains of antibodies are structurally divided into variable regions and constant regions according to the variability of the amino acid sequence. The constant region of the heavy chain is composed 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, and the light chain is composed of one constant region, LC. The heavy and light chains are linked by a single covalent disulfide bond with their variable and constant regions aligned side by side, and bind specifically to antigens through the light chain and the variable regions of the heavy and light chains. Since the entire antibody is composed of two pairs of heavy and light chains (HC / LC), one entire antibody molecule has a bivalent monospecificity that binds to the same two antigens through the two variable regions. The antibody variable region that binds to the antigen is called the antigen-binding site of the antibody, and the part of the antigen surface that is recognized by the antibody is called the epitope.

[0055] The variable region of an antibody, which contains the antigen-binding site, is subdivided into a framework region (FR) with low sequence variability and a complementary determining region (CDR), which is a hypervariable region with high sequence variability. 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. The CDR with the highest sequence variability within the variable region of an antibody is the site that directly binds to the antigen and is most important for the antigen specificity of the antibody.

[0056] 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, and the antibody may be an IgG, IgA, IgM, IgE, or IgD type antibody, and is particularly preferably an IgG antibody. The IgG subtypes include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. In addition, the antibody may be a monoclonal antibody derived from a single B cell, or a polyclonal antibody derived from a plurality of B cells, but is preferably a monoclonal antibody which is a group of antibodies in which the amino acid sequences of the heavy and light chains of the antibody are substantially the same. In addition, the antibody or fragment thereof of the present invention may be conjugated to an enzyme, a fluorescent substance, a radioactive substance, a protein, or the like, but is not limited thereto.

[0057] The antibody of the present invention may be derived from any animal, including mammals, birds, etc., including humans, and preferably may be derived from humans, or may be a chimeric antibody comprising a portion of an antibody derived from humans and a portion of an antibody derived from an animal of another species.

[0058]

[0059] Additionally, in the present invention, an antibody fragment means a fragment that maintains the antigen-specific binding ability of the entire antibody, and specifically, may be in the form of Fab, F(ab'), F(ab')2, Fv, scFv, diabody, or dsFv.

[0060] Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain of each heavy and light chain. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and has a structure in which two Fabs are linked by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to Fab, which is separated by reducing the disulfide bond of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment composed only of the variable domains of each heavy and light chain. scFv (single chain variable fragment) is a recombinant antibody fragment in which the heavy chain variable domain (VH) and the light chain variable domain (VL) are linked by a flexible peptide linker. Diabody refers to a fragment in which the VH and VL of scFv are connected by a very short linker and cannot bind to each other, and the VH and VL of another scFv of the same type bind to each other to form a dimer. dsFv refers to a polypeptide in which one amino acid residue in each of the VH and VL is replaced with a cysteine ​​residue, and the polypeptide is linked through an SS bond between the cysteine ​​residues. The amino acid residue replaced with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody according to a method known in the art by Reiter et al.

[0061]

[0062] In one embodiment of the present invention, the antibody according to the present invention uses the antibody disclosed in the applicant's prior patent registration 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 an amino acid sequence represented by SEQ ID NO: 1, a heavy chain complementarity determining region (VH-CDR2) comprising an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain complementarity determining region (VH-CDR3) comprising an amino acid sequence represented by SEQ ID NO: 3; and an antibody light chain variable region (VL) comprising a light chain complementarity determining region (VL-CDR1) comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain complementarity determining region (VL-CDR) comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain complementarity determining region (VL-CDR) comprising an amino acid sequence represented by SEQ ID NO: 6.

[0063]

[0064] However, as described above, the feature of the present invention that claudin-3 protein is an effective target for small cell lung cancer is not exerted by any specific antibody specific for claudin-3, but is due to the characteristic of claudin-3 protein itself in small cell lung cancer. Therefore, the effect can be obtained equally when using any antibody specific for claudin-3 protein, not just an antibody having the specific complementarity determining region sequence described above.

[0065]

[0066] Additionally, in some embodiments, antibodies according to the invention may be chemically modified (e.g., one or more moieties are attached to the antibody), glycosylated (e.g., hypofucosylated, afucosylated, or increased sialylated), or modified or produced in cell lines and / or cell culture conditions to alter one or more functional properties of the antibody.

[0067] In some embodiments, antibodies of the present invention comprise an Fc region with altered glycosylation to enhance ADCC activity. That is, in some embodiments, the Fc region of an antibody of the present invention may not contain a fucose sugar. Such afucosylated antibodies may be produced using a cell line expressing a heterologous enzyme that depletes the intracellular fucose pool, or may be produced using a host cell line lacking the endogenous α-1,6-fucosyltransferase (FUT8) gene, but are not limited thereto.

[0068]

[0069] In one embodiment of the present invention, when ADCC activity was measured using an anti-claudin-3 and anti-CD3 bispecific antibody, it was confirmed that it exhibited excellent anticancer activity in all tested cell lines. Therefore, the antibody according to the present invention may be a bispecific antibody. The term 'bispecific antibody' refers to an antibody having two binding sites specific for different antigens, and the antibody of the present invention may be a bispecific antibody that can specifically bind to CD3 in addition to the above-described claudin-3, but is not limited thereto, and in addition to CD3, any target known to be applicable to small cell lung cancer may be applied without limitation, and examples thereof include DLL3, B7H3, PD-L1, IGFR, VEGFR, FGFR, TROP2, and SEZ6.

[0070]

[0071] In one embodiment of the present invention, it was confirmed that when a small cell lung cancer cell line was treated with a chemotherapy drug, the expression level of the claudin-3 protein increased. This suggests that antibody-based drugs targeting the claudin-3 protein, such as the composition according to the present invention, can exhibit superior anticancer activity by increasing the binding opportunity. Therefore, the composition for treating small cell lung cancer according to the present invention is characterized by being administered to small cell lung cancer patients who have received chemotherapy.

[0072] In the present invention, the chemotherapy drug may be applied without limitation as long as it can be applied to patients with small cell lung cancer, but preferably, it may be cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

[0073]

[0074] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient.

[0075]

[0076] The present invention also provides a method for providing information for diagnosing small cell lung cancer, comprising the step of measuring the level of claudin-3 protein from a sample of a patient suspected of having small cell lung cancer.

[0077]

[0078] In the present invention, the method for providing information for diagnosing small cell lung cancer may further include, after the above step, a step of comparing the expression level of claudin-3 protein or a gene encoding the same in a sample isolated from a patient suspected of having small cell lung cancer with that of a healthy individual.

[0079] 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 a body fluid sample, including, but not limited to, blood, serum, plasma, lymph, breast milk, urine, feces, and saliva.

[0080] In the method according to the present invention, the claudin-3 expression level can be measured by at least one method selected from the group consisting of a method using 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, and a person skilled in the art can perform the method according to the present invention by using any method known in the art without limitation as long as it is a method capable of measuring the expression level of claudin-3 from a biological sample.

[0081]

[0082] The present invention also provides the use of an anti-claudin-3 specific antibody for preparing a composition for treating small cell lung cancer with reduced side effects.

[0083]

[0084] The 'effective amount' of the present invention refers to an amount that, when administered to an individual, exhibits an effect of improving, treating, detecting, diagnosing, or inhibiting or reducing small cell lung cancer, and the 'individual' may be an animal, preferably a mammal, particularly an animal including a human, and may also be a cell, tissue, organ, etc. derived from an animal.

[0085]

[0086] The 'treatment' of the present invention comprehensively refers to improving symptoms caused by small cell lung cancer, which may include curing, substantially preventing, or improving the condition of the disease, and includes, but is not limited to, alleviating, curing, or preventing one or most of the symptoms resulting from the disease.

[0087]

[0088] The present invention also provides a method for treating small cell lung cancer with reduced side effects, comprising administering an effective amount of a composition comprising anti-claudin-3 as an active ingredient to a subject in need thereof.

[0089]

[0090] The present invention also 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 receiving a chemotherapy drug.

[0091]

[0092] The above 'subject' may be a small cell lung cancer patient who has been administered a chemotherapy drug, and the chemotherapy drug may be cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

[0093]

[0094] In addition, the present invention provides 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 small cell lung cancer patient receiving a chemotherapy drug.

[0095]

[0096] The present invention relates to 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. When targeting claudin-3, the ADCC activity of the antibody is low in normal cells but significantly high in cancer cells, and therefore can be usefully used in the development of a small cell lung cancer-specific treatment with reduced side effects.

[0097]

[0098] Figures 1a to 1c show that among various cancer types, claudin-3 mRNA is expressed at the highest rate in small cell lung cancer.

[0099] Figure 2 shows the results of analyzing the correlation between claudin-3 protein and mRNA levels.

[0100] Figure 3 shows the results of analyzing the expression rate of claudin-3 protein in small cell lung cancer cell lines using flow cytometry.

[0101] Figures 4a to 4d show the results of analyzing the expression rate of claudin-3 protein in claudin-3 positive small cell lung cancer cell lines using immunochemical staining.

[0102] Figures 5a to 5i show the results of confirming the anticancer effect of a claudin-3 targeting monoclonal antibody in a claudin-3 positive small cell lung cancer cell line using the LDH method.

[0103] Figures 6a to 6f show the results of confirming the anticancer effect of a claudin-3 targeting monoclonal antibody in a claudin-3 positive normal cell line using the LDH method.

[0104] 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 chemotherapeutic drugs etoposide, cisplatin, and carboplatin, respectively.

[0105] Figures 8a to 8c show the results of confirming the anticancer effect of a claudin-3 targeting CD3 dual antibody in a claudin-3 positive small cell lung cancer cell line using the WST method.

[0106]

[0107] The present invention is described in detail below. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0108]

[0109] Example 1. Analysis of high expression of Claudin-3 mRNA in small cell lung cancer.

[0110] The distribution of claudin-3 mRNA levels in cancer cells by cancer type was compared using the DepMab database. As a result, it was confirmed that many cells with specifically high claudin-3 mRNA levels exist in small cell lung cancer (see Figures 1a to 1c).

[0111]

[0112] Example 2. Correlation Analysis between Claudin-3 Protein and mRNA

[0113] Considering that mRNA and protein expression levels of a specific gene do not always correspond, we analyzed the correlation between claudin-3 protein and mRNA expression in 51 cell lines. To this end, mRNA expression data for 51 cancer cell lines was obtained from the DepMab database, and claudin-3 protein expression in these cell lines was confirmed using flow cytometry according to the following method.

[0114] After culturing 51 cell lines, cells were harvested and 2 x 10 6 Cells / mL (100 μl) were treated with 10 μg / mL of anti-claudin-3 antibody (antibody discovered using a high-claudin-3 expression cell line) at 4°C for 45 minutes, and then washed three times with PBS / 1% FBS buffer. Subsequently, anti-human IgG-FITC antibody (Jackson Immunoresearch Laboratories) diluted 1:100 was treated for 45 minutes at 4°C, and washed three times with PBS / 1% FBS buffer before analysis using a flow cytometer (BD FACSCalibur). The correlation between claudin-3 protein and mRNA expression rates was then evaluated using the Pearson correlation coefficient. The result was confirmed to be r = 0.5816, confirming that there was a positive correlation between claudin-3 protein and mRNA expression rates (see Fig. 2).

[0115]

[0116] Example 3. Analysis of the expression level of claudin-3 protein in small cell lung cancer.

[0117] The expression of claudin-3 protein 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 protein expression rate evaluation method was performed in the same manner as in Example 2.

[0118] As a result, as can be confirmed in Fig. 3, claudin-3 positivity was confirmed in all 16 cell lines used in the analysis, and in particular, claudin-3 was confirmed to be expressed at a high level in all subtypes of small cell lung cancer except Type Y small cell lung cancer.

[0119]

[0120] Example 4. Analysis of the expression level of claudin-3 protein in claudin-3-positive small cell lung cancer cell lines.

[0121] Among the claudin-3 positive cell lines confirmed in Example 3 above, high / medium / low claudin-3 protein expression cell lines were selected, and the expression level of claudin-3 protein was evaluated through immunochemical staining.

[0122] First, based on the results of Example 3 and Fig. 3, 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 through immunohistochemical staining.

[0123]

[0124] As a result, as can be confirmed in Figures 4a to 4d, even in cells with relatively low expression of claudin-3 protein by flow cytometry, it was confirmed that expression was moderate or higher, indicating 2+ or 1+, by immunochemical staining.

[0125]

[0126] Example 5. Confirmation of anticancer activity of a monoclonal antibody targeting claudin-3 in claudin-3-positive small cell lung cancer cell lines.

[0127] Anticancer activity was confirmed using an anti-claudin-3 monoclonal antibody (aABN501, afucosylated) in a claudin-3-positive small cell lung cancer cell line and the NK92-MI-CD16a cell line expressing human CD16a.

[0128] Detailed information on the ABN501 antibody used in this example can be found in Patent Registration No. 10-2340989, and more specifically, the complementarity determining region (CDR) sequence of the antibody is as follows.

[0129] Division Amino Acid Sequence SEQ ID NO: VH-CDR1SYAMS1VH-CDR2IINPSGASTSHAQRFQ2VH-CDR3RYGRYGSFDI3VL-CDR1SGSTSNIGRNYVS4VL-CDR2DTSNKHF5VL-CDR3QSYDSSKVV6

[0130] Each cell line was divided into 1x10 6 After seeding in a 96-well plate at 10 cells / mL (50 μl), anti-claudin-3 antibody at 10 μg / mL to 0.001 ng / mL was treated for 30 minutes at room temperature, and 4x10 6 NK92-MI-CD16a cells were treated at a concentration of 25 μl / mL and cultured for 4 hours. The anticancer effect was then confirmed by measuring LDH (lactate dehydrogenase).

[0131] As a result, as can be seen in Figures 5a to 5i, effective anticancer activity was confirmed in all claudin-3 positive cell lines.

[0132]

[0133] Example 6. Confirmation of anticancer activity of a monoclonal antibody targeting claudin-3 in a claudin-3-positive normal cell line.

[0134] Anticancer activity was confirmed using an anti-claudin-3 monoclonal antibody (aABN501, afucosylated) in a normal cell line expressing claudin-3 and the NK92-MI-CD16a cell line expressing human CD16a.

[0135] Each cell line was divided into 2x10 5 After seeding cells / mL (100 μl) in a 96-well plate, culture until 100% confluency is reached. After removing the culture medium, treat with anti-claudin-3 antibody (75 μl) at 10 μg / mL to 0.001 ng / mL (final concentration) at room temperature for 30 minutes, and 4 x 10 6 NK92-MI-CD16a cells were treated at a concentration of 25 μl / mL and cultured for 4 hours. Toxicity was then confirmed by measuring LDH (lactate dehydrogenase).

[0136] As a result, as can be seen in Figures 6a to 6f, it was confirmed that apoptosis was not induced in normal Claudin 3-positive cells.

[0137] Comparing the results of Example 5 and FIGS. 5a to 5i, when treated with a high dose of 66.6 nM (10 μg / mL) antibody, ADCC activity of 30% to 80% was observed in all cancer cell lines, although there was some difference depending on the type of cell line and the claudin-3 expression rate. In contrast, normal cells exhibited a low ADCC activity of 7.9% or less. This value in normal cells is a value that appears at a dose that is on average 100 times lower than that in small cell lung cancer cell lines with a low claudin-3 expression rate.

[0138]

[0139] Example 7. Increased expression of claudin-3 by chemotherapy in small cell lung cancer.

[0140] NCI-H146 and NCI-H526 cells, small cell lung cancer cell lines, were cultured at 1x10 6 After seeding cells in 100π culture dice, lysates were obtained after treatment with etoposide (200, 400 nM), cisplatin (5 μM), or carboplatin (100 μM), which are chemotherapeutic drugs for small cell lung cancer, and cultured for 48 and 24 hours, respectively. The expression rates of claudin-3 and β-actin were then confirmed using Western blotting.

[0141] As a result, as can be seen in Figure 7, it was confirmed that the expression level of claudin-3 protein increased in small cell lung cancer cell lines due to chemotherapy. This suggests that the binding opportunity for antibody-based drugs targeting claudin-3 increased, which may contribute to enhanced anticancer activity.

[0142]

[0143] Example 8. Confirmation of anticancer activity of a claudin-3-targeting CD3 dual antibody in claudin-3-positive small cell lung cancer cell lines.

[0144] Anticancer activity was confirmed using anti-claudin-3 x CD3 dual antibody and human PBMC in claudin-3 positive small cell lung cancer cell lines. Each cell line was cultured at 4 x 10 5 After seeding in a U-shaped 96-well plate at 50 μl (cells / mL), 0.1 pM to 1 μM anti-claudin-3 x CD3 double antibody was added at 37°C in a 5% CO2 incubator for 30 min. Afterwards, 4 x 10 6 hPBMC cells (#LOT 238230218-C) were treated at 50 μl / mL (cells / mL) and cultured for 72 hours, and then the anticancer effect was confirmed using the WST (DoGENBio, EZ-Cytox) method.

[0145] As a result, as can be seen in Figures 8a to 8c, effective anticancer activity was confirmed in all claudin-3 positive cell lines.

[0146]

[0147] As described above, the present invention relates to 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. When targeting claudin-3, the ADCC activity of the antibody is low in normal cells but significantly high in cancer cells, and therefore, it can be usefully used in the development of a small cell lung cancer-specific treatment with reduced side effects.

Claims

1. 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.

2. A composition for preventing or treating small cell lung cancer with reduced side effects, characterized in that the composition in claim 1 does not exhibit cytotoxic activity against normal cell tissue.

3. A composition for preventing or treating small cell lung cancer with reduced side effects, characterized in that the side effect in paragraph 1 is any one selected from the group consisting of leukopenia, lymphopenia, fatigue, anemia, dyspnea, vomiting, stomatitis, cough, and reproductive dysfunction.

4. In the first paragraph, the antibody An antibody heavy chain variable region (VH) comprising a heavy chain complementarity determining region (VH-CDR1) comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain complementarity determining region (VH-CDR2) comprising an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain complementarity determining region (VH-CDR3) comprising an amino acid sequence represented by SEQ ID NO: 3; and An antibody comprising an antibody light chain variable region (VL) comprising a light chain complementarity determining region (VL-CDR1) comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain complementarity determining region (VL-CDR) comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain complementarity determining region (VL-CDR) comprising an amino acid sequence represented by SEQ ID NO: 6; A composition for preventing or treating small cell lung cancer with reduced side effects characterized by:

5. A composition for preventing or treating small cell lung cancer with reduced side effects, characterized in that the antibody in claim 1 is afucosylated.

6. A composition for preventing or treating small cell lung cancer with reduced side effects, characterized in that the antibody in claim 1 is an anti-claudin-3 and anti-CD3 dual specific antibody.

7. A composition for preventing or treating small cell lung cancer with reduced side effects, characterized in that the composition is administered to a small cell lung cancer patient who has received chemotherapy drugs in the first paragraph.

8. A composition for preventing or treating small cell lung cancer with reduced side effects, characterized in that the chemotherapy drug in paragraph 7 is cisplatin, carboplatin, etoposide, topotecan, cyclophosphamide, doxorubicin, vincristine, lurbinectedin, irinotecan, or a combination thereof.

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

10. Use of an anti-claudin-3 specific antibody for preparing a composition for treating small cell lung cancer with reduced side effects.

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

12. In the 10th paragraph, the antibody An antibody heavy chain variable region (VH) comprising a heavy chain complementarity determining region (VH-CDR1) comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain complementarity determining region (VH-CDR2) comprising an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain complementarity determining region (VH-CDR3) comprising an amino acid sequence represented by SEQ ID NO: 3; and An antibody comprising an antibody light chain variable region (VL) comprising a light chain complementarity determining region (VL-CDR1) comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain complementarity determining region (VL-CDR) comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain complementarity determining region (VL-CDR) comprising an amino acid sequence represented by SEQ ID NO: 6; Use of an anti-claudin-3 specific antibody characterized by:

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

14. Use of an anti-claudin-3 specific antibody according to claim 10, characterized in that the antibody is an anti-claudin-3 and anti-CD3 dual specific antibody.

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 a subject in need thereof.

16. A method for treating small cell lung cancer with reduced side effects, characterized in that the subject is a small cell lung cancer patient who has received chemotherapy drugs in paragraph 15.

17. A method for treating small cell lung cancer with reduced side effects, characterized in that the chemotherapy drug in claim 16 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 received a chemotherapy drug.

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

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