Anti-claudin 6 antibody and use thereof

Abstract

An anti-CLDN6 antibody and an antigen-binding fragment thereof are disclosed in the present disclosure. In addition, a therapeutic use of the anti-CLDN6 antibody or the antigen-binding fragment thereof is disclosed in the present disclosure.

Description

Anti-Claudin 6 Antibody and Uses thereof

[0001] The present disclosure relates to the field of antibody technology, and in particular to a novel antibody that specifically binds to the human Claudin 6 (CLDN6) protein and an antigen-binding fragment thereof. The present disclosure also relates to a method for producing said antibody or antigen-binding fragment. Furthermore, the present disclosure relates to the treatment of cancer and related diseases using a composition comprising said antibody.

[0002] Claudin proteins are a major component of tight junctions between cells and are known to be abnormally expressed in certain cancer cells. In particular, CLDN6 proteins are rarely expressed in normal adult cells but are reported to be expressed in various tumor cells. Accordingly, CLDN6 proteins have attracted attention as a target for cancer treatment. Although antibodies targeting CLDN6 proteins have been disclosed in some literature (e.g., WO 2019 / 056023 A2), reports on antibodies targeting CLDN6 proteins remain limited to date. Therefore, there is a need for the development of clinically useful antibodies.

[0003] The present disclosure aims to provide a novel antibody capable of binding to human CLDN6 protein and an antigen-binding fragment thereof, and further to provide a method for treating cancer, a cancer treatment agent, and a use of a related substance utilizing the same.

[0004] To solve the above technical problem, the present disclosure provides the sequence of a novel antibody capable of binding to the human CLDN6 protein. The inventors of the present disclosure confirmed through experiments that the novel antibody can bind to the human CLDN6 protein and can be internalized in cells expressing the human CLDN6 protein.

[0005] The novel antibody provided in the present disclosure has high binding affinity and high specificity for human CLDN6 protein. Therefore, it can be utilized in the novel cancer treatment method and cancer treatment of the present disclosure.

[0006] Figure 1 shows the results of analyzing the binding ability of a mouse anti-CLDN6 antibody obtained from a mouse to the human CLDN6 protein-VLP.

[0007] Figures 2 and 3 show the results of analyzing the binding ability of chimeric IgG 1C6 and benchmark antibody B1 to 293T cells or 3T3 cells expressing human CLDN6, CLDN9, CLDN4, or CLDN3.

[0008] Figure 4 shows the results of analyzing the binding ability of chimeric IgG 1C6 and benchmark antibody B1 to liver cancer cell lines HepG2 and Huh7, gastric cancer cell line AGS, and breast cancer cell line SK-BR-3.

[0009] Figure 5 shows the results of analyzing the internalization effects of chimeric IgG 1C6 and benchmark antibody B1 on the liver cancer cell line HepG2.

[0010] Figure 6 shows the results of analyzing the apoptotic effects of chimeric IgG 1C6 and DT3C conjugates.

[0011] Figure 7 shows the results of analyzing the binding ability of nine types of humanized antibodies to CHO-K1 cells that stably express human CLDN6 protein.

[0012] Figure 8 shows the results of analyzing the specific binding ability of nine types of humanized antibodies to human CLDN6 protein.

[0013] Figure 9 shows the results of a comparative evaluation of the binding ability of AB0976 and control antibodies AB0463 and AB0388 to human CLDN6 and CLDN9.

[0014] Figure 10 shows the results of evaluating the binding ability according to the change in the heavy chain invariant region of AB0976.

[0015] Figure 11 shows the results of evaluating the binding specificity of AB0976 to claudin-family proteins.

[0016] Figures 12 and 13 show the results of evaluating the binding ability of the antibody to eight cell lines.

[0017] Figure 14 shows the results of evaluating the binding specificity of B1 antibody, SC105 antibody, and KM3907 antibody to claudin-family proteins.

[0018] Figures 15, 16, and 17 show the results of evaluating the internalization activity of the antibody.

[0019] Figure 18 shows the results of evaluating the binding affinity of antibodies against human CLDN6 antigen-expressing cell lines with mutations.

[0020] Figures 19 and 20 show the results of analyzing the apoptotic effects of AB0976-37728 ADC and IgG1-37728 ADC.

[0021] Figure 21 shows the results of analyzing the bystander killing effect of AB0976-37728 ADC.

[0022] Figure 22 shows the results of analyzing the in vivo anti-tumor activity of AB0976-37728 ADC.

[0023] Figure 23 shows the results of evaluating the plasma stability of AB0976-37728 ADC.

[0024] Hereinafter, with reference to the attached drawings, the content of the invention will be described in more detail through specific embodiments and examples. It should be noted that the attached drawings include some embodiments of the invention, but not all embodiments. The content of the invention disclosed by this specification may be implemented in various ways and is not limited to the specific embodiments described herein. Such embodiments should be considered as provided to satisfy the legal requirements applicable to this specification. A person skilled in the art to which the invention disclosed in this specification belongs would be able to conceive of many variations and other embodiments of the content of the invention disclosed in this specification. Therefore, the content of the invention disclosed in this specification is not limited to the specific embodiments described herein, and variations and other embodiments thereof should be understood to be included within the scope of the claims.

[0025] It should be understood that the contents of the disclosure described exemplarily herein can be suitably practiced in the absence of any element(s) or limitation(s) not specifically presented in the disclosure. For example, any terms “comprising,” “essentially made,” and “made” in the disclosure may be replaced with any two other terms. Furthermore, the terms and expressions used in the disclosure should be understood as being used for illustrative purposes rather than as limitations.

[0026] All publications, patents, and other references mentioned in this disclosure are incorporated by reference in their entirety.

[0027]

[0028] Definition of Terms

[0029] Unless otherwise stated, all technical and scientific terms used in this disclosure have the meaning generally understood by those skilled in the art related to this disclosure.

[0030] The definitions of terms used in this disclosure are as follows.

[0031]

[0032] approximately

[0033] The term “about” as used in the present disclosure means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0034] Amino acid sequence notation

[0035] Unless otherwise stated, amino acid sequences described in this disclosure are written in the direction from the N-terminus to the C-terminus using either a single-letter or three-letter amino acid notation. For example, when denoted as RNVP, it signifies a peptide in which arginine, asparagine, valine, and proline are linked in sequence from the N-terminus to the C-terminus. As another example, when denoted as Thr-Leu-Lys, it signifies a peptide in which threonine, leucine, and lysine are linked in sequence from the N-terminus to the C-terminus. For amino acids that cannot be represented by the single-letter notation, other letters are used for notation and further explanation is provided.

[0036] The notation for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Val, V).

[0037] Nucleic acid sequence notation

[0038] The symbols A, T, C, G, and U used in this disclosure are to be interpreted as understood by a person skilled in the art. Depending on the context and description, they may be appropriately interpreted as bases, nucleosides, or nucleotides on DNA or RNA. For example, when referring to a base, it may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) itself, respectively; when referring to a nucleoside, it may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively; and when referring to a nucleotide in a sequence, it should be interpreted as a nucleotide containing each of the above nucleosides.

[0039] In the present disclosure, the symbol N may be appropriately interpreted as a base, nucleoside, or nucleotide on DNA or RNA depending on the context and description. For example, when referring to a base, it may be interpreted as any one of adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U); when referring to a nucleoside, it may be interpreted as any one of adenosine (A), thymidine (T), cytidine (C), guanosine (G), and uridine (U); and when referring to a nucleotide in a sequence, it should be interpreted as a nucleotide containing each of the above nucleosides.

[0040] vector

[0041] As used in this disclosure, the term "vector" refers collectively to any material capable of transporting genetic material into a cell, unless otherwise specified. For example, a vector may contain the target genetic material in the form of a nucleic acid sequence. For example, it may be a DNA molecule containing a nucleic acid encoding a protein (e.g., an antibody), an RNA molecule (e.g., an mRNA molecule), etc., but is not limited thereto. The above term encompasses all meanings recognizable by a person skilled in the art and may be interpreted appropriately according to the context.

[0042] Immunological binding and binding dissociation constants

[0043] The terms "immunological binding" and "immunological binding characteristics" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody) and the antigen to which that immunoglobulin exhibits specificity. The strength or affinity of an immunological binding interaction is the binding dissociation constant (K) of that binding. D　 It can be expressed in terms of ), where K D　The smaller the value, the greater the affinity. The immunological binding properties of selected immunoglobulin molecules can be quantified using methods well known in this field of technology.

[0044] For example, immunological binding characteristics can be quantified by measuring the complex formation and dissociation rates of antibodies and antigens. Here, each rate depends on the concentrations of the antibody and antigen, the affinity of the interaction, and geometric parameters, which equally influence the rates in both directions. Therefore, the binding rate constant (K a or K on ) and dissociation rate constant (K d or K off ) can be determined by calculating the concentrations of the antibody and antigen, and the actual rates of binding and dissociation. In this case, the binding-dissociation constant (K D　 ) is K d / K a As a ratio value, it refers to the value in which all parameters unrelated to affinity are offset. (See Nature 361: 186-87 (1993), and Davies et al. (1990) Annual Rev Biochem 59: 439-473).

[0045] The above bond dissociation constant (K D　 ) can be measured by radioligand binding analysis, surface plasmon resonance (SPR), flow cytometry binding analysis, or by analytical methods used by those skilled in this field.

[0046] Internalization activation

[0047] As used in this disclosure, "internalization activity" refers to the ability of an antibody to move into a cell by endocytosis through the cell membrane after binding to an antigen on the cell surface. Internalization activity can be confirmed, for example, through the intracellular accumulation of fluorescently labeled antibodies using flow cytometry (FACS), fluorescence microscopy, or quantitative chemiluminescence analysis.

[0048] Fc region-mediated cytotoxicity

[0049] As used in this disclosure, "Fc region-mediated cytotoxicity" refers to the function in which the Fc region of an antibody induces a killing effect on target cells by interacting with components of the immune system. Specific examples of such Fc region-mediated cytotoxicity include ADCC activity, ADCP activity, and CDC activity.

[0050] The above ADCC activity (Antibody-Dependent Cellular Cytotoxicity activity) refers to the ability of an antibody's Fc region to bind to immune cells (e.g., NK cells, macrophages, neutrophils) that express the Fcγ receptor (FcγR), thereby inducing the immune cells to specifically lyse or apoptotically kill the target cells to which the antibody is bound.

[0051] The aforementioned ADCP activity (Antibody-Dependent Cellular Phagocytosis activity) refers to the ability of an antibody's Fc domain to interact with Fcγ receptors on phagocytic cells, such as macrophages, monocytes, and dendritic cells, thereby mediating the phagocytosis of the target cells to which the antibody binds.

[0052] The aforementioned CDC activity (Complement-Dependent Cytotoxicity activity) refers to the ability of an antibody's Fc region to bind to a complement protein, initiate a complement activation chain reaction, and consequently form a membrane attack complex (MAC) that induces the lysis or death of the target cell to which the antibody is bound.

[0053] Sequence identity

[0054] The term “sequence identity” as used in this disclosure is a term used in relation to the degree of similarity between two or more sequences. For example, the term “sequence identity” is used in conjunction with a term referring to a reference sequence and a term indicating a ratio (e.g., a percentage). For example, the term “sequence identity” may be used to describe a sequence that is similar or substantially identical to a reference amino acid sequence. The method of calculating and / or determining the percentage of sequence identity is not otherwise limited and may be calculated and / or determined through reasonable methods or algorithms available to a person skilled in the art.

[0055] antibodies

[0056] As used in this disclosure, the term "antibody" refers collectively to proteins that specifically bind to a particular antigen. Antibodies may be proteins naturally produced in the immune system in response to stimulation by an antigen, or proteins artificially produced by chemical synthesis or recombinant technology, and there are no particular limitations on their types.

[0057] The antibody may comprise any isotype of full-length immunoglobulin and may also comprise an antigen-binding fragment capable of binding to an antigen such as the full-length antibody. Typically, the full-length antibody comprises at least two full-length heavy chains and two full-length light chains.

[0058] The antibodies or antigen-binding fragments of the present disclosure may be non-naturally generated and may be produced, for example, by recombinant DNA technology, chemical synthesis, hybridoma technology, or enzymatic or chemical cleavage of full-length antibodies. Unless otherwise noted, the term “antibody” in the present disclosure is used to include not only full-length antibodies comprising two full-length heavy chains and two full-length light chains, but also their derivatives, variants, fragments, mutants, etc.

[0059] Antibodies may be animal antibodies (e.g., mouse antibodies), chimeric antibodies (e.g., mouse-human chimeric antibodies), humanized antibodies, or human antibodies, and encompass both monospecific and multispecific (e.g., bispecific) antibodies. In addition, it includes not only monoclonal antibodies but also polyclonal antibodies.

[0060] For the antibodies provided in this disclosure, the defined antigen-binding fragment, heavy chain CDR and light chain CDR, or the remaining regions excluding the heavy chain variable region and light chain variable region may be derived from any isotype of immunoglobulin (e.g., IgG, IgA, IgM, IgE, IgD, etc.). For example, the light chain constant region or the heavy chain constant region may be derived from any isotype of immunoglobulin (e.g., IgG, IgA, IgM, IgE, IgD, etc.). Accordingly, the antibodies of this disclosure may be human IgG type antibodies (e.g., IgG1, IgG2, IgG3, IgG4), but are not limited thereto.

[0061]

[0062] Understanding Antibody Structure

[0063] In this section, the structure of antibodies is described in detail based on generally known facts in the art to aid in understanding antibody structures, and the scope of the present disclosure is not limited by the description in this section.

[0064] The aforementioned antibody contains a heavy chain and a light chain. Here, the heavy chain and light chain are concepts distinguished based on the type of chain constituting the antibody. Antibodies are generally known to contain two heavy chains and two light chains.

[0065] The above antibody includes a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). In this case, the Fab region and the Fc region are regions distinguished based on their function of binding to antigens.

[0066] The above antibody includes a variable region and a constant region. In this case, the variable region and the constant region are regions distinguished based on the variability of the antibody's amino acid sequence.

[0067] The above antibody further includes components including a hinge portion and a tail portion.

[0068] Antibody Structure 1 - Heavy Chain and Light Chain Regions

[0069] The above antibody includes a heavy chain and a light chain.

[0070] The above heavy chain has a polypeptide chain structure and includes four or five immunoglobulin domains. For example, the heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region includes, for example, CH1, CH2, and CH3 or CH1, CH2, CH3, and CH4. For example, the heavy chain constant region of an IgG-type antibody includes CH1, CH2, and CH3.

[0071] The light chain has a polypeptide chain structure and includes two immunoglobulin domains. For example, the light chain includes a light chain variable region (VL) and a light chain constant region (CL).

[0072] Antibody Structure 2 - Fragment Antigen-Binding and Crystallizable Region

[0073] The above heavy chain includes a portion of the fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). The above light chain includes a portion of the fragment antigen-binding region (Fab region) and does not include the fragment crystallizable region.

[0074] Functionally, the fragment antigen-binding region of the heavy chain and the fragment antigen-binding region of the light chain have the function of binding to antigens.

[0075] The above Fab region is a portion that includes a part that binds to an antigen (antigen binding portion), and the above Fc region is a portion that can bind to an Fc receptor.

[0076] Meanwhile, the aforementioned fragment crystallizable region has the function of binding to the Fc receptor.

[0077] Antibody Structure 3 - Variable and Invariant Regions

[0078] The Fab region of the above heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1). The Fc region of the above heavy chain includes a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3), or includes CH2, CH3, and a heavy chain constant region 4 (CH4). In this case, the entire heavy chain constant region of the antibody is referred to as CH. For example, the entire combined region of CH1, CH2, and CH3 of IgG1 can be expressed as CH.

[0079] The Fab region of the light chain includes a light chain variable region (VL) and a light chain constant region (CL). The light chain does not have an Fc region. VH, CH1, CH2, CH3, CH4, VL, and CL are each one immunoglobulin domain.

[0080] Antibody Structure 4 - Connection Relationships

[0081] The immunoglobulin domains included in the heavy chain are located in the order of VH, CH1, CH2, and CH3 or VH, CH1, CH2, CH3, and CH4 from the N-terminus to the C-terminus. The immunoglobulin domains included in the light chain are located in the order of VL and CL from the N-terminus to the C-terminus.

[0082] The heavy chain and the light chain are connected by a disulfide bond. Specifically, they are connected by a disulfide bond between CH1 of the heavy chain and CL of the light chain. The Fab region and the Fc region are connected by a hinge region. Specifically, the C-terminus of CH1 of the heavy chain region and the N-terminus of CH2 are connected by a hinge region.

[0083] Characteristics of the variable region

[0084] The above variable regions (VH and VL) are regions that include antigen binding portions. Therefore, unlike the above constant regions (CH1, CH2, CH3, CH4, and CL), the sequence of the above variable regions may differ depending on the type of antigen to be bound and / or the binding site (epitope).

[0085] Even within the aforementioned variable region, there is a hypervariable region, which is called the complementarity-determining region (CDR).

[0086] The above VH includes three CDRs, and the three CDRs included in the VH are respectively called HCDR1, HCDR2, or HCDR3. The CDRs in the VH are located in the order of HCDR1, HCDR2, and HCDR3 from the N-terminus to the C-terminus.

[0087] The above VL includes three CDRs, and the three CDRs included in the VL are respectively called LCDR1, LCDR2, or LCDR3. The CDRs in the above VH are located in the order of LCDR1, LCDR2, and LCDR3 from the N-terminus to the C-terminus.

[0088] These CDRs can provide major contact residues for antibodies to bind to antigens or epitopes. In one embodiment, the complementarity determining sites (CDRs) described in this disclosure may be determined based on the definition of a CDR according to the kabat system, but the scope of the present invention is not limited to sequences derived by a specific CDR definition system, and functionally identical CDR sequences that can be obtained by applying ordinary definition systems (IMGT, Chothia, AbM, AHo, etc.) other than kabat should also be considered to be within the scope of the present invention.

[0089] The portion of the variable region excluding the CDR is called the framework region (FR). The VH contains four FRs, and the four FRs included in the VH are respectively called HFR1, HFR2, HFR3, or HFR4. The FRs in the VH are located in the order of HFR1, HFR2, HFR3, and HFR4 from the N-terminus to the C-terminus.

[0090] The above VL includes four FRs, and the four FRs included in the VL are each called LFR1, LFR2, LFR3, or LFR4. The FRs in the VL are located in the order of LFR1, LFR2, LFR3, and LFR4 from the N-terminus to the C-terminus.

[0091] The above VHs are located in the order FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, and FRH4 from the N-terminus to the C-terminus. The above VLs are located in the order FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 from the N-terminus to the C-terminus.

[0092] Characteristics of the invariant domain

[0093] The above constant region may interact with cells or molecules of the immune system. In one embodiment, the above constant region may interact (be bound or connected) with the cell membrane of an immune cell (e.g., lymphocyte, neutrophil, dendritic cell, and / or macrophage, etc.). Specifically, the CH2 portion of the above constant region may bind to a receptor (e.g., Fc εRⅢ) on the cell membrane of the immune cell. In another embodiment, the above constant region may bind to FcRn.

[0094] The above-mentioned heavy chain constant region (hereinafter referred to as "heavy chain constant region") is broadly classified into five classes (or isotypes): alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ). At this time, the type of the heavy chain constant region is not determined individually for CH1, CH2, CH3, and CH4, but is determined by considering all heavy chain constant regions (CH1, CH2, and CH3; or CH1, CH2, CH3, and CH4) included in the antibody.

[0095] The above-mentioned invariant region of the light chain (hereinafter referred to as the "light chain invariant region") has two types, and the two types are lambda (λ) and kappa (κ).

[0096] Antibody classes

[0097] There are five main types of antibodies (class or isotype). The above five types are determined by the type of heavy chain constant region.

[0098] There are five types of antibodies: Immunoglobulin M (IgM), Immunoglobulin D (IgD), Immunoglobulin G (IgG), Immunoglobulin A (IgA), and Immunoglobulin E (IgE). If the heavy chain constant region of the antibody is alpha, the type of antibody is IgA. If the heavy chain constant region of the antibody is gamma, the type of antibody is IgG. If the heavy chain constant region of the antibody is delta, the type of antibody is IgD. If the heavy chain constant region of the antibody is epsilon, the type of antibody is IgE. If the heavy chain constant region of the antibody is mu, the type of antibody is IgM.

[0099] Among the five types of antibodies mentioned above, IgG is classified into a more subdivided subclass. When the antibody is a human antibody, if the type of the heavy chain constant region of the antibody is gamma 1 (γ1), the type of antibody is IgG1; if it is gamma 2 (γ2), the type of antibody is IgG2; if it is gamma 3 (γ3), the type of antibody is IgG3; and if it is gamma 4 (γ4), the type of antibody is IgG4.

[0100] Immunoglobulin G (IgG), one of the types of antibodies, comprises two heavy chains and two light chains. In one embodiment, the two heavy chains included in the IgG may be identical heavy chains. Specifically, the two heavy chains included in the IgG may consist of the same amino acid sequence. In another embodiment, the two heavy chains included in the IgG may be different heavy chains. Specifically, the amino acid sequences of the two heavy chains included in the IgG may be different from each other.

[0101] In one embodiment, the two light chains included in the IgG may be the same light chain. Specifically, the two light chains included in the IgG may be composed of the same amino acid sequence. In another embodiment, the two light chains included in the IgG may be different light chains. Specifically, the amino acid sequences of the two light chains included in the IgG may be different from each other.

[0102] For convenience in explaining the structure of IgG, the two heavy chains and two light chains contained in IgG in the <<Classes of Antibodies>> section are referred to as the first heavy chain, the second heavy chain, the first light chain, and the second light chain, respectively. In the IgG, one heavy chain may be connected to one light chain through at least one disulfide bond. For example, the first heavy chain is connected to the first light chain by a disulfide bond, and the second heavy chain is connected to the second light chain by a disulfide bond. The IgG forms a Y-shaped quaternary structure overall.

[0103] When the subclass of IgG is IgG1, any one or more of the sequences of CH1, CH2, CH3, and the hinge portion may be known amino acid sequences known in the art. For example, when the subclass of IgG is IgG1, CH1, CH2, CH3, and the hinge portion may each be known amino acid sequences known in the art.

[0104] When the subclass of IgG is IgG2, any one or more of the sequences of CH1, CH2, CH3, and the hinge portion may be known amino acid sequences known in the art. For example, when the subclass of IgG is IgG2, CH1, CH2, CH3, and the hinge portion may each be known amino acid sequences known in the art.

[0105] When the subclass of IgG is IgG3, any one or more of the sequences of CH1, CH2, CH3, and the hinge portion may be known amino acid sequences known in the art. For example, when the subclass of IgG is IgG3, CH1, CH2, CH3, and the hinge portion may each be known amino acid sequences known in the art.

[0106] When the subclass of IgG is IgG4, any one or more of the sequences of CH1, CH2, CH3, and the hinge portion may be known amino acid sequences known in the art. For example, when the subclass of IgG is IgG4, CH1, CH2, CH3, and the hinge portion may each be known amino acid sequences known in the art.

[0107]

[0108] Claudine 6

[0109] Claudin 6 (hereinafter "CLDN6") protein belongs to the claudin family, a group of small transmembrane proteins, and human CLDN6 protein is known to consist of 220 amino acids (see UniProt Accession P56747). CLDN6 protein penetrates the cell membrane four times and contains two extracellular loops. Both the N-terminus (7 amino acids) and the C-terminus (19 amino acids) of CLDN6 protein are located in the cytoplasm.

[0110] Claudin proteins are one of several proteins that form tight junctions and play an important role in regulating tight junction permeability, barrier function, and the maintenance of cell polarity, as well as the proliferation and differentiation of epithelial and endothelial cells. This claudin family includes various proteins such as CLDN3, CLDN4, CLDN6, and CLDN9, and most claudins are widely expressed in various tissues. However, the CLDN6 protein is specifically expressed only in embryonic cells and pluripotent stem cells, and is rarely expressed in normal adult tissues.

[0111] In contrast, CLDN6 protein is expressed at high levels in various malignant tumors, including ovarian cancer, endometrial cancer, cervical cancer, lung cancer, gastric cancer, and testicular cancer, and it is known to be involved in the development, proliferation, and metastasis of some cancers. Furthermore, increased CLDN6 expression in tumor tissue has been reported as an independent prognostic factor associated with reduced survival rates in cancer patients. Due to these differences in expression between normal and tumor tissues, CLDN6 is attracting attention as a promising target for cancer treatment, and several anti-CLDN6 antibodies are currently undergoing clinical trials as candidates for anticancer therapeutics.

[0112] However, CLDN6 has very high structural or sequence similarity to other claudin proteins widely expressed in normal tissues, such as CLDN3 (Sequence No. 83, see UniProt Accession O15551), CLDN4 (Sequence No. 84, see UniProt Accession O14493), and CLDN9 (Sequence No. 85, see UniProt Accession O95484). In particular, the extracellular domain of CLDN6 differs from that of CLDN9 by only three amino acids. Specifically, there are only one amino acid difference in the first extracellular loop (M29 / L29) and two amino acid differences in the second loop (R145 / Q145, Q156 / L156) (McDermott MSJ, O'Brien NA, Hoffstrom B, et al., Clin Cancer Res. 2023;29(11):2131-2143, doi:10.1158 / 1078-0432.CCR-22-2981).

[0113] Due to this high homology, anti-CLDN6 antibodies may exhibit cross-reactivity with Claudin proteins in normal tissues, including CLDN3, CLDN4, or CLDN9. Indeed, many anti-CLDN6 antibodies reported to date show cross-reactivity not only with CLDN6 but also with other Claudin family proteins (CLDN3, CLDN4, CLDN9), particularly with CLDN9. This can lead to toxic side effects resulting from the antibody binding to normal tissues, which may cause a reduction in the therapeutic index—the safe dosage range. Furthermore, competition with normal tissues during antibody administration may reduce the amount delivered to cancer tissues, thereby decreasing drug efficacy. This serves as a significant limitation in the utilization of CLDN6 as a target for cancer treatment.

[0114] CLDN6 is described in detail in the literature [Du, H., Yang, X., Fan, J., & Du, X. (2021). Claudin 6: Therapeutic prospects for tumors, and mechanisms of expression and regulation. Molecular medicine reports, 24(3), 677.], the entire contents of which are incorporated by reference into the present disclosure.

[0115] The present disclosure relates to a novel anti-CLDN6 antibody and its antigen-binding fragment that can overcome these problems.

[0116]

[0117] 1. Anti-CLDN6 antibody

[0118] 1.1. Overview of Anti-CLDN6 Antibodies

[0119] In one aspect of the present disclosure, an antibody that binds to a Claudin 6 protein or an antigen-binding fragment thereof is disclosed. The “antibody that binds to a Claudin 6 protein” may be referred to as an anti-Claudin 6 antibody or an anti-CLDN6 antibody. The anti-CLDN6 antibody may be an isolated antibody. The anti-CLDN6 antibody may be a recombinant antibody.

[0120] The anti-CLDN6 antibody or its antigen-binding fragment may bind to the human CLDN6 protein. In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment binds to the extracellular loop of the human CLDN6 protein. In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment binds to the extracellular domain of the human CLDN6 protein. For example, the anti-CLDN6 antibody or its antigen-binding fragment may bind to the extracellular loop comprising residues M29 to R81 or residues W138 to L160 of the human CLDN6 protein of SEQ ID NO. 56. In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment specifically binds to the human CLDN6 protein.

[0121] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment has an antigen-binding portion (or antigen binding site). The antigen-binding portion may enable the anti-CLDN6 antibody or its antigen-binding fragment to bind to a human CLDN6 protein. In some embodiments, the antigen-binding portion includes a complementarity-determining region (CDR). In some embodiments, the antigen-binding portion may include six complementarity-determining regions (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3). In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment may include one or more antigen-binding portions. Preferably, the anti-CLDN6 antibody or its antigen-binding fragment may include two antigen-binding portions. In some embodiments, the antigen-binding portion may include one heavy chain variable region and one light chain variable region.

[0122] The above anti-CLDN6 antibody may include a heavy chain and a light chain. The above anti-CLDN6 antibody may include a fragment antigen-binding region (Fab region) having the function of binding to an antigen and a fragment crystallizable region (Fc region) having little relation to the antigen-binding function. The above anti-CLDN6 antibody may include a variable region with high variability in amino acid sequence and a constant region with low variability. The antibody may further include a hinge portion in addition to the variable region and the constant region, but is not limited thereto. The antibody may further include a tail portion for the variable region and the constant region, but is not limited thereto.

[0123] The class of the above anti-CLDN6 antibody may be IgG. In some embodiments, the subclass of the above anti-CLDN6 antibody is IgG1, IgG2, IgG3, or IgG4.

[0124] The anti-CLDN6 antibody may be a humanizing antibody, a human antibody, a chimeric antibody, or a non-human antibody. In some embodiments, the anti-CLDN6 antibody is a humanizing antibody.

[0125] The above anti-CLDN6 antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the above anti-CLDN6 antibody is a monoclonal antibody.

[0126] 1.2. Targets of Anti-CLDN6 Antibodies

[0127] The anti-CLDN6 antibody of the present disclosure can bind to a human CLDN6 protein. In this case, the amino acid sequence of the human CLDN6 protein may be MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEAQKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (Sequence No. 56).

[0128] In addition, the anti-CLDN6 antibody can bind to a specific domain of the human CLDN6 protein or to cells in which the human CLDN6 protein is expressed. For example, the anti-CLDN6 antibody can bind to the extracellular domain of the human CLDN6 protein. For example, the anti-CLDN6 antibody can bind to the human CLDN6 protein expressed in cells. For example, the anti-CLDN6 antibody can bind to cells in which the human CLDN6 protein is expressed. For example, the anti-CLDN6 antibody can bind to cancer cells or tumor cells in which the human CLDN6 protein is expressed. Generally, it is known that the human CLDN6 protein is expressed at high levels in various malignant tumors, such as ovarian cancer, endometrial cancer, cervical cancer, lung cancer, gastric cancer, testicular cancer, liver cancer, and bladder cancer. For example, in cells in which the CLDN6 protein is expressed, CLDN6 is located on the cell membrane while penetrating it, and the extracellular domain (or extracellular loop) of CLDN6 may be exposed to the extracellular space.

[0129] 1.3. Variable range of anti-CLDN6 antibody

[0130] 1.3.1. Overview of Variable Regions

[0131] The antibody includes a variable region with high variability in its amino acid sequence and a constant region with low variability. The variable region includes a heavy chain variable region (VH) and a light chain variable region (VL). Within the variable region, there is a hypervariable region with the highest variability, and this part is called the Complementarity Determining Region (CDR). The part of the variable region excluding the CDR is called the Framework Region (FR).

[0132] In some embodiments, the anti-CLDN6 antibody may include one or more CDRs. For example, the anti-CLDN6 antibody may include HCDR1, HCDR2, and HCDR3. For example, the anti-CLDN6 antibody may include LCDR1, LCDR2, and LCDR3. For example, the anti-CLDN6 antibody may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. For example, the anti-CLDN6 antibody may include six CDRs. The variable region of the anti-CLDN6 antibody may include six CDRs.

[0133] In some embodiments, the anti-CLDN6 antibody of the present disclosure may comprise a heavy chain variable region and a light chain variable region. For example, the anti-CLDN6 antibody of the present disclosure may comprise two heavy chain variable regions and two light chain variable regions. In some embodiments, one heavy chain variable region and one light chain variable region may form an antigen-binding portion.

[0134] The above heavy-chain variable region includes three CDRs. The three CDRs included in the above heavy-chain variable region are respectively called the first heavy-chain complementarity determining region (HCDR1; heavy-chain CDR1), the second heavy-chain complementarity determining region (HCDR2; heavy-chain CDR2), and the third heavy-chain complementarity determining region (HCDR3; heavy-chain CDR3). The CDRs in the above heavy-chain variable region are located in the order of HCDR1, HCDR2, and HCDR3 in the direction from the N-terminus to the C-terminus.

[0135] The above light-chain variable region includes three CDRs. The three CDRs included in the light-chain variable region are respectively called the first light-chain complementarity determining region (LCDR1; light-chain CDR1), the second light-chain complementarity determining region (LCDR2; light-chain CDR2), and the third light-chain complementarity determining region (LCDR3; light-chain CDR3). The CDRs in the light-chain variable region are located in the order of LCDR1, LCDR2, and LCDR3 in the direction from the N-terminus to the C-terminus.

[0136] In some specific examples, the variable region may further include a framework region. The framework region may refer to the portion of the variable region excluding the CDR.

[0137]

[0138] The above heavy-chain variable region may further include four FRs. The four FRs included in the above heavy-chain variable region are respectively referred to as the first heavy-chain framework region (Heavy-chain Framework region 1; HFR1; heavy-chain FR1), the second heavy-chain framework region (Heavy-chain Framework region 2; HFR2; heavy-chain FR2), the third heavy-chain framework region (Heavy-chain Framework region 3; HFR3; heavy-chain FR3), and the fourth heavy-chain framework region (Heavy-chain Framework region 4; HFR4; heavy-chain FR4). The FRs in the above heavy-chain variable region may be located in the order of HFR1, HFR2, HFR3, and HFR4 in the direction from the N-terminus to the C-terminus.

[0139] The above light-chain variable region may further include four FRs. The four FRs included in the above light-chain variable region are respectively referred to as the first light-chain framework region (Light-chain Framework region 1; LFR1; light-chain FR1), the second light-chain framework region (Light-chain Framework region 2; LFR2; light-chain FR2), the third light-chain framework region (Light-chain Framework region 3; LFR3; light-chain FR3), and the fourth light-chain framework region (Light-chain Framework region 4; LFR4; light-chain FR4). The FRs in the above light-chain variable region may be located in the order of LFR1, LFR2, LFR3, and LFR4 in the direction from the N-terminus to the C-terminus.

[0140] In some embodiments, the heavy chain variable region consists of HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4. HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4 may be positioned in order from the N-terminus to the C-terminus of the heavy chain variable region. In some embodiments, the light chain variable region consists of LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4. LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4 may be positioned in order from the N-terminus to the C-terminus of the light chain variable region.

[0141] 1.3.2. HCDR1

[0142] The amino acid sequence of HCDR1 of the above anti-CLDN6 antibody may be the same as the sequence of SEQ ID NO. 1, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 1, or may include the same.

[0143] In some embodiments, the amino acid sequence of HCDR1 of the anti-CLDN6 antibody is SEQ ID NO. 1. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR1 of SEQ ID NO. 1. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain CDR1 sequence identical to SEQ ID NO. 1. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR1 represented by the amino acid sequence of SEQ ID NO. 1.

[0144] 1.3.3. HCDR2

[0145] The amino acid sequence of HCDR2 of the above anti-CLDN6 antibody may be the same as sequence No. 2, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 2, or may include the same.

[0146] In some embodiments, the amino acid sequence of HCDR2 of the anti-CLDN6 antibody is SEQ ID NO. 2. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR2 of SEQ ID NO. 2. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain CDR2 sequence identical to SEQ ID NO. 2. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR2 represented by the amino acid sequence of SEQ ID NO. 2.

[0147] 1.3.4. HCDR3

[0148] The amino acid sequence of HCDR3 of the above anti-CLDN6 antibody may be the same as sequence No. 3, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 3, or may include the same.

[0149] In some embodiments, the amino acid sequence of HCDR3 of the anti-CLDN6 antibody is SEQ ID NO. 3. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR3 of SEQ ID NO. 3. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain CDR3 sequence identical to SEQ ID NO. 3. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR3 represented by the amino acid sequence of SEQ ID NO. 3.

[0150] 1.3.5. LCDR1

[0151] The amino acid sequence of LCDR1 of the above anti-CLDN6 antibody may be the same as sequence No. 4, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 4, or may include the same.

[0152] In some embodiments, the amino acid sequence of LCDR1 of the anti-CLDN6 antibody is SEQ ID NO. 4. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises the LCDR1 of SEQ ID NO. 4. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain CDR1 sequence identical to SEQ ID NO. 4. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises an LCDR1 represented by the amino acid sequence of SEQ ID NO. 4.

[0153] 1.3.6. LCDR2

[0154] The amino acid sequence of LCDR2 of the above anti-CLDN6 antibody may be the same as sequence No. 5, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 5, or may include the same.

[0155] In some embodiments, the amino acid sequence of LCDR2 of the anti-CLDN6 antibody is SEQ ID NO. 5. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises the LCDR2 of SEQ ID NO. 5. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain CDR2 sequence identical to SEQ ID NO. 5. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises an LCDR2 represented by the amino acid sequence of SEQ ID NO. 5.

[0156] 1.3.7. LCDR3

[0157] The amino acid sequence of LCDR3 of the above anti-CLDN6 antibody may be the same as sequence number 6, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence number 6, or may include the same.

[0158] In some embodiments, the amino acid sequence of LCDR3 of the anti-CLDN6 antibody is SEQ ID NO. 6. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises the LCDR3 of SEQ ID NO. 6. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain CDR3 sequence identical to SEQ ID NO. 6. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises an LCDR3 represented by the amino acid sequence of SEQ ID NO. 6.

[0159] 1.3.8. Combination of HCDRs

[0160] The heavy chain variable region of the above anti-CLDN6 antibody includes three complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO. 1, the amino acid sequence of HCDR2 is SEQ ID NO. 2, and the amino acid sequence of HCDR3 is SEQ ID NO. 3.

[0161] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3.

[0162] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody includes the heavy chain CDR1 sequence identical to SEQ ID NO. 1, the heavy chain CDR2 sequence identical to SEQ ID NO. 2, and the heavy chain CDR3 sequence identical to SEQ ID NO. 3.

[0163] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR1 represented by the amino acid sequence of SEQ ID NO. 1, HCDR2 represented by the amino acid sequence of SEQ ID NO. 2, and HCDR3 represented by the amino acid sequence of SEQ ID NO. 3.

[0164] 1.3.9. LCDR Combination

[0165] The light chain variable region of the above anti-CLDN6 antibody includes three complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is SEQ ID NO. 4, the amino acid sequence of LCDR2 is SEQ ID NO. 5, and the amino acid sequence of LCDR3 is SEQ ID NO. 6.

[0166] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6.

[0167] In some embodiments, the light chain variable region of the anti-CLDN6 antibody includes the light chain CDR1 sequence identical to SEQ ID NO. 4, the light chain CDR2 sequence identical to SEQ ID NO. 5, and the light chain CDR3 sequence identical to SEQ ID NO. 6.

[0168] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LCDR1 represented by the amino acid sequence of SEQ ID NO. 4, LCDR2 represented by the amino acid sequence of SEQ ID NO. 5, and LCDR3 represented by the amino acid sequence of SEQ ID NO. 6.

[0169] 1.3.10. Combination of HCDR and LCDR

[0170] The variable region of the above anti-CLDN6 antibody comprises six complementarity-determining regions (CDRs) including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO. 1, the amino acid sequence of HCDR2 is SEQ ID NO. 2, the amino acid sequence of HCDR3 is SEQ ID NO. 3, the amino acid sequence of LCDR1 is SEQ ID NO. 4, the amino acid sequence of LCDR2 is SEQ ID NO. 5, and the amino acid sequence of LCDR3 may be SEQ ID NO. 6.

[0171] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3; and the light chain variable region comprises LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6.

[0172] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain CDR1 sequence identical to SEQ ID NO. 1, a heavy chain CDR2 sequence identical to SEQ ID NO. 2, and a heavy chain CDR3 sequence identical to SEQ ID NO. 3; and the light chain variable region comprises a light chain CDR1 sequence identical to SEQ ID NO. 4, a light chain CDR2 sequence identical to SEQ ID NO. 5, and a light chain CDR3 sequence identical to SEQ ID NO. 6.

[0173] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HCDR1 represented by the amino acid sequence of SEQ ID NO. 1, HCDR2 represented by the amino acid sequence of SEQ ID NO. 2, and HCDR3 represented by the amino acid sequence of SEQ ID NO. 3; and the light chain variable region comprises LCDR1 represented by the amino acid sequence of SEQ ID NO. 4, LCDR2 represented by the amino acid sequence of SEQ ID NO. 5, and LCDR3 represented by the amino acid sequence of SEQ ID NO. 6.

[0174] 1.3.11. HFR1

[0175] The amino acid sequence of HFR1 of the above anti-CLDN6 antibody may be the same as the sequence of SEQ ID NO. 7, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 7, or may include the same.

[0176] In some embodiments, the amino acid sequence of HFR1 of the anti-CLDN6 antibody is SEQ ID NO. 7. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR1 of SEQ ID NO. 7. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain FR1 sequence identical to SEQ ID NO. 7. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR1 represented by the amino acid sequence of SEQ ID NO. 7.

[0177] 1.3.12. HFR2

[0178] The amino acid sequence of HFR2 of the above anti-CLDN6 antibody is the same as sequence No. 8 or No. 9; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 8 or No. 9, or may include the same. Sequence No. 9 is a sequence in which the arginine (R) at position 3 of sequence No. 8 is substituted with lysine (K).

[0179] In some embodiments, the amino acid sequence of HFR2 of the anti-CLDN6 antibody is SEQ ID NO. 8 or SEQ ID NO. 9. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR2 of SEQ ID NO. 8 or SEQ ID NO. 9. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain FR2 sequence identical to SEQ ID NO. 8 or SEQ ID NO. 9. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR2 represented by the amino acid sequence of SEQ ID NO. 8 or SEQ ID NO. 9.

[0180] In some embodiments, the amino acid sequence of HFR2 of the anti-CLDN6 antibody is WVXQAPGQRLEWIG (SEQN 77). In SEQN 77, X at position 3 is arginine (R) or lysine (K).

[0181] 1.3.13. HFR3

[0182] The amino acid sequence of HFR3 of the above anti-CLDN6 antibody is the same as sequence No. 10, sequence No. 11, or sequence No. 12; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 10, sequence No. 11, or sequence No. 12, or may include the same. Sequence No. 11 is a sequence in which the tyrosine (Y) at position 29 of sequence No. 10 is substituted with phenylalanine (F). Sequence No. 12 is a sequence in which the valine (V) at position 2 of Sequence No. 10 is substituted with alanine (A), and the tyrosine (Y) at position 29 is substituted with phenylalanine (F).

[0183] In some embodiments, the amino acid sequence of HFR3 of the anti-CLDN6 antibody is SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR3 of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain FR3 sequence identical to SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR3 represented by the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12.

[0184] In some embodiments, the amino acid sequence of HFR3 of the anti-CLDN6 antibody is RXTITADTSASTAYMELSSLRSEDTAVYXCAR (SEQ ID NO. 78). In SEQ ID NO. 78, X at position 2 is valine (V) or alanine (A). In SEQ ID NO. 78, X at position 29 is tyrosine (Y) or phenylalanine (F).

[0185] 1.3.14. HFR4

[0186] The amino acid sequence of HFR4 of the above anti-CLDN6 antibody may be the same as the sequence of SEQ ID NO. 13, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 13, or may include the same.

[0187] In some embodiments, the amino acid sequence of HFR4 of the anti-CLDN6 antibody is SEQ ID NO. 13. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR4 of SEQ ID NO. 13. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain FR4 sequence identical to SEQ ID NO. 13. In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR4 represented by the amino acid sequence of SEQ ID NO. 13.

[0188] 1.3.15. LFR1

[0189] The amino acid sequence of LFR1 of the above anti-CLDN6 antibody may be the same as sequence No. 14 or sequence No. 15; or may be an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 14 or sequence No. 15, or may include the same. Sequence No. 14 is a sequence in which the serine (S) at position 22 of sequence No. 15 is substituted with threonine (T).

[0190] In some embodiments, the amino acid sequence of LFR1 of the anti-CLDN6 antibody is SEQ ID NO. 14 or SEQ ID NO. 15. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR1 of SEQ ID NO. 14 or SEQ ID NO. 15. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR1 sequence identical to SEQ ID NO. 14 or SEQ ID NO. 15. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR1 represented by the amino acid sequence of SEQ ID NO. 14 or SEQ ID NO. 15.

[0191] In some embodiments, the amino acid sequence of LFR1 of the anti-CLDN6 antibody is DIVLTQSPASLAVSPGQRATIXC (SEQ ID NO. 79). In SEQ ID NO. 79, X at position 22 is serine (S) or threonine (T).

[0192] 1.3.16. LFR2

[0193] The amino acid sequence of LFR2 of the above anti-CLDN6 antibody is the same as sequence No. 16 or sequence No. 17; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 16 or sequence No. 17, or may include the same. Sequence No. 16 is a sequence in which the phenylalanine (F) at position 2 of sequence No. 17 is substituted with tyrosine (Y).

[0194] In some embodiments, the amino acid sequence of LFR2 of the anti-CLDN6 antibody is SEQ ID NO. 16 or SEQ ID NO. 17. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR2 of SEQ ID NO. 16 or SEQ ID NO. 17. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR2 sequence identical to SEQ ID NO. 16 or SEQ ID NO. 17. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR2 represented by the amino acid sequence of SEQ ID NO. 16 or SEQ ID NO. 17.

[0195] In some embodiments, the amino acid sequence of LFR2 of the anti-CLDN6 antibody is WXQQKPGQPPKLLIY (SEQ No. 80). In SEQ No. 80, X at position 2 is phenylalanine (F) or tyrosine (Y).

[0196] 1.3.17. LFR3

[0197] The amino acid sequence of LFR3 of the above anti-CLDN6 antibody may be the same as sequence No. 18 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 18, or may include the same.

[0198] In some embodiments, the amino acid sequence of LFR3 of the anti-CLDN6 antibody is SEQ ID NO. 18. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR3 of SEQ ID NO. 18. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR3 sequence identical to SEQ ID NO. 18. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR3 represented by the amino acid sequence of SEQ ID NO. 18.

[0199] 1.3.18. LFR4

[0200] The amino acid sequence of LFR4 of the above anti-CLDN6 antibody may be the same as the sequence of SEQ ID NO. 19, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 19, or may include the same.

[0201] In some embodiments, the amino acid sequence of LFR4 of the anti-CLDN6 antibody is SEQ ID NO. 19. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR4 of SEQ ID NO. 19. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR4 sequence identical to SEQ ID NO. 19. In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR4 represented by the amino acid sequence of SEQ ID NO. 19.

[0202] 1.3.19. Combination of HFRs

[0203] The heavy chain variable region of the above anti-CLDN6 antibody comprises four framework regions (FR) HFR1, HFR2, HFR3, and HFR4, wherein the amino acid sequence of HFR1 is SEQ ID NO. 7, the amino acid sequence of HFR2 is SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 77, the amino acid sequence of HFR3 is SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 78, and the amino acid sequence of HFR4 may be SEQ ID NO. 13.

[0204] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR1 of SEQ ID NO. 7; HFR2 of SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 77; HFR3 of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 78; and HFR4 of SEQ ID NO. 13.

[0205] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises the heavy chain FR1 sequence identical to SEQ ID NO. 7, the heavy chain FR2 sequence identical to SEQ ID NO. 77, the heavy chain FR3 sequence identical to SEQ ID NO. 78, and the heavy chain FR4 sequence identical to SEQ ID NO. 13.

[0206] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises the heavy chain FR1 sequence identical to SEQ ID NO. 7, the heavy chain FR2 sequence identical to SEQ ID NO. 8, the heavy chain FR3 sequence identical to SEQ ID NO. 10, and the heavy chain FR4 sequence identical to SEQ ID NO. 13.

[0207] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises the heavy chain FR1 sequence identical to SEQ ID NO. 7, the heavy chain FR2 sequence identical to SEQ ID NO. 8, the heavy chain FR3 sequence identical to SEQ ID NO. 11, and the heavy chain FR4 sequence identical to SEQ ID NO. 13.

[0208] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises the heavy chain FR1 sequence identical to SEQ ID NO. 7, the heavy chain FR2 sequence identical to SEQ ID NO. 9, the heavy chain FR3 sequence identical to SEQ ID NO. 12, and the heavy chain FR4 sequence identical to SEQ ID NO. 13.

[0209] 1.3.20. Combination of LFRs

[0210] The light chain variable region of the above anti-CLDN6 antibody comprises four framework regions (FR) LFR1, LFR2, LFR3, and LFR4, wherein the amino acid sequence of LFR1 is SEQ ID NO. 14, SEQ ID NO. 15, or SEQ ID NO. 79, the amino acid sequence of LFR2 is SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 80, the amino acid sequence of LFR3 is SEQ ID NO. 18, and the amino acid sequence of LFR4 may be SEQ ID NO. 19.

[0211] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises LFR1 of SEQ ID NO. 14, SEQ ID NO. 15, or SEQ ID NO. 79; LFR2 of SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 80; LFR3 of SEQ ID NO. 18; and LFR4 of SEQ ID NO. 19.

[0212] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR1 sequence identical to SEQ ID NO. 79, a light chain FR2 sequence identical to SEQ ID NO. 80, a light chain FR3 sequence identical to SEQ ID NO. 18, and a light chain FR4 sequence identical to SEQ ID NO. 19.

[0213] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR1 sequence identical to SEQ ID NO. 14, a light chain FR2 sequence identical to SEQ ID NO. 16, a light chain FR3 sequence identical to SEQ ID NO. 18, and a light chain FR4 sequence identical to SEQ ID NO. 19.

[0214] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR1 sequence identical to SEQ ID NO. 14, a light chain FR2 sequence identical to SEQ ID NO. 17, a light chain FR3 sequence identical to SEQ ID NO. 18, and a light chain FR4 sequence identical to SEQ ID NO. 19.

[0215] In some embodiments, the light chain variable region of the anti-CLDN6 antibody comprises a light chain FR1 sequence identical to SEQ ID NO. 15, a light chain FR2 sequence identical to SEQ ID NO. 17, a light chain FR3 sequence identical to SEQ ID NO. 18, and a light chain FR4 sequence identical to SEQ ID NO. 19.

[0216] 1.3.21. Combination of HFR and LFR

[0217] The variable region of the above anti-CLDN6 antibody comprises eight framework regions (FR) including HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, wherein the amino acid sequence of HFR1 is SEQ ID NO. 7, the amino acid sequence of HFR2 is SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 77, the amino acid sequence of HFR3 is SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 78, the amino acid sequence of HFR4 is SEQ ID NO. 13, the amino acid sequence of LFR1 is SEQ ID NO. 14, SEQ ID NO. 15, or SEQ ID NO. 79, the amino acid sequence of LFR2 is SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 80, the amino acid sequence of LFR3 is SEQ ID NO. 18, and the amino acid sequence of LFR4 may be SEQ ID NO. 19.

[0218] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises HFR1 of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, or HFR2 of SEQ ID NO. 77, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or HFR3 of SEQ ID NO. 78, and HFR4 of SEQ ID NO. 13; and the light chain variable region comprises LFR1 of SEQ ID NO. 14, SEQ ID NO. 15, or LFR1 of SEQ ID NO. 79, SEQ ID NO. 16, SEQ ID NO. 17, or LFR2 of SEQ ID NO. 80, LFR3 of SEQ ID NO. 18, and LFR4 of SEQ ID NO. 19.

[0219] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain FR1 sequence identical to SEQ ID NO. 7, a heavy chain FR2 sequence identical to SEQ ID NO. 77, a heavy chain FR3 sequence identical to SEQ ID NO. 78, and a heavy chain FR4 sequence identical to SEQ ID NO. 13; and the light chain variable region comprises a light chain FR1 sequence identical to SEQ ID NO. 79, a light chain FR2 sequence identical to SEQ ID NO. 80, a light chain FR3 sequence identical to SEQ ID NO. 18, and a light chain FR4 sequence identical to SEQ ID NO. 19.

[0220] In a preferred example, the heavy chain variable region of the anti-CLDN6 antibody comprises a heavy chain FR1 sequence identical to SEQ ID NO. 7, a heavy chain FR2 sequence identical to SEQ ID NO. 8, a heavy chain FR3 sequence identical to SEQ ID NO. 10, and a heavy chain FR4 sequence identical to SEQ ID NO. 13; and the light chain variable region comprises a light chain FR1 sequence identical to SEQ ID NO. 15, a light chain FR2 sequence identical to SEQ ID NO. 17, a light chain FR3 sequence identical to SEQ ID NO. 18, and a light chain FR4 sequence identical to SEQ ID NO. 19.

[0221] 1.3.22. Heavy Chain Variable Region (VH)

[0222] The heavy chain variable region of the above anti-CLDN6 antibody may be the same as sequence No. 20, sequence No. 21, or sequence No. 22; or may be an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 20, sequence No. 21, or sequence No. 20. Sequence No. 21 is a sequence in which the tyrosine (Y) at position 95 of sequence No. 20 is substituted with phenylalanine (F). Sequence No. 22 is a sequence in which the arginine (R) at position 38 of Sequence No. 20 is substituted with lysine (K), the valine (V) at position 68 is substituted with alanine (A), and the tyrosine (Y) at position 95 is substituted with phenylalanine (F).

[0223] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 20. In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 21. In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 22.

[0224] In some embodiments, the anti-CLDN6 antibody has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22, and comprises a heavy chain variable region including HCDR1 of SEQ ID NO. 1; HCDR2 of SEQ ID NO. 2; and HCDR3 of SEQ ID NO. 3.

[0225] In a specific embodiment, the anti-CLDN6 antibody has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 20, and comprises a heavy chain variable region including HCDR1 of SEQ ID NO. 1; HCDR2 of SEQ ID NO. 2; and HCDR3 of SEQ ID NO. 3. For example, the heavy chain variable region of the anti-CLDN6 antibody may include an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 20, wherein HCDR1 of SEQ ID NO. 1 is included at the 31st to 35th position relative to SEQ ID NO. 20, HCDR2 of SEQ ID NO. 2 is included at the 50th to 66th position relative to SEQ ID NO. 20, and HCDR3 of SEQ ID NO. 3 is included at the 99th to 112th position relative to SEQ ID NO. 20.

[0226] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; an HCDR1 corresponding to the 31st to 35th amino acid sequences of SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; and an HCDR2 corresponding to the 50th to 66th amino acid sequences of SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22. It includes HCDR3 corresponding to the 99th to 112th amino acid sequence of SEQ ID NO 20, SEQ ID NO 21, or SEQ ID NO 22.

[0227] In a specific embodiment, the anti-CLDN6 antibody comprises a heavy chain variable region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 20, and the heavy chain variable region comprises HCDR1 corresponding to the 31st to 35th amino acid sequence of SEQ ID NO. 20; HCDR2 corresponding to the 50th to 66th amino acid sequence of SEQ ID NO. 20; and HCDR3 corresponding to the 99th to 112th amino acid sequence of SEQ ID NO. 20.

[0228] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-CLDN6 antibody is QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFNMHWVXQAPGQRLEWIGYIYPLNGGTKYNQKFRGRXTITADTSASTAYMELSSLRSEDTAVYXCARWGFYDYSTFYAMDYWGQGTLVTVSS (SEQ No. 81). In SEQ No. 81, X at position 38 is arginine (R) or lysine (K). In SEQ No. 81, X at position 68 is valine (V) or alanine (A). In SEQ No. 81, X at position 95 is tyrosine (Y) or phenylalanine (F).

[0229] 1.3.23. Light chain variable region (VL)

[0230] The light chain variable region of the above anti-CLDN6 antibody may be a sequence identical to SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25; or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25, or may include the same. SEQ ID NO. 23 is a sequence in which the serine (S) at position 22 of SEQ ID NO. 25 is substituted with threonine (T), and the phenylalanine (F) at position 40 is substituted with tyrosine (Y). Sequence No. 24 is a sequence in which the serine (S) at position 22 of Sequence No. 25 is substituted with threonine (T).

[0231] In some embodiments, the anti-CLDN6 antibody comprises a light chain variable region sequence identical to SEQ ID NO. 23. In some embodiments, the anti-CLDN6 antibody comprises a light chain variable region sequence identical to SEQ ID NO. 24. In some embodiments, the anti-CLDN6 antibody comprises a light chain variable region sequence identical to SEQ ID NO. 25.

[0232] In some embodiments, the anti-CLDN6 antibody has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25, and comprises a light chain variable region including LCDR1 of SEQ ID NO. 4; LCDR2 of SEQ ID NO. 5; and LCDR3 of SEQ ID NO. 6.

[0233] In a specific embodiment, the anti-CLDN6 antibody has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 25, and comprises a light chain variable region including LCDR1 of SEQ ID NO. 4; LCDR2 of SEQ ID NO. 5; and LCDR3 of SEQ ID NO. 6. For example, the light chain variable region of the anti-CLDN6 antibody may include an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 25, wherein it includes an LCDR1 of SEQ ID NO. 4 at the 24th to 38th position relative to SEQ ID NO. 25, an LCDR2 of SEQ ID NO. 5 at the 54th to 60th position relative to SEQ ID NO. 25, and an LCDR3 of SEQ ID NO. 6 at the 93rd to 101st position relative to SEQ ID NO. 25.

[0234] In some embodiments, the anti-CLDN6 antibody comprises a light chain variable region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25, and the light chain variable region comprises an LCDR1 corresponding to the 24th to 38th amino acid sequence of SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25; an LCDR2 corresponding to the 54th to 60th amino acid sequence of SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25; and an LCDR3 corresponding to the 93rd to 101st amino acid sequence of SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25.

[0235] In a specific embodiment, the anti-CLDN6 antibody comprises a light chain variable region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 25, wherein the light chain variable region comprises an LCDR1 corresponding to the 24th to 38th amino acid sequence of SEQ ID NO. 25; an LCDR2 corresponding to the 54th to 60th amino acid sequence of SEQ ID NO. 25; and an LCDR3 corresponding to the 93rd to 101st amino acid sequence of SEQ ID NO. 25.

[0236] In some embodiments, the amino acid sequence of the light chain variable region of the anti-CLDN6 antibody is DIVLTQSPASLAVSPGQRATIXCRASKSVSTSGYSYFHWXQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQHSRELPFTFGGGTKLEIK (SEQ ID NO. 82). In SEQ ID NO. 82, X at position 22 is serine (S) or threonine (T). In SEQ ID NO. 82, X at position 40 is phenylalanine (F) or tyrosine (Y).

[0237] 1.3.24. Combination of VH and VL

[0238] The variable region of the above anti-CLDN6 antibody includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, or SEQ ID NO. 81, and the amino acid sequence of the light chain variable region may be SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, or SEQ ID NO. 82.

[0239] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 81 and a light chain variable region sequence identical to SEQ ID NO. 82.

[0240] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 20 and a light chain variable region sequence identical to SEQ ID NO. 23.

[0241] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 20 and a light chain variable region sequence identical to SEQ ID NO. 24.

[0242] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 20 and a light chain variable region sequence identical to SEQ ID NO. 25.

[0243] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 21 and a light chain variable region sequence identical to SEQ ID NO. 23.

[0244] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 21 and a light chain variable region sequence identical to SEQ ID NO. 24.

[0245] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 21 and a light chain variable region sequence identical to SEQ ID NO. 25.

[0246] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 22 and a light chain variable region sequence identical to SEQ ID NO. 23.

[0247] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 22 and a light chain variable region sequence identical to SEQ ID NO. 24.

[0248] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 22 and a light chain variable region sequence identical to SEQ ID NO. 25.

[0249] As a preferred example, the anti-CLDN6 antibody comprises a heavy chain variable region sequence identical to SEQ ID NO. 20 and a light chain variable region sequence identical to SEQ ID NO. 25.

[0250] 1.3.25. Table 1

[0251] Table 1 below relates to the CDR sequence, FR sequence, heavy chain variable region sequence, and light chain variable region sequence of the anti-CLDN6 antibodies of the present disclosure.

[0252] [Table 1] Amino acid sequences of CDR, FR, heavy chain variable region, and light chain variable region

[0253]

[0254]

[0255]

[0256] 1.4. Constant Region of Anti-CLDN6 Antibody

[0257] 1.4.1. Overview of Invariants

[0258] The constant region of the antibody includes a heavy chain constant region (CH) and a light chain constant region (CL).

[0259] In some embodiments, the anti-CLDN6 antibody of the present disclosure comprises a heavy chain constant region. For example, the anti-CLDN6 antibody of the present disclosure comprises a heavy chain, wherein the heavy chain may further comprise a heavy chain constant region in addition to a heavy chain variable region.

[0260] The above heavy chain constant region may include three immunoglobulin domains. The three immunoglobulin domains included in the heavy chain constant region are respectively referred to as heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3). The immunoglobulin domains in the heavy chain constant region are located in the order of CH1, CH2, and CH3 from the N-terminus to the C-terminus. Here, the N-terminus of the heavy chain constant region 1 (CH1) is connected to the C-terminus of the heavy chain constant region.

[0261] In some embodiments, the anti-CLDN6 antibody of the present disclosure comprises a light chain constant region. For example, the anti-CLDN6 antibody of the present disclosure comprises a light chain, wherein the light chain may further comprise a light chain constant region in addition to a light chain variable region. The light chain constant region comprises one immunoglobulin domain. The N-terminus of the light chain constant region may be connected to the C-terminus of the light chain variable region.

[0262] 1.4.2. Heavy Chain Invariant Region (CH)

[0263] The heavy chain constant region of the anti-CLDN6 antibody may be the constant region of human IgG. In some embodiments, the heavy chain constant region of the anti-CLDN6 antibody is the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4; or a variant sequence thereof. In some embodiments, the type of the heavy chain constant region of the anti-CLDN6 antibody is gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), or gamma4 (γ4).

[0264] In some embodiments, the heavy chain constant region of the anti-CLDN6 antibody is the heavy chain constant region of human IgG1. For example, the anti-CLDN6 antibody may include the heavy chain constant region of SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, or SEQ ID NO. 30, or a heavy chain constant region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, or SEQ ID NO. 30. In a preferred example, the anti-CLDN6 antibody may include the heavy chain constant region of SEQ ID NO. 26.

[0265] SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, or SEQ ID NO. 30 are sequences of the heavy chain constant region of antibodies commonly used in the art. SEQ ID NO. 26 is the heavy chain constant region sequence of wild-type human IgG1 (see UniProt Accession P01857). SEQ ID NO. 27 corresponds to the heavy chain constant region sequence of the known IgG1 antibody Herceptin. SEQ ID NO. 28 corresponds to the heavy chain constant region sequence of the known IgG1 antibody atezolizumab. SEQ ID NO. 29 corresponds to the heavy chain constant region sequence of the known IgG1 antibody durvalumab. SEQ ID NO. 30 is an IgG1 sequence in which the N297A substitution (EU numbering) is introduced into SEQ ID NO. 26, which is also well known in the art and which is known to inhibit FcγR binding ability.

[0266] In some embodiments, the heavy chain constant region of the anti-CLDN6 antibody is the heavy chain constant region of human IgG2. For example, the anti-CLDN6 antibody may include the heavy chain constant region of SEQ ID NO. 31 or a heavy chain constant region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 31.

[0267] In some embodiments, the heavy chain constant region of the anti-CLDN6 antibody is the heavy chain constant region of human IgG3. For example, the anti-CLDN6 antibody may include the heavy chain constant region of SEQ ID NO. 32 or a heavy chain constant region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 32.

[0268] In some embodiments, the heavy chain constant region of the anti-CLDN6 antibody is the heavy chain constant region of human IgG4. For example, the anti-CLDN6 antibody may include the heavy chain constant region of SEQ ID NO. 33 or SEQ ID NO. 34 or a heavy chain constant region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 33 or SEQ ID NO. 34.

[0269] 1.4.3. Light Chain Invariant Region (CL)

[0270] The type of light chain constant region of the above anti-CLDN6 antibody may be kappa (κ) type or lambda (λ) type.

[0271] In some embodiments, the light chain constant region of the anti-CLDN6 antibody is a human IgG kappa light chain. For example, the anti-CLDN6 antibody may include the kappa light chain constant region of SEQ ID NO. 35 or a kappa light chain constant region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 35.

[0272] In some embodiments, the light chain constant region of the anti-CLDN6 antibody is a human IgG lambda light chain. For example, the anti-CLDN6 antibody may include the lambda light chain constant region of SEQ ID NO. 36 or a lambda light chain constant region having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 36.

[0273] 1.4.4. Table 2

[0274] Table 2 below relates to the heavy chain constant region and light chain constant region sequences of the anti-CLDN6 antibodies of the present disclosure.

[0275] [Table 2] Amino acid sequences of the heavy chain invariant region and the light chain invariant region

[0276]

[0277]

[0278] 1.5. Heavy and Light Chains of Anti-CLDN6 Antibody

[0279] 1.5.1. Overview of Heavy and Light Chains

[0280] Antibodies contain a heavy chain and a light chain that are separated into different chains, and generally contain two heavy chains and two light chains.

[0281] In some embodiments, the anti-CLDN6 antibody of the present disclosure comprises a heavy chain and a light chain. For example, the anti-CLDN6 antibody of the present disclosure may comprise two heavy chains and two light chains. Each heavy chain has a polypeptide chain structure and may comprise four immunoglobulin domains (VH, CH1, CH2, and CH3). Each light chain has a polypeptide chain structure and may comprise two immunoglobulin domains (VL and CL). The heavy chains and light chains may be connected by a disulfide bond. Specifically, they may be connected by a disulfide bond between the CH1 of the constant region of the heavy chain and the constant region of the light chain.

[0282] 1.5.2. Reprint

[0283] The amino acid sequence of the heavy chain of the above anti-CLDN6 antibody is the same as sequence No. 37, sequence No. 38, or sequence No. 39; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence No. 37, sequence No. 38, or sequence No. 39, or may include the same.

[0284] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 37. In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 38. In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 39.

[0285] 1.5.3. Light Chain

[0286] The amino acid sequence of the light chain of the above anti-CLDN6 antibody is the same as sequence number 40, sequence number 41, or sequence number 42; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence number 40, sequence number 41, or sequence number 42, or may include the same.

[0287] In some embodiments, the anti-CLDN6 antibody comprises a light chain sequence identical to SEQ ID NO. 40. In some embodiments, the anti-CLDN6 antibody comprises a light chain sequence identical to SEQ ID NO. 41. In some embodiments, the anti-CLDN6 antibody comprises a light chain sequence identical to SEQ ID NO. 42.

[0288] 1.5.4. Combination of Heavy and Light Chains

[0289] The above anti-CLDN6 antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39, and the amino acid sequence of the light chain may be SEQ ID NO. 40, SEQ ID NO. 41, or SEQ ID NO. 42.

[0290] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 37 and a light chain sequence identical to SEQ ID NO. 40.

[0291] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 37 and a light chain sequence identical to SEQ ID NO. 41.

[0292] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 37 and a light chain sequence identical to SEQ ID NO. 42.

[0293] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 38 and a light chain sequence identical to SEQ ID NO. 40.

[0294] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 38 and a light chain sequence identical to SEQ ID NO. 41.

[0295] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 38 and a light chain sequence identical to SEQ ID NO. 42.

[0296] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 39 and a light chain sequence identical to SEQ ID NO. 40.

[0297] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 39 and a light chain sequence identical to SEQ ID NO. 41.

[0298] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 39 and a light chain sequence identical to SEQ ID NO. 42.

[0299] As a preferred example, the anti-CLDN6 antibody comprises a heavy chain sequence identical to SEQ ID NO. 37 and a light chain sequence identical to SEQ ID NO. 42.

[0300] 1.5.5. Table 3

[0301] Table 3 below relates to the heavy chain and light chain sequences of the anti-CLDN6 antibodies of the present disclosure.

[0302] [Table 3] Amino acid sequences of heavy and light chains

[0303]

[0304]

[0305]

[0306] 1.6. Fab and Fc of Anti-CLDN6 Antibody

[0307] 1.6.1. Overview of Fab and Fc Regions

[0308] Antibodies include a fragment antigen-binding region (Fab region) that has the function of binding to antigens, and a fragment crystallizable region (Fc region) that is less related to antigen binding function.

[0309] In some embodiments, the anti-CLDN6 antibody of the present disclosure may comprise a fragment antigen-binding region. In some embodiments, the anti-CLDN6 antibody of the present disclosure may further comprise a fragment crystallizable region in addition to the fragment antigen-binding region.

[0310] The above Fab region is a portion that includes a part that binds to an antigen (antigen binding portion). The above Fab region includes a heavy chain variable region, CH1 of a heavy chain constant region, a light chain variable region, and a light chain constant region. Here, CH1 of the heavy chain variable region and the heavy chain constant region included in the Fab region may be referred to as the heavy chain portion of the Fab region. The light chain variable region and the light chain constant region included in the Fab region may be referred to as the light chain portion of the Fab region.

[0311] The above Fc region is a portion capable of binding to the Fc receptor. The above Fc region includes CH2 and CH3 of the heavy chain constant region.

[0312] The above Fab region and Fc region are connected by a hinge region. Specifically, the C-terminus of the heavy chain portion of the Fab region and the N-terminus of the Fc region may be connected by a hinge region. In some embodiments, the hinge region may be described as being included in the Fab region and is not otherwise limited. In some embodiments, the hinge region may be described as being included in the Fc region and is not otherwise limited. In some embodiments, a portion of the N-terminus of the hinge region may be described as being included in the Fab region and a portion of the C-terminus may be described as being included in the Fc region and is not otherwise limited.

[0313] 1.6.2. Fab Area

[0314] The Fab region of the above anti-CLDN6 antibody includes a heavy chain portion and a light chain portion, the amino acid sequence of the heavy chain portion of the Fab region is SEQ ID NO. 43, SEQ ID NO. 44, or SEQ ID NO. 45, and the amino acid sequence of the light chain portion of the Fab region may be SEQ ID NO. 40, SEQ ID NO. 41, or SEQ ID NO. 42.

[0315] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 43 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 40.

[0316] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 43 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 41.

[0317] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 43 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 42.

[0318] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 44 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 40.

[0319] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 44 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 41.

[0320] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 44 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 42.

[0321] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 45 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 40.

[0322] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 45 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 41.

[0323] In some embodiments, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 45 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 42.

[0324] In a preferred example, the anti-CLDN6 antibody comprises a heavy chain portion sequence of the Fab region identical to SEQ ID NO. 43 and a light chain portion sequence of the Fab region identical to SEQ ID NO. 42.

[0325] 1.6.3. Fc Region

[0326] The Fc region of the above anti-CLDN6 antibody may have an amino acid sequence identical to sequence number 46, sequence number 47, sequence number 48, sequence number 49, sequence number 50, sequence number 51, sequence number 52, or sequence number 53; or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with sequence number 46, sequence number 47, sequence number 48, sequence number 49, sequence number 50, sequence number 51, sequence number 52, or sequence number 53, or may include such a sequence.

[0327] As a preferred example, the anti-CLDN6 antibody includes the same Fc region as SEQ ID NO. 46.

[0328] 1.6.4. Table 4

[0329] [Table 4] Amino acid sequences of the Fab and Fc regions

[0330]

[0331]

[0332]

[0333] 1.7. Hinge region of anti-CLDN6 antibody

[0334] In some embodiments, the anti-CLDN6 antibody of the present disclosure may include a hinge region. In some embodiments, the hinge region connects the Fab region and the Fc region. In some embodiments, the hinge region connects CH1 and CH2. For example, the hinge region connects the C-terminus of CH1 and the N-terminus of CH2.

[0335] In some embodiments, the anti-CLDN6 antibody comprises a hinge region of SCDKTHTCPPCPAPELLGG (Sequence No. 55).

[0336] As a preferred example, the anti-CLDN6 antibody includes a hinge region sequence identical to SEQ ID NO. 55.

[0337] 1.8. Class of Anti-CLDN6 Antibodies

[0338] The class of anti-CLDN6 antibodies of the present disclosure may be immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), or immunoglobulin E (IgE).

[0339] Preferably, the class of the anti-CLDN6 antibody may be IgG. In some embodiments, the subclass of the anti-CLDN6 antibody may be IgG1, IgG2, IgG3, or IgG4. For example, if the type of heavy chain constant region of the anti-CLDN6 antibody is gamma1 (γ1), the subclass of the antibody is IgG1. For example, if the type of heavy chain constant region of the anti-CLDN6 antibody is gamma2 (γ2), the subclass of the antibody is IgG2. For example, if the type of heavy chain constant region of the anti-CLDN6 antibody is gamma3 (γ3), the subclass of the antibody is IgG3. For example, if the type of heavy chain constant region of the anti-CLDN6 antibody is gamma4 (γ4), the subclass of the antibody is IgG4.

[0340] In a preferred example, the class of the anti-CLDN6 antibody is IgG. In a preferred example, the subclass of the anti-CLDN6 antibody is IgG1. In a preferred example, the subclass of the anti-CLDN6 antibody is human IgG1.

[0341] 1.9. Types of Anti-CLDN6 Antibodies

[0342] The anti-CLDN6 antibody of the present disclosure may be a humanized antibody; a chimeric antibody; a human antibody; or a non-human antibody.

[0343]

[0344] In some specific embodiments, the anti-CLDN6 antibody may be a humanized antibody. Generally, antibodies obtained from non-human animals may cause immunogenicity issues when developing therapeutic agents for humans. To mitigate these problems, a method for producing humanized antibodies may be utilized by employing a humanization technique in which a portion of the sequence of a non-human animal antibody is replaced with the sequence of a human antibody (or the germline sequence of a human antibody). A representative method for producing humanized antibodies is CDR grafting, in which it is common practice to maintain the CDR sequence, which determines antigen binding specificity, while replacing the remaining sequence (framework sequence, constant region sequence, etc.) with the germline sequence of a human antibody.

[0345] The anti-CLDN6 antibody of the present disclosure may be a humanized antibody in which only the CDR sequence of a non-human antibody (e.g., a murine antibody) that binds to a human CLDN6 protein is preserved, and other sequences (e.g., sequences other than the CDR within a variable region) are replaced with germline sequences for the human antibody. In some embodiments, a humanization method for obtaining the humanized antibody (anti-CLDN6 antibody) of the present disclosure may include the following steps (1) to (3):

[0346] (1) Obtain the light chain variable region sequence and the light chain variable region sequence of a non-human antibody (e.g., murine antibody) or chimeric antibody that binds to human CLDN6 protein. Obtain the CDR sequence and framework sequence from the light chain variable region sequence and the light chain variable region sequence using Kabat, Chothia, IMGT numbering system, etc.

[0347] (2) Refer to a public DNA database or literature disclosing germline antibody gene sequences to select a human antibody framework sequence that is structurally similar to the framework sequence of the non-human antibody or chimeric antibody that binds.

[0348] (3) Transplant the CDR sequence of the murine anti-CLDN6 antibody into the selected human antibody framework sequence.

[0349] In the above humanization method, a back mutation process may be additionally performed to reintroduce into the human framework a residue of the murine anti-CLDN6 antibody that plays an important role in antigen binding. Generally, this back mutation process may be performed after (3) of the above-described process, but is not limited thereto.

[0350] The humanization method may further include the step of obtaining a non-human antibody that binds to the human CLDN6 protein that is the target of humanization in the method. In some embodiments, the humanization method may further include the step of obtaining an anti-CLDN6 antibody from the plasma of a non-human animal after immunizing the non-human animal with the human CLDN6 protein. Specifically, the humanization method may further include the step of administering the human CLDN6 protein to a murine or mouse, collecting plasma after a certain period of time, and isolating a murine antibody capable of binding to the human CLDN6 protein from the plasma.

[0351] In this case, the humanized antibody may have the constant region sequence and the variable region FR sequence derived from a human immunoglobulin sequence, and the variable region CDR sequence derived from a non-human animal (e.g., murine) immunoglobulin sequence.

[0352] In this case, the constant region sequence of the humanized anti-CLDN6 antibody may be one of the known constant region sequences of human IgG1, IgG2, IgG3, or IgG4.

[0353] Humanization methods are well known in the art, and references may be made to the literature in which the full contents of each are incorporated herein by reference [Riechmann, L., Clark, M., Waldmann, H., & Winter, G. (1988). Reshaping human antibodies for therapy. Nature, 332(6162), 323-327.]; [U.S. Patent No. 5,530,101]; [U.S. Patent No. 6,180,370]; [Almagro, JC, & Fransson, J. (2008). Humanization of antibodies. Front Biosci, 13(1), 1619-33.]; and [Safdari, Y., Farajnia, S., Asgharzadeh, M., & Khalili, M. (2013). Antibody humanization methods-a review and update. Biotechnology and Genetic Engineering Reviews, 29(2), 175-186.].

[0354] In some embodiments, one or more of the other regions excluding the CDRs of anti-CLDN6 (e.g., the framework region of the heavy chain variable region, the framework region of the light chain variable region, CH1, CH2, CH3, CL) may be of human origin. In one embodiment, one or more of the other regions excluding the CDRs of anti-CLDN6 may be of human origin.

[0355] In some embodiments, the anti-CLDN6 antibody may be a human antibody. The human antibody may have an amino acid sequence in which all amino acids constituting the antibody are derived from a human immunoglobulin sequence.

[0356] In some embodiments, the anti-CLDN6 antibody may be a non-human antibody. The non-human antibody may have an amino acid sequence in which all amino acid sequences constituting the antibody are derived from a non-human immunoglobulin sequence. For example, the sequence of the non-human antibody may be a mouse antibody derived from a mouse immunoglobulin sequence.

[0357] In some embodiments, the anti-CLDN6 antibody may be a chimeric antibody. The chimeric antibody may have a variable region sequence derived from a non-human animal immunoglobulin sequence and a constant region derived from a human immunoglobulin sequence.

[0358] In some embodiments, the anti-CLDN6 antibody may not be a human antibody.

[0359] In some embodiments, the anti-CLDN6 antibody may not be a non-human antibody.

[0360] In some embodiments, the anti-CLDN6 antibody may not be a chimeric antibody.

[0361]

[0362] In a preferred embodiment, the anti-CLDN6 antibody may be a humanized antibody. Specifically, the constant region sequence and the variable region FR sequence of the anti-CLDN6 antibody may be derived from a human immunoglobulin sequence, and the variable region CDR sequence may be derived from a murine immunoglobulin sequence. In this case, the variable region FR sequence may contain a substitution mutation in the sequence derived from the human immunoglobulin sequence.

[0363] In some embodiments, the sequence of the region excluding the complementarity-determining region (CDR) in the Fab region of the anti-CLDN6 antibody is humanized. Specifically, the sequence of the region excluding the complementarity-determining region in the Fab region of the anti-CLDN6 antibody is derived from a human immunoglobulin sequence. In this case, the complementarity-determining region of the anti-CLDN6 antibody may be derived from a murine immunoglobulin sequence.

[0364] In some specific examples, the antigen-binding portion of the anti-CLDN6 antibody may be humanized.

[0365] In some embodiments, the heavy chain variable region of the anti-CLDN6 antibody may be a humanized heavy chain variable region. In some embodiments, the light chain variable region of the anti-CLDN6 may be a humanized light chain variable region.

[0366] In some embodiments, the sequence of the region excluding the complementary-determining region in the variable region of the anti-CLDN6 antibody is humanized. Specifically, the sequence of the region excluding the complementary-determining region in the variable region of the anti-CLDN6 antibody is derived from a human immunoglobulin sequence. In this case, the complementary-determining region of the anti-CLDN6 antibody may be derived from a murine immunoglobulin sequence.

[0367] In some embodiments, the sequences of the framework regions (HFR1, HFR2, HFR3, HFR4) of the heavy chain variable region of the anti-CLDN6 antibody are humanized. Specifically, the sequences of the framework regions of the heavy chain variable region of the anti-CLDN6 antibody are derived from human immunoglobulin sequences.

[0368] In some embodiments, the sequences of the framework regions (LFR1, LFR2, LFR3, LFR4) of the light chain variable region of the anti-CLDN6 antibody are humanized. Specifically, the sequences of the framework regions of the light chain variable region of the anti-CLDN6 antibody are derived from human immunoglobulin sequences.

[0369] In some embodiments, the constant region sequence of the anti-CLDN6 antibody is humanized. Specifically, the constant region sequence of the anti-CLDN6 antibody is derived from a human immunoglobulin sequence.

[0370] In some embodiments, the anti-CLDN6 antibody may be a humanized antibody obtained by applying a CDR grafting method based on the sequence of an anti-CLDN6 antibody obtained from a mouse. For example, the variable region sequence of the anti-CLDN6 antibody may be obtained by performing CDR grafting using a human framework sequence available from a public DNA database or literature disclosing germline antibody gene sequences (e.g., Tomlinson, IM et al., J. Mol. Biol. 227:776-798, 1992; Cox, JPL et al., Eur. J. Immunol. 24:827-836, 1994).

[0371]

[0372] 1.10. Binding ability to human CLDN6 protein

[0373] The anti-CLDN6 antibody of the present disclosure can bind to the human CLDN6 protein.

[0374] In some embodiments, the anti-CLDN6 antibody can specifically bind to the human CLDN6 protein.

[0375] In some embodiments, the anti-CLDN6 antibody may be a monoclonal antibody capable of binding to the human CLDN6 protein.

[0376] In some embodiments, the anti-CLDN6 antibody may be a polyclonal antibody capable of binding to human CLDN6 protein.

[0377] 1.11. Antigen-binding fragment of anti-CLDN6 antibody

[0378] In one aspect of the present disclosure, an antigen-binding fragment of an anti-CLDN6 antibody is disclosed. The antigen-binding fragment of the anti-CLDN6 antibody comprises a portion of the anti-CLDN6 antibody of the present disclosure, and the antigen-binding fragment of the anti-CLDN6 antibody may have any type of form capable of binding to a human CLDN6 protein. Preferably, the antigen-binding fragment of the anti-CLDN6 antibody may comprise an antigen-binding portion of the anti-CLDN6 antibody of the present disclosure. The antigen-binding fragment of the anti-CLDN6 antibody of the present disclosure comprises a portion of any one of the anti-CLDN6 antibodies described in sections <<1.1. Overview of Anti-CLDN6 Antibodies>> through <<1.10. Binding Ability to Human CLDN6 Protein>> above. Below, examples of structures that the antigen-binding fragment of the anti-CLDN6 antibody may have are described, but are not limited thereto.

[0379] In some embodiments, the antigen-binding fragment of the anti-CLDN6 antibody may include six CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3). The variable region of the antigen-binding fragment of the anti-CLDN6 antibody may include six CDRs.

[0380] In some embodiments, the antigen-binding fragment of the anti-CLDN6 antibody may include a heavy chain variable region and a light chain variable region. For example, the antigen-binding fragment of the anti-CLDN6 antibody may include one heavy chain variable region and one light chain variable region. For example, the antigen-binding fragment of the anti-CLDN6 antibody may include two heavy chain variable regions and two light chain variable regions. In this case, the heavy chain variable region of the antigen-binding fragment may include three CDRs (e.g., HCDR1, HCDR2, HCDR3). In this case, the light chain variable region of the antigen-binding fragment may include three CDRs (e.g., LCDR1, LCDR2, LCDR3).

[0381] In some embodiments, the antigen-binding fragment of the anti-CLDN6 antibody may be selected from the group consisting of F(ab), F(ab'), F(ab')2, F(ab')2, single-chain variable fragment (scFv), and scFv-Fc.

[0382] The above F(ab) means having a structure that includes the Fab region of the antibody and has only one antigen binding site.

[0383] The above F(ab') means having a structure including the Fab region of the antibody, and further including a hinge region containing one or more cysteine ​​residues at the C-terminus of the Fab region heavy chain portion compared to the Fab region.

[0384] The above F(ab')2 means that two F(ab') are connected. In this case, the connection of the two F(ab') is due to a disulfide bond of a cysteine ​​residue in the hinge region, and the two connected F(ab') may be identical or different. The above F(ab')2 means that the antigen-binding portions of the two connected F(ab') of F(ab')2 can bind to the same antigen.

[0385] The above single-chain variable fragment (scFv) refers to a fragment comprising a heavy chain variable region and a light chain variable region of an antibody, and does not include an immunoglobulin domain belonging to the constant region of the antibody. In this case, the heavy chain variable region and the light chain variable region included in the scFv are generally covalently bonded to each other through a peptide linker. Specifically, the peptide linker connects the C-terminus of the heavy chain variable region with the N-terminus of the light chain variable region, or connects the N-terminus of the heavy chain variable region with the C-terminus of the light chain variable region. When two or three scFvs are linked, there may be types such as tandem di-scFv, diabody, tandem tri-scFv, and tribody. The above scFv-Fc refers to a fragment comprising the scFv and the Fc region of the antibody.

[0386] 1.12. A molecule comprising an anti-CLDN6 antibody or its antigen-binding fragment

[0387] In one aspect of the present disclosure, a molecule that binds to a human CLDN6 protein is disclosed, comprising the anti-CLDN6 antibody of the present disclosure or an antigen-binding fragment thereof. The molecule that binds to the human CLDN6 protein comprises the anti-CLDN6 antibody of the present disclosure or a portion thereof, and the molecule that binds to the human CLDN6 protein may have any form capable of binding to the human CLDN6 protein. In this case, the molecule that binds to the human CLDN6 protein may comprise any one of the anti-CLDN6 antibody or the antigen-binding fragments thereof described in the sections <<1.1. Overview of Anti-CLDN6 Antibody>> to <<1.11. Antigen-binding Fragment of Anti-CLDN6 Antibody>> above.

[0388] In some embodiments, the molecule binding to the human CLDN6 protein comprises the anti-CLDN6 antibody in which some amino acid residues are modified.

[0389]

[0390] In some embodiments, the molecule binding to the human CLDN6 protein is an immunoconjugate or a conjugate. In some embodiments, the immunoconjugate comprises the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment. In some embodiments, the immunoconjugate comprises the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment and an effector molecule. The immunoconjugate refers to an immunoconjugate in which an effector molecule is directly or indirectly conjugated (or linked) to the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment. Such conjugation may be achieved by chemical or recombinant means. In some embodiments, the conjugation is a chemical linkage in which a covalent bond is formed between two molecules by a reaction between the antibody portion and the effector molecule to form a single molecule. In some embodiments, a linker may be included between the antibody and the effector molecule. In some embodiments, the linker may include, but is not limited to, a peptide linker (short peptide sequence). In some embodiments, the effector molecule may be a detectable label, immunotoxin, cytokine, chemokine, or (che)therapeutic agent.

[0391] Examples of the above immunotoxins include abrin, lysine, Pseudomonas exotoxin (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, colix toxin, or modified toxins thereof; or other toxic substances that directly or indirectly inhibit cell growth or cause cell death, but are not limited thereto.

[0392] Examples of the above cytokines include interferons (e.g., IFN-α, IFN-β, and IFN-γ); members of the tumor necrosis factor superfamily (TNFSF); human growth hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle-stimulating hormone (FSH); thyroid-stimulating hormone (TSH); luteinizing hormone (LH); hepatic growth factor; prostaglandins, fibroblast growth factor; prolactin; placental lactogen, OB protein; TNF-α; TNF-β; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; TGF-α; TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); or colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); interleukin (IL-1 to IL-21); FLT-3; angiostatin; thrombospondin or endostatin are included, but not limited to, these.

[0393] Examples of the above chemokines include, but are not limited to, RANTES, MCAF, MCP-1, and fractalkines.

[0394] Examples of the above therapeutic agents (or chemotherapy agents) include auristatin, dolastatin, AFP, mytansinoids AEB, AEVB, doxorubicin, daunorubicin, methotrexate, melphalan, chlorambucil, vinca alkaloids, 5-fluorouridine, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, nitrogen mustard, cyclosan, etoposide, BCNU, topoisomerase I inhibitors bleomycin, idarubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel; or their salts, solvents, and derivatives are included, but not limited to.

[0395] The effector molecule may be linked to the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment using various means known to those skilled in the art. Covalent and / or non-covalent means may be used for conjugation. The method of conjugating the effector molecule to the antibody depends on the chemical structure of the effector molecule. If the effector molecule is a polypeptide, it generally contains various functional groups such as a carboxylic acid group (-COOH), a free amine group (-NH2), or a sulfhydryl group (-SH), and these functional groups may react with the functional groups of the antibody to induce the binding of the effector molecule. Alternatively, the antibody may be derivatized to expose or attach additional reactive functional groups. Derivatization may involve attaching various linker molecules, such as the linker molecules sold by Pierce Chemical Company, Rockford, Ill. The linker may be any molecule used to link the antibody to the effector molecule. The linker may also form covalent bonds to both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linker may be connected to an amino acid residue of the polypeptide through a side chain (e.g., a disulfide bond to a cysteine ​​residue) or to an alpha-carbon amino group or a carboxyl group of the terminal amino acid of the polypeptide.

[0396] In some embodiments, an effector molecule may be detached from the antibody when the immunoconjugate reaches the target site. In such cases, the immunoconjugate may include a linker that can be cleaved near the target site. The cleavage of the linker to detach the effector molecule from the antibody may be facilitated by enzymatic activity or conditions that the immunoconjugate receives inside the target cell or near the target site.

[0397] In some embodiments, the immunoconjugate maintains the immunoreactivity of the anti-CLDN6 antibody or its antigen-binding fragment. For example, the ability of the anti-CLDN6 antibody or its antigen-binding fragment to bind to the antigen before and after conjugation may be nearly the same or slightly reduced.

[0398] The immunoconjugate of the present disclosure may be an antibody-drug conjugate (ADC). The antibody-drug conjugate (ADC) combines the binding specificity of an antibody with the efficacy of a drug, such as a cytotoxic agent, anticancer agent, or immunosuppressant. While the use of the antibody-drug conjugate enables target-specific delivery of the drug, administering the unconjugated drug may result in unacceptable levels of toxicity to normal cells. The mechanism of the antibody-drug conjugate involves the antibody recognizing and binding to a specific antigen to trigger a series of reactions, after which it enters the cytoplasm via endocytosis. The linker is cleaved by intracellular lysosomal enzymes, separating the highly cytotoxic antibody and potentially killing cancer cells. Unlike conventional chemotherapy, which causes indiscriminate damage to both cancer cells and normal tissues, targeted drug delivery allows the drug to act specifically on cancer cells, thereby reducing damage to normal cells.

[0399] In some embodiments, the antibody-drug conjugate of the present disclosure has the following chemical formula: Ab-(LD)n, where Ab is the anti-CLDN6 antibody of the present disclosure, L is the linker, D is the drug portion, and n is an integer from 1 to 8.

[0400]

[0401] In some embodiments, the molecule binding to the human CLDN6 protein is a bispecific molecule or a multispecific molecule. The bispecific molecule or multispecific molecule means a molecule that is derivatized or linked to another functional molecule, for example, another peptide or protein (e.g., another antibody or ligand against a receptor), to the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment, and is capable of binding to at least two different binding sites or target molecules.

[0402] For example, the bispecific molecule may be a bispecific antibody. In some embodiments, the bispecific antibody comprises the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment. In some embodiments, the bispecific antibody may further comprise another antibody or its antigen-binding fragment in addition to the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment. The other antibody or its antigen-binding fragment may have binding specificity to an antigen other than CLDN6. For example, the bispecific antibody may further comprise an antibody or antigen-binding fragment that binds to an antigen other than CLDN6 in addition to the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment. For example, the bispecific antibody may have binding specificity to at least two different antigens. For example, it may have binding specificity to at least two different epitopes.

[0403]

[0404] In some embodiments, the molecule binding to the human CLDN6 protein comprises a variable region of the anti-CLDN6 antibody.

[0405] In some embodiments, the molecule binding to the human CLDN6 protein comprises the CDR sequence of the anti-CLDN6 antibody.

[0406] In some embodiments, the molecule binding to the human CLDN6 protein comprises one selected from the group consisting of (ab), F(ab'), F(ab')2, F(ab')2 single-chain variable fragment (scFv), and scFv-Fc.

[0407] In some embodiments, the molecule binding to the human CLDN6 protein is F(ab), F(ab'), F(ab')2, F(ab') 2, It was selected from the group consisting of single-chain variable fragments (scFv) and scFv-Fc.

[0408] 1.13. Composition comprising an anti-CLDN6 antibody or an antigen-binding fragment thereof

[0409] In one aspect of the present disclosure, a composition or kit is provided comprising: the anti-CLDN6 antibody of the present disclosure; an antigen-binding fragment of the anti-CLDN6 antibody; or a molecule comprising the anti-CLDN6 antibody or the antigen-binding fragment of the anti-CLDN6 antibody. The composition (or kit) may further comprise any carrier.

[0410] In some embodiments, the composition (or kit) comprises a plurality of anti-CLDN6 antibodies. In this case, the plurality of anti-CLDN6 antibodies may be the anti-CLDN6 antibodies of the present disclosure and may be the same antibody.

[0411] In some embodiments, the composition comprises a plurality of antigen-binding fragments of an anti-CLDN6 antibody. In this case, the plurality of antigen-binding fragments of the anti-CLDN6 antibody may be antigen-binding fragments of the anti-CLDN6 antibody of the present disclosure and may be identical antigen-binding fragments.

[0412] In some embodiments, the composition comprises a molecule comprising a plurality of anti-CLDN6 antibodies. In this case, the molecule comprising the plurality of anti-CLDN6 antibodies may be the molecule comprising the anti-CLDN6 antibody of the present disclosure and may be identical to each other.

[0413] In some embodiments, the composition comprises a molecule comprising a plurality of antigen-binding fragments of an anti-CLDN6 antibody. In this case, the molecule comprising the antigen-binding fragments of the anti-CLDN6 antibody may be the molecule comprising the antigen-binding fragments of the anti-CLDN6 antibody of the present disclosure, and may be identical to each other.

[0414] In some embodiments, the composition may be used to produce an anti-CLDN6 antibody or a molecule (e.g., an immunoconjugate) comprising an antigen-binding fragment thereof.

[0415] In some embodiments, the carrier is one that is conventionally used in formulations and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other conventional additives such as antioxidants, buffers, etc., as needed. In some embodiments, the carrier is a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax; a sweetener; a flavoring agent; a syrup; a liquid carrier such as a fatty oil; a sterile aqueous solution; propylene glycol; polyethylene glycol; an injectable ester such as ethyl oleate; It may include suspensions; emulsions; freeze-dried preparations; topical agents; stabilizers; buffers; animal oils; vegetable oils; waxes; paraffins; starches; tracanths; cellulose derivatives; polyethylene glycol; silicones; bentonite; silica; talc; zinc oxide, or combinations thereof.

[0416]

[0417] 2. Advantages of Anti-CLDN6 Antibodies

[0418] 2.1. Overview of the Advantages of Antibodies

[0419] The anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may bind to the human Claudin-6 protein. In this case, the amino acid sequence of the human Claudin-6 protein may be SEQ ID NO. 56.

[0420] In particular, the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may bind relatively strongly to the human Claudin 6 protein represented by SEQ ID NO. 56, whereas may bind relatively weakly to the human Claudin 3 protein, human Claudin 4 protein, or human Claudin 9 protein. That is, the anti-CLDN6 antibody of the present disclosure has binding specificity or selective binding to the human Claudin 6 protein.

[0421] In addition, the anti-CLDN 6 antibody of the present disclosure or its antigen-binding fragment can be internalized into a cell expressing human CLDN6 protein.

[0422] Additionally, the anti-CLDN 6 antibody of the present disclosure or its antigen-binding fragment may have a limited cytotoxic effect mediated by the Fc region. As a result, the anti-CLDN 6 antibody of the present disclosure or its antigen-binding fragment may not, alone, exhibit direct anticancer activity in vivo. In one embodiment, the anti-CLDN 6 antibody of the present disclosure or its antigen-binding fragment may not, alone, exhibit anticancer activity in vivo in humans or murines (e.g., mice or rats). In one embodiment, the fact that the anti-CLDN 6 antibody of the present disclosure does not substantially exhibit anticancer activity in vivo may be due to the CLDN 6 antibody of the present disclosure not having Fc region-mediated cytotoxicity or having a low level of Fc region-mediated cytotoxicity, but is not otherwise limited.

[0423] 2.2. Binding ability to CLDN6

[0424] The anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment can bind to the human CLDN6 protein. In other words, the anti-CLDN6 antibody or its antigen-binding fragment can recognize or immunologically bind to the human CLDN6 protein.

[0425]

[0426] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment has a high binding affinity for the human CLDN6 protein. Here, high binding affinity means the binding dissociation constant (K between the antibody and the antigen) D　 This could mean that ) is lower.

[0427] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment has a binding dissociation constant (K) for the human CLDN6 protein D ) is approximately 1.00×10 -9 , 1.50×10 -9 , 2.00×10 -9 , 2.50×10 -9 , 3.00×10 -9 , 3.10×10 -9 , 3.20×10 -9 , 3.25×10 -9 , 3.30×10 -9 , 3.31×10 -9 , 3.32×10 -9 , 3.33×10 -9 , 3.34×10 -9 , 3.35×10 -9 , 3.36×10 -9 , 3.37×10 -9 , 3.38×10 -9 , 3.39×10 -9 , 3.40×10 -9 , 3.41×10 -9 , 3.42×10 -9 , 3.43×10 -9 , 3.44×10 -9 , 3.45×10 -9 , 3.46×10 -9 , 3.47×10 -9 , 3.48×10 -9 , 3.49×10 -9 , 3.50×10 -9 , 3.51×10 -9 , 3.52×10 -9 , 3.53×10 -9 , 3.54×10 -9 , 3.55×10 -9, 3.56×10 -9 , 3.57×10 -9 , 3.58×10 -9 , 3.59×10 -9 , 3.60×10 -9 , 3.61×10 -9 , 3.62×10 -9 , 3.63×10 -9 , 3.64×10 -9 , 3.65×10 -9 , 3.66×10 -9 , 3.67×10 -9 , 3.68×10 -9 , 3.69×10 -9 , 3.70×10 -9 , 3.71×10 -9 , 3.72×10 -9 , 3.73×10 -9 , 3.74×10 -9 , 3.75×10 -9 , 3.76×10 -9 , 3.77×10 -9 , 3.78×10 -9 , 3.79×10 -9 , 3.80×10 -9 , 3.81×10 -9 , 3.82×10 -9 , 3.83×10 -9 , 3.84×10 -9 , 3.85×10 -9 , 3.90×10 -9 , 4.00×10 -9 , 4.50×10 -9 , 5.00×10 -9 , 5.50×10 -9 , 6.00×10 -9 , 7.00×10 -9 , 8.00×10 -9 , or 9.00×10 -9 (M) or may be within the range of two numbers selected from the above numbers. For example, the anti-CLDN6 antibody or its antigen-binding fragment has a binding dissociation constant (K) for human CLDN6 protein. D ) is 3.5×10 -9(M) to 3.6×10 -9 (M) may be. As a preferred example, an anti-CLDN6 antibody or its antigen-binding fragment has a binding dissociation constant (K) for human CLDN6 protein D ) is 3.54×10 -9 (M) could be.

[0428] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment may have excellent binding affinity. Having excellent binding affinity means, for example, that the anti-CLDN6 antibody has a higher binding affinity for the same target than a specific comparison antibody. For example, the binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment for human CLDN6 may be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 times greater than the binding affinity of the comparison antibody.

[0429]

[0430] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment binds to human CLDN6 protein-expressing cells. For example, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to the human CLDN6 protein 50 The value may be low.

[0431] Here, cell binding EC 50The value refers to the concentration of an antibody that can produce a response corresponding to 50% of the maximum binding signal from a dose-response curve obtained by curve fitting the measured mean fluorescence intensity (MFI) to the antibody concentration, after contacting the antibody with the cell (or VLP) to measure the ability of a specific antibody to bind to a cell expressing an antigen or a virus-like particle(s); VLP) on the surface of which the antibody is present, and then measuring the amount of antibody bound to the cell surface (or VLP) using flow cytometry or an equivalent analysis method.

[0432] In some embodiments, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to the human CLDN6 protein 50 The value may be approximately 0.900, 0.950, 1.000, 1.050, 1.100, 1.150, 1.200, 1.250, 1.300, 1.350, 1.400, 1.450, 1.500, 1.550, 1.600, 1.650, 1.700, 1.750, 1.800, 1.850, 1.900, 1.950, 2.000, 2.050, 2.100, 2.150, or 2.200 (nM), or may be within the range of two numbers selected from the above numbers. For example, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein 50 The value may be 0.9 (nM) to 2.1 (nM). At this time, the cell binding EC 50The value is 1 x 10⁶ per well for cell lines expressing human CLDN6 protein (e.g., Claudin-6 CHO Cell Line (BPS Bioscience, 78527-M), Claudin-9 CHO Cell Line (BPS Bioscience, 78687), PA-1 (ATCC, CRL-1572), SNU-119 (KCLB, 00119), OVCAR-3 (ATCC, HTB-161), OV-90 (ATCC, CRL-3585), BeWo (ATCC, CCL-98), or OUMS-23 (JCRB, JCRB1022)). 5 After seeding cells, the cells were incubated with anti-CLDN6 antibody (concentration range of 100 nM to 0.38 pM) in FACS buffer (1X PBS containing 2% FBS) at 4°C for 1 hour; after washing the cells twice with FACS buffer, they were treated with a 1:1,000 ratio fluorescently conjugated secondary antibody diluted with FACS buffer and incubated at 4°C under dark conditions for 30 minutes; after washing the cells twice with FACS buffer and resuspending them in 200 μL of cold FACS buffer, the EC based on the results of analyzing the samples using a BD LSRFortessa™ X-20 flow cytometer (BD biosciences) 50 It can be a value.

[0433] In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing CHO-K1 cells 50 The value may be 1.2 to 1.3 (nM) or 1.262 (nM). In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing PA-1 cells 50The value may be 1.5 to 1.6 (nM) or 1.572 (nM). In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing SNU-119 cells 50 The value may be 2 to 2.1 (nM) or 2.080 (nM). In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing OVCAR-3 cells 50 The value may be 1.1 to 1.2 (nM) or 1.157 (nM). In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing OV-90 cells 50 The value may be 0.9 to 1 (nM) or 0.923 (nM). In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing BeWo cells 50 The value may be 1.6 to 1.7 (nM) or 1.642 (nM). In one embodiment, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to human CLDN6 protein-expressing OUMS-23 cells 50 The value may be 1.3 to 1.4 (nM) or 1.335 (nM).

[0434]

[0435] In some embodiments, the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may have excellent target cell binding affinity. Having excellent target cell binding affinity means, for example, that the target cell binding affinity of the anti-CLDN6 antibody is higher than that of a specific comparison antibody. Target cell binding affinity can be measured through cell binding EC50, cell binding MFI, etc., as described above. For example, a lower cell binding EC50 value indicates higher target cell binding affinity. For example, a higher cell binding MFI indicates higher target cell binding affinity. Target cells are, for example, cells expressing human CLDN6. For example, target cells may be any one selected from CHO-K1 cells, PA-1 cells, SNU-119 cells, OVCAR-3 cells, OV-90 cells, BeWo cells, and OUMS-23 cells expressing human CLDN6.

[0436] In some embodiments, the target cell binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment may be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 times greater than the target cell binding affinity of the comparative antibody.

[0437] Here, cell binding MFI refers to the mean fluorescence intensity (MFI) measured by flow cytometry or an equivalent analysis method after contacting the antibody with the cell (or VLP) to measure the ability of a specific antibody to bind to a cell expressing an antigen (or a virus-like particle (VLP) on which an antigen is presented on the surface).

[0438] 2.3. Specificity for CLDN6

[0439] The above anti-CLDN6 antibody or its antigen-binding fragment may specifically bind to human CLDN6 protein. In the present disclosure, "specifically binds to human CLDN6 protein" means that the level of binding to human CLDN6 protein is significantly higher compared to binding to other antigens, or that it binds selectively only to human CLDN6 protein.

[0440] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment does not bind to cells that do not express or substantially do not express the human CLDN6 protein.

[0441] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment does not bind to normal cells in human adults. In this case, the normal cells may be cells that do not express or substantially do not express the human CLDN6 protein.

[0442] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment binds to cancer cells expressing human CLDN6 protein in human adults, whereas it does not bind to normal cells that do not express or substantially do not express human CLDN6 protein.

[0443]

[0444] In some embodiments, the binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment for the human CLDN6 protein is higher than the binding affinity for other antigens other than the human CLDN6 protein. For example, the binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment for the human CLDN6 protein may be higher than the binding affinity for the human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein.

[0445] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment has a lower binding dissociation constant (K) for human CLDN6 protein compared to antigens other than human CLDN6 protein. D　 ) indicates. For example, the anti-CLDN6 antibody or its antigen-binding fragment has a lower binding dissociation constant (K) for human CLDN6 protein compared to human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein. D　 ) may represent. Specifically, the binding dissociation constant between the anti-CLDN6 antibody or its antigen-binding fragment and the human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 10 times the binding dissociation constant between the anti-CLDN6 antibody or its antigen-binding fragment and the human CLDN6 protein. 2 Pear, 10 3 Pear, 10 4 Pear, 10 5 Pear, or 10 6 It can be more than double.

[0446]

[0447] In some embodiments, the binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment to cells expressing human CLDN6 protein is higher than the binding affinity to cells not expressing human CLDN6 protein. For example, the binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment to cells expressing human CLDN6 protein may be higher than the binding affinity to cells not expressing human CLDN6 protein but expressing human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein. Specifically, the binding affinity of the anti-CLDN6 antibody or its antigen-binding fragment to the extracellular loop of human CLDN6 protein may be higher than the binding affinity to the extracellular loop of human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein.

[0448] Generally, when the binding affinity of an antibody to an antigen-expressing cell is higher, cell binding EC 50 (cell binding EC 50 The value may be lower.

[0449] In some embodiments, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to cells expressing human CLDN6 protein 50 (cell binding EC 50 The ) value is the cell binding EC to cells expressing antigens other than human CLDN6 protein. 50 It may be lower than the value. For example, the cell binding EC of the above anti-CLDN6 antibody or its antigen-binding fragment to cells expressing human CLDN6 protein 50 The value is the cell binding EC for cells expressing human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein. 50The value may be lower than. In some embodiments, cells expressing human CLDN3 protein, cells expressing human CLDN4 protein, or cells expressing human CLDN9 protein may not express or substantially not express human CLDN6 protein. Specifically, the cell binding EC of the anti-CLDN6 antibody or its antigen-binding fragment to cells expressing human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein 50 The value is the cell binding EC for cells expressing human CLDN6 protein. 50 Approximately 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 10 2 Pear, 10 3 Pear, 10 4 Pear, 10 5 Pear, or 10 6 It can be more than double.

[0450] 2.4. Cross-reactivity to the Claudine family

[0451] Various types of the claudin family are known, including CLDN1, CLDN2, CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN9, CLDN10, CLDN11, CLDN12, CLDN14, CLDN15, CLDN16, CLDN18, CLDN19, and CLDN23. Among these, CLDN3, CLDN4, and CLDN9 proteins have structural or sequence similarities with the CLDN6 protein; consequently, many of the antibodies reported to date that bind to CLDN6 have shown cross-reactivity with CLDN3, CLDN4, and / or CLDN9.

[0452] In some embodiments, the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may bind specifically or selectively only to the Claudin 6 protein of the Claudin family. For example, the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may bind specifically or selectively only to the human Claudin 6 protein among the human Claudin 6 protein, human Claudin 3 protein, human Claudin 4 protein, and human Claudin 9 protein.

[0453] The anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may not exhibit, or substantially not exhibit, cross-reactivity with other Claudin family proteins other than CLDN6. The anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may not exhibit, or substantially not have, cross-reactivity with other Claudin family proteins other than CLDN6. The anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may not have, or substantially not have, cross-reactivity with CLDN3 protein, CLDN4 protein, and CLDN9 protein.

[0454] For example, the anti-CLDN6 antibody or its antigen-binding fragment may bind to the human CLDN6 protein, but may not bind or may not substantially bind to the human CLDN3 protein, human CLDN4 protein, and human CLDN9 protein. For example, the anti-CLDN6 antibody or its antigen-binding fragment may bind more strongly to the human CLDN6 protein than to the human CLDN3 protein, human CLDN4 protein, and human CLDN9 protein.

[0455] For example, the anti-CLDN6 antibody or its antigen-binding fragment may not bind to or substantially not bind to the human CLDN3 protein. For example, the anti-CLDN6 antibody or its antigen-binding fragment may not bind to or substantially not bind to the human CLDN3 protein expressed on the cell membrane.

[0456] For example, the anti-CLDN6 antibody or its antigen-binding fragment may not bind to or substantially not bind to the human CLDN4 protein. For example, the anti-CLDN6 antibody or its antigen-binding fragment may not bind to or substantially not bind to the human CLDN4 protein expressed on the cell membrane.

[0457] For example, the anti-CLDN6 antibody or its antigen-binding fragment may not bind to or substantially not bind to the human CLDN9 protein. For example, the anti-CLDN6 antibody or its antigen-binding fragment may not bind to or substantially not bind to the human CLDN9 protein expressed on the cell membrane.

[0458] The characteristic of the antibody binding better to CLDN6 compared to CLDN3, CLDN4, and CLDN9, which have high structural or sequence similarity to CLDN6, is very useful for selectively targeting "cancer cells", considering that CLDN3, CLDN4, and CLDN9 are more likely to be expressed in "normal cells" than CLDN6, and furthermore, considering that CNLD6 is specifically expressed in cancer cells compared to CLDN3, CLDN4, and CLDN9.

[0459] In particular, the extracellular domain of the human CLDN6 protein is known to differ from the human CLDN9 protein by only three amino acids. Specifically, it is known that there is only one amino acid difference (M29 / L29) in the first extracellular loop and two amino acids (R145 / Q145, Q156 / L156) in the second extracellular loop; for this reason, the extracellular domains of the human CLDN6 protein and the human CLDN9 protein have very high sequence and structural similarities. Due to this similarity, although antibodies that generally bind to human CLDN6 protein are likely to bind to human CLDN9 protein, the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment binds strongly to human CLDN6 protein (or cells expressing it) while substantially not binding to human CLDN9 protein (or cells expressing it, e.g., cells that do not express human CLDN6 but express human CLDN9), so it is very useful for selectively targeting only cancer cells expressing human CLDN6 protein in the body.

[0460] 2.5. Internalization of CLDN6-expressing cells

[0461] The anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment not only binds to cells expressing human CLDN6 but can also be internalized into the cells expressing human CLDN6. The internalization activity of such antibodies is an important characteristic when antibodies are utilized in a form bound to various substances, and the greater the internalization activity, the more the efficiency of delivering substances (e.g., proteins, nucleic acids, drugs, etc.) into the cell can be maximized. In other words, the excellent internalization activity of the anti-CLDN6 antibody or its antigen-binding fragment suggests the possibility that the anti-CLDN6 antibody of the present disclosure can be used as a component of an immunoconjugate.

[0462] 2.6. Fc-region mediated cytotoxicity

[0463] The anti-CLDN6 antibody of the present disclosure may have low toxicity to cells when used alone. Specifically, the anti-CLDN6 antibody may have limited or low Fc region-mediated cytotoxicity against cells expressing human CLDN6 protein.

[0464] For example, the above anti-CLDN6 antibody may have limited or low ADCC activity (Antibody-Dependent Cellular Cytotoxicity activity) against human CLDN6 protein-expressing cells, and accordingly, the killing effect of human CLDN6-expressing cancer cells through ADCC activity may be low.

[0465] For example, the above anti-CLDN6 antibody may have limited or low ADCP activity (Antibody-Dependent Cellular Phagocytosis activity) against human CLDN6 protein-expressing cells, and accordingly, the killing effect of human CLDN6-expressing cancer cells through ADCP activity may be low.

[0466] For example, the above anti-CLDN6 antibody may have limited or low complement-dependent cytotoxicity activity (CDC) on human CLDN6 protein-expressing cells, and accordingly, the killing effect of human CLDN6-expressing cancer cells through CDC activity may be low.

[0467] When the class of the antibody is IgG, the immunoconjugate containing the IgG antibody may bind to the Fcγ receptor of myeloid cells and be internalized into the myeloid cells. In this case, the immunoconjugate containing the antibody, which generally has Fc region-mediated cytotoxicity such as ADCC activity, may cause hematological toxicity due to internalization into the myeloid cells.

[0468] Although the class of the anti-CLDN6 antibody of the present disclosure is IgG, since the Fc region-mediated cytotoxicity caused by ADCC activity, etc. is limited or low, there is an advantage that the toxicity to normal bone marrow cells, etc., may be low even when the anti-CLDN6 antibody of the present disclosure is used as an immunoconjugate.

[0469] In other words, since the anti-CLDN6 antibody of the present disclosure exhibits low Fc region-mediated cytotoxicity, there is a low likelihood of unexpected or uncontrollable toxicity occurring even when utilized in combination with other substances (e.g., proteins, nucleic acids, drugs, etc.). Therefore, the low Fc-mediated cytotoxicity of the anti-CLDN6 antibody suggests the possibility that the anti-CLDN6 antibody of the present disclosure can be used as a component of a therapeutic complex.

[0470]

[0471] 3. Epitope

[0472] A specific part of an antigen that antibodies recognize is called an epitope, which is also referred to as an antigenic determinant.

[0473] The epitope of the human CLDN6 protein recognized by the anti-CLDN6 antibody of the present disclosure or its antigen-binding fragment may comprise one or more (e.g., one, two, or three or more) amino acid residues of the extracellular domain. In some embodiments, the epitope may comprise one or more amino acid residues of a first extracellular loop of the human CLDN6 protein represented by SEQ ID NO. 56. The first extracellular loop refers to an extracellular loop comprising residues M29 to R81 of the human CLDN6 protein represented by SEQ ID NO. 56. In some embodiments, the epitope may comprise one or more second extracellular amino acid residues of the human CLDN6 protein represented by SEQ ID NO. 56. The second extracellular loop refers to an extracellular loop comprising residues W138 to L160 of the human CLDN6 protein represented by SEQ ID NO. 56.

[0474] In some embodiments, the epitope of the human Claudin 6 protein recognized by the anti-CLDN6 antibody or its antigen-binding fragment may comprise one or more amino acid residues of the first extracellular loop and one or more amino acid residues of the second extracellular loop. In other expressions, the anti-CLDN6 antibody or its antigen-binding fragment may recognize or bind to one or more amino acid residues of the first extracellular loop and one or more amino acid residues of the second extracellular loop.

[0475] In some embodiments, the epitope of the human CLDN6 protein recognized by the anti-CLDN6 antibody or its antigen-binding fragment may include a glutamine (Q) residue at position 156 of the human CLDN6 protein represented by SEQ ID NO. 56. In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment may recognize or bind to an extracellular domain comprising a glutamine (Q) residue at position 156 of the human CLDN6 protein represented by SEQ ID NO. 56.

[0476] In some embodiments, the epitope of the human CLDN6 protein recognized by the anti-CLDN6 antibody or its antigen-binding fragment may include a methionine (M) residue at position 29 of the human CLDN6 protein represented by SEQ ID NO. 56. In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment may recognize or bind to an extracellular domain comprising a methionine (M) residue at position 29 of the human CLDN6 protein represented by SEQ ID NO. 56.

[0477] In some embodiments, the epitope for the CLDN6 protein of the anti-CLDN6 antibody or its antigen-binding fragment may include a glutamine residue at position 156 and a methionine residue at position 29 based on the human CLDN6 protein represented by SEQ ID NO. 56.

[0478] In some embodiments, the epitope of the anti-CLDN6 antibody or its antigen-binding fragment may be a discontinuous epitope. In some embodiments, the epitope of the anti-CLDN6 antibody or its antigen-binding fragment may be a stereochemical epitope.

[0479] In some embodiments, the epitope of the human Claudin 6 protein recognized by the anti-CLDN6 antibody or its antigen-binding fragment may be a conformational epitope or a discontinuous epitope comprising one or more amino acid residues of the first extracellular loop and one or more amino acid residues of the second extracellular loop. In some embodiments, the epitope may not be a linear epitope comprising only one of i) some amino acid residues of the first extracellular loop and ii) some amino acid residues of the second extracellular loop.

[0480]

[0481] 4. Production of anti-CLDN6 antibodies

[0482] 4.1. Overview of Antibody Production

[0483] One aspect of the present disclosure discloses a method for producing an anti-CLDN6 antibody or an antigen-binding fragment thereof. The anti-CLDN6 antibody or the antigen-binding fragment thereof is the anti-CLDN6 antibody or the antigen-binding fragment thereof described in <1. Anti-CLDN6 Antibody>.

[0484] The above production method comprises producing a recombinant protein (antibody or fragment thereof) using a nucleic acid encoding the anti-CLDN6 antibody or its antigen-binding fragment, or a vector containing said nucleic acid.

[0485] The above production method comprises: i) preparing a nucleic acid encoding the anti-CLDN6 antibody or its antigen-binding fragment, or a vector containing said nucleic acid; and ii) delivering said prepared vector to an antibody-producing cell. said antibody-producing cell is transformed by said vector.

[0486] 4.2. Nucleic acids encoding antibodies

[0487] The present disclosure discloses an anti-CLDN6 antibody or a nucleic acid encoding an antigen-binding fragment thereof as described in <1. Anti-CLDN6 Antibody>.

[0488] In some embodiments, the sequence of nucleic acid encoding the heavy chain of the anti-CLDN6 antibody is a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO. 34, SEQ ID NO. 35, and SEQ ID NO. 36. In some embodiments, the sequence of nucleic acid encoding the light chain of the anti-CLDN6 antibody is a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO. 37, SEQ ID NO. 38, and SEQ ID NO. 39. In this case, the sequence of nucleic acid encoding the heavy chain or light chain of the anti-CLDN6 antibody may be codon optimized to suit the characteristics of the cell.

[0489] In some embodiments, the sequence of the nucleic acid encoding the anti-CLDN6 antibody is a sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, and SEQ ID NO. 62.

[0490] 4.3. Vector containing nucleic acid encoding an antibody

[0491] The present disclosure discloses a vector into which a nucleic acid encoding the anti-CLDN6 antibody or its antigen-binding fragment is inserted. The vector may amplify or express the inserted nucleic acid and, upon expressing the inserted nucleic acid, may produce the anti-CLDN6 antibody or its antigen-binding fragment. In some embodiments, the nucleic acid encoding the anti-CLDN6 antibody or its antigen-binding fragment in the vector is operably linked to a promoter.

[0492] In some embodiments, the vector may further include expression control elements, selection elements, etc. The expression control elements may be a promoter, enhancer, polyadenylation signal, Kozak consensus sequence, inverted terminal repeat (ITR), long terminal repeat (LTR), terminator, internal ribosome entry site (IRES), 2A self-cleaving peptides, or replication origin, etc. The selection elements may be a fluorescent protein gene, tag, reporter gene, antibiotic resistance gene, etc.

[0493] In some embodiments, the vector may be a plasmid or a viral vector. In one embodiment, the viral vector may be one selected from the group consisting of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vacciniaviruses, poxviruses, and herpes simplex viruses. In some embodiments, the vector may be a nonviral vector. In some embodiments, the nonviral vector may be one or more selected from the group consisting of plasmids, phages, naked DNA, DNA complexes, and mRNA. In some embodiments, the plasmid may be selected from the group consisting of the pcDNA series, pS456, p326, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19. In some embodiments, the phage may be selected from the group consisting of λgt4λB, λ-Charon, λ△z1, and M13. In some embodiments, the coding nucleic acid may be a PCR amplicon.

[0494] 4.4. Cells for Antibody Production

[0495] The present disclosure provides a cell capable of producing the anti-CLDN6 antibody described in <1. Anti-CLDN6 Antibody> above.

[0496] In some embodiments, the cell may be a cell in which a nucleic acid encoding the heavy chain of the anti-CLDN6 antibody and a nucleic acid encoding the light chain of the anti-CLDN6 antibody are inserted into the cell genome, thereby enabling the cell to stably express the anti-CLDN6 antibody even after cell division. For example, the cell may be one in which the nucleic acid encoding the heavy chain of the anti-CLDN6 antibody and the nucleic acid encoding the light chain of the anti-CLDN6 antibody are inserted at specific locations within the genome or randomly integrated (e.g., CHO cells, HEK293 cells, etc.). Such cells can continuously produce the anti-CLDN6 antibody during culture. The cell may be used in the method for producing the anti-CLDN6 antibody of the present disclosure.

[0497] In some embodiments, the cell may be a host cell that transiently produces an anti-CLDN6 antibody by introducing into the cell an expression vector comprising a nucleic acid encoding the heavy chain of the anti-CLDN6 antibody and a nucleic acid encoding the light chain of the anti-CLDN6 antibody. For example, the cell may be one into which an expression vector encoding the anti-CLDN6 antibody of the present disclosure (e.g., pcDNA3.4, pCEP4, pCI-neo, etc.) is introduced by methods such as lipofection, electroporation, calcium phosphate precipitation, or polyethylenimine (PEI)-mediated delivery. In this case, the expression vector introduced into the cell generates antibody proteins through transcription and translation processes within the cytoplasm, and the cell may secrete antibodies into the culture supernatant. The cell may be used in the method for producing the anti-CLDN6 antibody of the present disclosure.

[0498] 4.5. Examples of Antibody Production Methods

[0499] In some embodiments, the method for producing the anti-CLDN6 antibody may include the step of transfecting a host cell with a nucleic acid encoding the anti-CLDN6 antibody and then purifying the antibody from the cell culture medium.

[0500] In some specific examples, the method for producing the anti-CLDN6 antibody may include the following:

[0501] Synthesizing a DNA fragment having a nucleic acid sequence encoding the heavy chain of the above-mentioned anti-CLDN6 antibody and a DNA fragment having a nucleic acid sequence encoding the light chain;

[0502] Inserting the above-mentioned synthesized DNA fragment into a vector (e.g., pcDNA3.4 vector) using a restriction enzyme and cloning it;

[0503] Transfecting the above vector into a host cell (e.g., CHO cell) that can be used for recombinant protein expression;

[0504] Culturing the above-mentioned transfected cells under conditions that allow antibody formation; and

[0505] After culturing the above-mentioned transfected cells, the antibody is purified from the culture supernatant using a separate purification device (e.g., Protein A affinity chromatography).

[0506]

[0507] 5. Uses of antibodies or conjugates

[0508] In one aspect of the present disclosure, a use is provided for the production of an immunoconjugate (or antibody-drug conjugate) of the anti-CLDN6 antibody of the present disclosure and its antigen-binding fragment. The anti-CLDN6 antibody of the present disclosure and its antigen-binding fragment may be used in the production of an immunoconjugate or an antibody-drug conjugate. The anti-CLDN6 antibody of the present disclosure and its antigen-binding fragment may be incorporated into an immunoconjugate or an antibody-drug conjugate and used for cancer treatment.

[0509] The above-mentioned immunoconjugate or antibody-drug conjugate may be used for cancer treatment, and related embodiments are provided. For example, in one embodiment of the present disclosure, a pharmaceutical composition for cancer treatment comprising the above-mentioned immunoconjugate or antibody-drug conjugate is provided. In one embodiment of the present disclosure, a method for cancer treatment using the above-mentioned immunoconjugate or antibody-drug conjugate is provided. The cancer treatment method comprises administering the above-mentioned immunoconjugate or antibody-drug conjugate to a treatment subject. In one embodiment of the present disclosure, a use of the above-mentioned immunoconjugate or antibody-drug conjugate for treating cancer is provided. In one embodiment of the present disclosure, a use for manufacturing a pharmaceutical product for cancer treatment using the above-mentioned immunoconjugate or antibody-drug conjugate is provided. The cancer may be a CLDN6-positive cancer. For example, the cancer may be a cancer in which human CLDN6 protein is expressed (e.g., overexpressed).

[0510]

[0511]

[0512] [Numbered Examples (Enumerated Embodiments)]

[0513] Hereinafter, various numbered embodiments (or modes) are described sequentially to aid in understanding the invention. However, the present invention should not be interpreted as being limited to the embodiments listed below, but should be understood to include all variations, equivalents, and additional combinations that a person skilled in the art can easily derive from this specification, the accompanying drawings, and the claims.

[0514]

[0515] Anti-CLDN6 antibody

[0516] Example 1. Antibody binding to CLDN6

[0517] Anti-CLDN6 antibody that binds to Claudin 6 protein.

[0518] Example 2. Human CLDN6 protein

[0519] In Example 1, the claudin 6 protein is a human claudin 6 protein, and the amino acid sequence of the human claudin 6 protein is SEQ ID NO. 56, an anti-CLDN6 antibody.

[0520] Example 3. Variable region

[0521] In any one of Examples 1 to 2 selected, the anti-CLDN6 antibody comprises a heavy chain variable region and a light chain variable region, and

[0522] The above heavy chain variable region includes a Heavy Chain Complementarity Determining Region (HCDR), and

[0523] The above light chain variable region includes a Light Chain Complementarity Determining Region (LCDR),

[0524] Anti-CLDN6 antibody.

[0525] Example 4. Sequence of heavy chain CDR

[0526] In Example 3, the heavy chain variable region includes a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), and a third heavy chain complementarity determining region (HCDR3), and

[0527] The amino acid sequence of the above HCDR1 is SEQ ID NO. 1, the amino acid sequence of the above HCDR2 is SEQ ID NO. 2, and the amino acid sequence of the above HCDR3 is SEQ ID NO. 3,

[0528] Anti-CLDN6 antibody.

[0529] Example 5. Heavy-print framework

[0530] In any one of Examples 3 to 4 selected, the heavy chain variable region further comprises a first heavy chain framework region (HFR1), a second heavy chain framework region (HFR2), a third heavy chain framework region (HFR3), and a fourth heavy chain framework region (HFR4), and

[0531] The amino acid sequence of the above HFR1 is the same as the sequence of SEQ ID NO. 7, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 7, or comprises the same,

[0532] The amino acid sequence of the above HFR2 is the same as the sequence of SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 77, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 77, or comprises such a sequence, and

[0533] The amino acid sequence of the above HFR3 is the same as the sequence of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 78, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 78, or comprises the same, and

[0534] The amino acid sequence of the above HFR4 is the same as the sequence of SEQ ID NO. 13, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 13, or comprises the same.

[0535] Anti-CLDN6 antibody.

[0536] Example 6. Sequence of the variable heavy chain region

[0537] In any one selected from Examples 3 to 5, the amino acid sequence of the medium chain variable region is

[0538] A sequence identical to SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, or SEQ ID NO. 81, or a sequence containing the same; or

[0539] An amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, or SEQ ID NO. 81, and is an amino acid sequence that essentially comprises HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3, or is a sequence comprising the same.

[0540] Anti-CLDN6 antibody.

[0541] Example 7. Sequence of light chain CDR

[0542] In any one of Examples 3 to 6 selected, the light chain variable region comprises a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), and

[0543] The amino acid sequence of the LCDR1 is SEQ ID NO. 4, the amino acid sequence of the LCDR2 is SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is SEQ ID NO. 6,

[0544] Anti-CLDN6 antibody.

[0545] Example 8. Light chain framework

[0546] In any one of Examples 3 to 7 selected, the light chain variable region further comprises a first light chain framework region (LFR1), a second light chain framework region (LFR2), a third light chain framework region (LFR3), and a fourth light chain framework region (LFR4).

[0547] The amino acid sequence of the above LFR1 is the same as the sequence of SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 79, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 79, or comprises the same,

[0548] The amino acid sequence of the above LFR2 is the same as the sequence of SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 80, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 80, or comprises such a sequence, and

[0549] The amino acid sequence of the above LFR3 is the same as the sequence of SEQ ID NO. 18 or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 18, or comprises the same, and

[0550] The amino acid sequence of the above LFR4 is the same as the sequence of SEQ ID NO. 19, or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 19, or comprises the same.

[0551] Anti-CLDN6 antibody.

[0552] Example 9. Sequence of the light chain variable region

[0553] In any one selected from Examples 3 to 8, the amino acid sequence of the light chain variable region is

[0554] A sequence identical to SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, or SEQ ID NO. 82, or a sequence containing the same; or

[0555] An amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, or SEQ ID NO. 82, and is an amino acid sequence that essentially comprises HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3, or is a sequence comprising the same.

[0556] Anti-CLDN6 antibody.

[0557] Example 10. Type of heavy chain invariant region

[0558] In any one selected from Examples 1 to 9, the anti-CLDN6 antibody comprises a heavy chain constant region, wherein the heavy chain constant region is a constant region of human IgG (or human IgG1, 2, 3, or 4).

[0559] Anti-CLDN6 antibody.

[0560] Example 11. Sequence of the heavy chain invariant region

[0561] In any one selected from Examples 1 to 10, the anti-CLDN6 antibody comprises a heavy chain constant region, and

[0562] The amino acid sequence of the above heavy chain invariant region is the same as SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, or SEQ ID NO. 34; or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, or SEQ ID NO. 34, or comprising the same,

[0563] Anti-CLDN6 antibody.

[0564] Example 12. Type of light chain invariant region

[0565] In any one selected from Examples 1 to 11, the anti-CLDN6 antibody comprises a light chain constant region, wherein the light chain constant region is a kappa (κ) type or lambda (λ) type constant region.

[0566] Anti-CLDN6 antibody.

[0567] Example 13. Sequence of the light chain invariant region

[0568] In any one selected from Examples 1 to 12, the anti-CLDN6 antibody comprises a light chain constant region, and

[0569] The amino acid sequence of the light chain invariant region is the same as the sequence of SEQ ID NO. 35 or SEQ ID NO. 36; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 35 or SEQ ID NO. 36, or comprises the same.

[0570] Anti-CLDN6 antibody.

[0571] Example 14. Type of Fc region

[0572] In any one selected from Examples 1 to 13, the anti-CLDN6 antibody comprises a fragment crystallizable region (Fc region), wherein the Fc region is the Fc region of human IgG (or human IgG1, 2, 3, or 4).

[0573] Anti-CLDN6 antibody.

[0574] Example 15. Sequence of the Fc region

[0575] In any one selected from Examples 1 to 14, the anti-CLDN6 antibody comprises a fragment crystallizable region (Fc region), and

[0576] The amino acid sequence of the Fc region is the same as SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, or SEQ ID NO. 53; or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, or SEQ ID NO. 53, or comprising the same.

[0577] Anti-CLDN6 antibody.

[0578] Example 16. Sequence of heavy chain

[0579] In any one selected from Examples 1 to 15, the anti-CLDN6 antibody comprises a heavy chain, and

[0580] The amino acid sequence of the heavy chain above is the same as the sequence of SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39, or comprises the same.

[0581] Anti-CLDN6 antibody.

[0582] Example 17. Sequence of light chains

[0583] In any one selected from Examples 1 to 16, the anti-CLDN6 antibody comprises a light chain, and

[0584] The amino acid sequence of the light chain is the same as the sequence of SEQ ID NO. 40, SEQ ID NO. 41, or SEQ ID NO. 42; or is an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with SEQ ID NO. 40, SEQ ID NO. 41, or SEQ ID NO. 42, or comprises the same.

[0585] Anti-CLDN6 antibody.

[0586] Example 18. Class of antibodies

[0587] In any one selected from Examples 1 to 17, the class of the anti-CLDN6 antibody is Immunoglobulin G (IgG), and

[0588] The above immunoglobulin G (IgG) is IgG1, IgG2, IgG3, or IgG4,

[0589] Anti-CLDN6 antibody.

[0590] Example 19. Type of antibody

[0591] In any one selected from Examples 1 to 18, the anti-CLDN6 antibody is a humanized antibody; a chimeric antibody; a human antibody; or a non-human antibody,

[0592] Anti-CLDN6 antibody.

[0593] Example 20. Humanized variable area

[0594] In any one selected from Examples 1 to 19, the heavy chain variable region is a humanized heavy chain variable region, and the light chain variable region is a humanized light chain variable region.

[0595] Anti-CLDN6 antibody.

[0596] Antigen-binding fragment

[0597] Example 21. Antigen-binding fragment of anti-CLDN6 antibody

[0598] Antigen-binding fragment of any one of the anti-CLDN6 antibodies selected from Examples 1 to 20.

[0599] Example 22. HCDR of antigen-binding fragment

[0600] In Example 21, the antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 of any one of the anti-CLDN6 antibodies selected from Examples 1 to 20.

[0601] Antigen-binding fragment.

[0602] Example 23. Variable region of the heavy chain of the antigen-binding fragment

[0603] In any one selected from Examples 21 to 22, the antigen-binding fragment comprises a heavy chain variable region of any one of Examples 1 to 20 anti-CLDN6 antibody.

[0604] Antigen-binding fragment.

[0605] Example 24. LCDR of antigen-binding fragment

[0606] In any one selected from Examples 21 to 23, the antigen-binding fragment comprises LCDR1, LCDR2, and LCDR3 of the anti-CLDN6 antibody selected from any one of Examples 1 to 20.

[0607] Antigen-binding fragment.

[0608] Example 25. Variable region of the light chain of the antigen-binding fragment

[0609] In any one selected from Examples 21 to 24, the antigen-binding fragment comprises a light chain variable region of any one of Examples 1 to 20 anti-CLDN6 antibody.

[0610] Antigen-binding fragment.

[0611] Example 26. Types of antigen-binding fragments

[0612] In any one of Examples 21 to 25, the antigen-binding fragment is selected from the group consisting of F(ab), F(ab'), F(ab')2, F(ab')2, single-chain variable fragment (scFv), and scFv-Fc.

[0613] Antigen-binding fragment.

[0614] Advantages of the anti-CLDN6 antibody or its antigen-binding fragment

[0615] Example 27. Specificity for CLDN6

[0616] In any one selected from Examples 1 to 26, the anti-CLDN6 antibody or its antigen-binding fragment specifically binds to the human CLDN6 protein, the extracellular domain of the human CLDN6 protein, the human CLDN6 protein expressed on a cell membrane, a cell expressing the human CLDN6 protein, and / or a cancer cell expressing the human CLDN6 protein.

[0617] Anti-CLDN6 antibody or its antigen-binding fragment.

[0618] Example 28. Cross-reactivity

[0619] In any one selected from Examples 1 to 27, the anti-CLDN6 antibody or its antigen-binding fragment does not exhibit cross-reactivity or substantially bind to human CLDN3 protein, human CLDN4 protein, human CLDN9 protein, cells expressing human CLDN3 protein, cells expressing human CLDN4 protein, and / or cells expressing human CLDN9 protein.

[0620] Anti-CLDN6 antibody or its antigen-binding fragment.

[0621] Example 29. Bond dissociation constant

[0622] In any one selected from Examples 1 to 28, the binding dissociation constant (K) of the anti-CLDN6 antibody or its antigen-binding fragment to the human CLDN6 protein D ) is approximately 1.00×10 -9 , 1.50×10 -9 , 2.00×10 -9 , 2.50×10 -9 , 3.00×10 -9 , 3.10×10 -9 , 3.20×10 -9 , 3.25×10 -9 , 3.30×10 -9 , 3.31×10 -9 , 3.32×10 -9 , 3.33×10 -9 , 3.34×10 -9 , 3.35×10 -9 , 3.36×10 -9 , 3.37×10 -9 , 3.38×10 -9 , 3.39×10 -9 , 3.40×10 -9 , 3.41×10 -9 , 3.42×10 -9 , 3.43×10 -9 , 3.44×10 -9 , 3.45×10 -9 , 3.46×10 -9 , 3.47×10 -9 , 3.48×10 -9 , 3.49×10 -9 , 3.50×10 -9 , 3.51×10 -9 , 3.52×10 -9 , 3.53×10 -9 , 3.54×10 -9 , 3.55×10 -9 , 3.56×10 -9 , 3.57×10 -9 , 3.58×10 -9 , 3.59×10 -9 , 3.60×10 -9, 3.61×10 -9 , 3.62×10 -9 , 3.63×10 -9 , 3.64×10 -9 , 3.65×10 -9 , 3.66×10 -9 , 3.67×10 -9 , 3.68×10 -9 , 3.69×10 -9 , 3.70×10 -9 , 3.71×10 -9 , 3.72×10 -9 , 3.73×10 -9 , 3.74×10 -9 , 3.75×10 -9 , 3.76×10 -9 , 3.77×10 -9 , 3.78×10 -9 , 3.79×10 -9 , 3.80×10 -9 , 3.81×10 -9 , 3.82×10 -9 , 3.83×10 -9 , 3.84×10 -9 , 3.85×10 -9 , 3.90×10 -9 , 4.00×10 -9 , 4.50×10 -9 , 5.00×10 -9 , 5.50×10 -9 , 6.00×10 -9 , 7.00×10 -9 , 8.00×10 -9 , or 9.00×10 -9 (M) or within the range of 2 numbers selected from the above numbers,

[0623] Anti-CLDN6 antibody or its antigen-binding fragment.

[0624] Example 30. Difference in bond dissociation constant

[0625] In any one selected from Examples 1 to 30, the binding dissociation constant between the anti-CLDN6 antibody or its antigen-binding fragment and the human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 10 times the binding dissociation constant between the anti-CLDN6 antibody or its antigen-binding fragment and the human CLDN6 protein. 2 Pear, 10 3 Pear, 10 4 Pear, 10 5 Pear, or 10 6 more than twice,

[0626] Anti-CLDN6 antibody or its antigen-binding fragment.

[0627] Molecules that bind to human CLDN6 protein

[0628] Example 31. Molecule binding to human CLDN6 protein

[0629] Any one of the anti-CLDN6 antibody selected from Examples 1 to 30 or its antigen-binding fragment; or an anti-CLDN6 antibody or its antigen-binding fragment comprising a modified amino acid residue of any one of the anti-CLDN6 antibody selected from Examples 1 to 30 or its antigen-binding fragment,

[0630] A molecule that binds to human CLDN6 protein.

[0631] Example 32. Immunoconjugate

[0632] In Example 31, the molecule binding to the human CLDN6 protein is an immunoconjugate comprising an effector molecule selected from the group consisting of detectable labels, immunotoxins, cytokines, chemokines, and (che)therapeutic agents.

[0633] A molecule that binds to human CLDN6 protein.

[0634] Example 33. Antibody-drug conjugate

[0635] In any one selected from Examples 31 to 32, the molecule binding to the human CLDN6 protein is an antibody-drug conjugate (ADC) containing a drug,

[0636] A molecule that binds to human CLDN6 protein.

[0637] Example 34. Chemical formula

[0638] In Example 33, the antibody-drug conjugate has the following chemical formula:

[0639] Ab-(LD)n,

[0640] Here, Ab is any one of the anti-CLDN6 antibodies selected from Examples 1 to 16; an anti-CLDN6 antibody in which some amino acid residues of any one of the anti-CLDN6 antibodies selected from Examples 1 to 16 are modified; any one of the antigen-binding fragments selected from Examples 17 to 22; or an antigen-binding fragment in which some amino acid residues of any one of the antigen-binding fragments selected from Examples 17 to 22 are modified, L is a linker, D is a drug portion, and n is an integer from 1 to 8,

[0641] A molecule that binds to human CLDN6 protein.

[0642] Example 35. Bispecific molecule

[0643] In Example 31, the molecule binding to the human CLDN6 protein is a bispecific molecule, a multispecific molecule, a bispecific antibody, or a multispecific antibody.

[0644] A molecule that binds to human CLDN6 protein.

[0645] Production of anti-CLDN6 antibody or its antigen-binding fragment

[0646] Example 36. Isolated polynucleotide

[0647] An isolated polynucleotide comprising a nucleic acid sequence encoding any one of the anti-CLDN6 antibody selected from Examples 1 to 30 or an antigen-binding fragment thereof.

[0648] Example 37. Expression Vector

[0649] An expression vector comprising the isolated polynucleotide of Example 36.

[0650] Example 38. Cells

[0651] Cells comprising the isolated polynucleotide of Example 36 and / or the expression vector of Example 37.

[0652] Example 39. Method for producing antibodies

[0653] A method for producing antibodies including the following:

[0654] Culturing the cells of Example 38 under conditions that allow antibody formation.

[0655]

[0656]

[0657] [Experimental Examples]

[0658] The invention provided by this specification will be described in more detail below through experimental examples and embodiments. These embodiments are intended solely to illustrate the contents disclosed by this specification, and it will be obvious to those skilled in the art that the scope of the contents disclosed by this specification is not to be interpreted as being limited by these embodiments.

[0659]

[0660] Experimental Example 1. Mouse antibody production

[0661] Human CLDN6 protein-VLP (product name: CLD-HM006) was purchased from Kactus. The amino acid sequence of the human CLDN6 protein on the surface of the above human CLDN6 protein-VLP (hereinafter referred to as 'CLDN6-VLP') is SEQ ID NO. 56.

[0662] The characteristics of CLDN6-VLP were evaluated by ELISA. Briefly, plates were coated with CLDN6-VLP, and mouse anti-CLDN6 antibody (SC105) was added to each well at various concentrations. A secondary antibody (goat-derived anti-mouse IgG HRP) and the color substrate TMB were used for staining, and the plates were read at an OD of 450 nm. A graph of the read results is shown in Figure 1. The EC50 value of the mouse anti-CLDN6 antibody against CLDN6-VLP was calculated using GraphPad software. The EC50 value was 1.18 nM.

[0663] Female BaIb / c and C57BI / 6 mice were administered 100 µg of CLDN6-VLP intraperitoneally (ip) three times (every 2 weeks) to immunize them with CLDN6-VLP. After final boosting was performed on the mice at week 9, splenocytes collected from the mice were fused with the myeloma cell line SP2 / 0 to obtain hybridoma cells, after which the hybridoma supernatant was screened for antigen binding and FACS binding.

[0664] Finally, six mAbs were selected from the initial screening for subcloning (limitation dilution method) and further evaluation. The specific methods are as follows: Hybridomas were cultured in roller bottles using IMDM medium, and the supernatant was collected for antibody production. The mAbs were purified by Protein A affinity chromatography. SDS-PAGE Coomassie staining results showed an estimated purity of over 90%. Secondary screening of the six purified mAbs included the following analyses: binding assay (ELISA) to human CLDN6-VLP; Cells stably transformed with CT26-CLDN6, cells stably transformed with CT26-CLDN9, cells stably transformed with 293T-CLDN6, cells stably transformed with 293T-CLDN9, cells stably transformed with 293T-CLDN3, cells stably transformed with 293T-CLDN4, binding analysis (flow cytometry (FACS)) for Huh7 and HepG2 cancer cells; and internalization analysis for HepG2 cells using flow cytometry (FACS).

[0665] Based on the analysis results, mouse antibody 1C6 was selected and sequencing was performed. The specific sequencing method is as follows. Total RNA was extracted from frozen hybridoma cells according to the technical manual for TRIzol® Reagent. Total RNA was analyzed by agarose gel electrophoresis. According to the technical manual for the PrimeScript™ 1st Strand cDNA Synthesis Kit, total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers. Then, PCR was performed to amplify the variable regions (heavy chain and light chain) of the antibody, after which the antibodies were separately cloned into a standard cloning vector and sequenced.

[0666] Selected mouse antibody 1C6 includes the heavy chain variable region of SEQ ID NO. 71 and the light chain variable region of SEQ ID NO. 72.

[0667]

[0668] Experimental Example 2. Production and Evaluation of Chimeric Antibodies

[0669] Using the heavy chain variable region sequence (HCVR) and light chain variable region sequence (LCVR) of the mouse antibody 1C6, a murine-human chimeric IgG1 (hereinafter "Chimeric IgG 1C6") containing the heavy chain of SEQ ID NO. 73 and the light chain of SEQ ID NO. 74 was prepared. The specific method is as follows. A nucleic acid encoding the heavy chain variable region of SEQ ID NO. 71 linked with a leader sequence (SEQ ID NO. 75) and a nucleic acid encoding the light chain variable region of SEQ ID NO. 72 linked with a leader sequence (SEQ ID NO. 76) were amplified and inserted into a plasmid to construct a plasmid expressing the full length of the heavy and light chains. The heavy and light chain expressing plasmids were co-transfected into 100 mL of HEK293 cells. The recombinant IgG secreted into the medium by the transfected cells was purified using protein A affinity chromatography. In SDS-PAGE, purified IgG shifted to the ~150 kDa band under non-reducing conditions and to the ~55 kDa and ~30 kDa bands under reducing conditions.

[0670] The specificity of chimeric IgG 1C6 for members of the claudin family was evaluated by flow cytometry. In this experiment, human CLDN6, CLDN9, CLDN3, and CLDN4 stable cell lines were constructed. Specifically, human CLDN6, CLDN9, CLDN3, or CLDN4 genes were stably transfected into human HEK-293 or 3T3 fibroblasts. These stable cells were seeded into plates and cultured overnight. Next, chimeric IgG 1C6 or benchmark B1 antibody (anti-claudin-6 / 9 mouse IgG chimeric antibody, US Publication No. 2022 / 0168440) appropriately diluted in FACS buffer was added to the cells, and they were incubated at 37°C for 1 hour. After incubation, the cells were washed three times with FACS buffer to remove unbound antibodies. Then, cells were separated using trypsin, stained with a fluorescently conjugated secondary antibody (goat anti-human IgG secondary antibody), placed on ice in the dark for 30 minutes, and analyzed using a Beckman flow cytometer. The fluorescence intensity at each time point was analyzed using Beckman flow cytometry software.

[0671] The analysis results graphs are shown in Figures 2 and 3. As can be seen in Figure 2, the benchmark B1 antibody bound to HEK-293T and 3T3 cells overexpressing human CLDN6 protein or human CLDN9 protein, but did not bind to cells expressing human CLDN3 protein or human CLDN4 protein.

[0672] Figure 3 showed the results for chimeric IgG 1C6. Chimeric IgG 1C6 bound to HEK-293T and 3T3 cells overexpressing human CLDN6 protein, but did not show significant binding to cells overexpressing human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein.

[0673]

[0674] The binding of chimeric IgG 1C6 to cancer cell lines was evaluated using a flow cytometer. Liver cancer cell lines HepG2 and Huh7, gastric cancer cell line AGS, and breast cancer cell line SK-BR-3 were collected. Chimeric IgG 1C6 or benchmark B1 antibodies diluted to a concentration of 10,000 ng / ml in FACS buffer were added to the cells, and they were incubated at 4°C for 40 minutes. After incubation, the cells were washed twice with PBS to remove unbound antibodies. Prior to analysis using a Beckman flow cytometer, the cells were stained with a fluorescently conjugated secondary antibody (goat anti-human IgG secondary antibody) on ice for 30 minutes. Fluorescence intensity at each time point was analyzed using Beckman flow cytometry software.

[0675] The analysis results graph is shown in Figure 4. As can be seen in Figure 4, chimeric IgG 1C6 bound to HepG2, Huh-7, and AGS cells, but did not bind to SK-BR-3 cells. This binding pattern was consistent with the B1 antibody. Cell binding EC of chimeric IgG 1C6 and B1 to HepG2 cells. 50 The values ​​are presented in Figure 4. The affinity of chimeric IgG 1C6 for HepG2 cells was similar to that of B1. The maximum binding affinity of B1 was higher than that of chimeric IgG 1C6, which may be due to the additional binding of B1 to CLDN9 expressed in HepG2 cells.

[0676]

[0677] Chimeric IgG 1C6 was further evaluated through an internalization assay using the CLDN6-positive hepatocellular carcinoma cell line HepG2. The specific method is as follows. 1 x 10⁶ HepG2 cells were used per well. 5 The cells were aliquoted. Next, chimeric IgG 1C6 or benchmark B1 antibodies were added to the cells in FACS buffer, and the cells were incubated at 4°C for 2 hours. After incubation, the cells were washed to remove unbound antibodies. Then, the cells were incubated at 37°C. After 2 hours, the cells were detached, stained with a fluorescently conjugated secondary antibody (goat anti-mouse IgG), incubated on ice in the dark for 30 minutes, and then analyzed using a Beckman flow cytometer. Fluorescence intensity and cell binding rates at each time point were analyzed using Beckman flow cytometry software. The amount of chimeric IgG 1C6 internalized into the cells was measured using the following formula: Internalization rate (%) = 100 x (MFI 4℃ - MFI 37℃ ) / MFI 4℃ .

[0678] A graph of the analysis results is shown in Fig. 5. As can be seen in Fig. 5, chimeric IgG 1C6 can be internalized in cells expressing CLDN6 with an internalization rate of approximately 80%. The internalization rate of chimeric IgG 1C6 was higher than that of the benchmark antibody B1.

[0679]

[0680] The internalization and apoptotic effects of chimeric IgG 1C6 were evaluated via a DT3C-mediated cytotoxicity assay. In this experiment, HepG2 cells were plated in 96-well plates at a density of 4,000 cells / well. Cells were incubated overnight at 37°C in a humidified environment of 5% CO2. DT3C was diluted to 1 µg / ml and mixed with chimeric IgG 1C6 or B1 antibodies. The antibody concentration was 10 µg / ml. After incubation at room temperature for 30 minutes, the mixture was added to the cells and cultured for 5 days. DT3C alone, chimeric IgG 1C6 alone, B1 alone, and negative control antibodies alone or mixed with DT3C were included as controls. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot#PL701) was added to the wells and treated at 37°C for 1–4 hours. Subsequently, absorbance was measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices) and SoftMax Pro 5.4.1 software. The inhibition rate was calculated using the following formula: (Average absorbance of treated samples / Average absorbance of control samples) × 100.

[0681] A graph of the analysis results is shown in Fig. 6. According to the results, treatment with chimeric IgG 1C6 alone did not affect HepG2 cell viability (top graph of Fig. 6), but the chimeric IgG 1C6-DT3C conjugate was internalized in cells expressing CLDN6 and was able to significantly reduce cell viability (bottom graph of Fig. 6). The degree of cell death at this time was similar to that of the B1 antibody.

[0682]

[0683] Experimental Example 3. Screening of Humanized Antibodies

[0684] A humanized anti-CLDN6 mAb derived from chimeric IgG 1C6 (hereinafter 'humanized IgG 1C6') was prepared using CDR grafting and the back mutation method. The specific method is as follows. The structure of the parental antibody (chimeric IgG 1C6) was modeled using an MOE homology modeling program, and the humanized antibody was designed using the CDR grafting method. Briefly, the CDR sequence of chimeric IgG 1C6 was transplanted into a human receptor to obtain humanized light and heavy chains derived from chimeric IgG 1C6. PTM analysis was performed to identify a single DS isomerization hotspot in the FR3 region. To improve protein stability, a DT mutation was introduced to replace the DS (Asp-Ser) motif, which is more easily isomerized than the DT (Asp-Thr) motif. Three humanized heavy chains selected for use in screening humanized antibodies were named 1C6 VH1, 1C6 VH2, and 1C6 VH3, respectively, and each include the heavy chain variable region sequence of SEQ ID NO. 20, the heavy chain variable region sequence of SEQ ID NO. 21, and the heavy chain variable region sequence of SEQ ID NO. 22. Three humanized light chains selected for use in screening humanized antibodies were named 1C6 VL1, 1C6 VL2, and 1C6 VL3, respectively, and each include the light chain variable region sequence of SEQ ID NO. 23, the light chain variable region sequence of SEQ ID NO. 24, and the light chain variable region sequence of SEQ ID NO. 24.

[0685] Humanized antibodies against a total of nine combinations were expressed in ExpiCHO-S cells: the combination of 1C6 VH1 and 1C6 VL1; the combination of 1C6 VH1 and 1C6 VL2; the combination of 1C6 VH1 and 1C6 VL3; the combination of 1C6 VH2 and 1C6 VL1; the combination of 1C6 VH2 and 1C6 VL2; the combination of 1C6 VH2 and 1C6 VL3; the combination of 1C6 VH3 and 1C6 VL1; the combination of 1C6 VH3 and 1C6 VL2; and the combination of 1C6 VH3 and 1C6 VL3. Specifically, a full-length IgG expression plasmid was prepared by synthesizing a nucleic acid encoding one of the heavy chains of 1C6 VH1, 1C6 VH2, and 1C6 VH3 and one of the light chains of 1C6 VL1, 1C6 VL2, and 1C6 VL3, and inserting it into the pcDNA3.4 vector. The heavy and light chain expression plasmids were co-expressed in ExpiCHO-S cells. The recombinant IgG secreted into the cell medium was purified using a protein A affinity column. The purified antibodies were buffered with PBS using a PD-10 desalting column. The concentration and purity of the purified proteins were measured by OD280 and SDS-PAGE, respectively. The amino acid sequences of the heavy and light chain variable regions of the nine humanized anti-CLDN6 antibodies are shown in Table 5.

[0686] [Table 5] Sequences of 9 Humanized Antibodies

[0687]

[0688]

[0689] Nine types of humanized IgG were evaluated using a cell binding assay on CHO-K1 cells stably expressing human CLDN6 protein. The experimental method was the same as the cell binding assay in Experimental Example 2, and the results are shown in Figure 7.

[0690]

[0691] Experimental Example 4. Expression and Purification of Antibodies

[0692] DNA fragments containing heavy chain DNA sequences encoding the heavy chain of the anti-Claudin-6 antibody and DNA fragments containing light chain DNA sequences were synthesized. The synthesized DNA fragments were inserted into and cloned into the pcDNA3.4 vector (Thermofisher) using restriction enzymes.

[0693] The cloned vectors are ExpiCHO-S TM ExpiFectamine on Thermofisher (A29127) cells TM Transient expression was induced using the CHO kit (Thermofisher, A29129). Cells were induced using ExpiCHO TM They were cultured in expression medium (Thermofisher, A2910002) at 37°C under CO2 incubator (including rotary shaker) conditions for 7 days. Plasmid DNA HC and LC (1:2) (200 and 400 μg, respectively) in 8 mL and ExpiFectamine TM 8 mL of CHO reagent (640 μL) was mixed into Opti-MEM® medium (final volume 16 mL) and reacted at room temperature for 5 minutes. This mixed solution was 1.2 x 10⁻⁶ 9 Dog ExpiCHO TM Cells (200 mL ExpiCHO TM It was added to (during culture in expression medium) and gently mixed in a shaker incubator at 37°C under an 8% CO2 humidified atmosphere. 18 hours after transfection, ExpiFectamine TM CHO Transfection Enhancer 1.2 mL and ExpiFectamine TM 48 mL of CHO Transfection Feed was added to each flask. Seven days after transfection, cells were harvested, and the supernatant was used for purification.

[0694] Monoantibodies were obtained from the cell culture supernatant via recombinant Protein A affinity chromatography (MabSelect TMSuRe TM The protein was purified by gel filtration chromatography using a HiLoad 26 / 600 Superdex-200 prep grade column (Cytiva, 28-9893-36). To confirm the size and purity of the purified protein, SDS-PAGE (NuPAGE 4-12% Bis-Tris gel, NP0321) and size exclusion HPLC (Agilent, 1200 series) were analyzed using a SE-HPLC column (TSKgel G3000SWXL, inner diameter 7.8 mm, length 30 cm, particle size 5 μm, TOSOH, 0008541). The purified protein was exchanged and concentrated with 20 mM Histidine-HCl, pH 6.0, 8% (w / v) Sucrose (buffer) using an Amicon Ultra-15, 30K centrifugal filter (Millipore, UFC903096). Subsequently, protein concentration was measured using a Nanodrop One (Thermo Fisher).

[0695] Information on the obtained monoantibodies is described in Table 6 below. These monoantibodies were used in subsequent experiments described below.

[0696] [Table 6] Information on obtained antibodies

[0697]

[0698]

[0699] Here, AB1522 is the antibody corresponding to VH1 + VL1 in Table 6. AB1523 is the antibody corresponding to VH1 + VL2 in Table 6. AB0976 is the antibody corresponding to VH1 + VL3 in Table 6. AB1519 is the antibody corresponding to VH2 + VL1 in Table 6. AB1520 is the antibody corresponding to VH2 + VL2 in Table 6. AB1521 is the antibody corresponding to VH2 + VL3 in Table 6. AB1524 is the antibody corresponding to VH3 + VL1 in Table 6. AB1525 is the antibody corresponding to VH3 + VL2 in Table 6. AB1526 is the antibody corresponding to VH3 + VL3 in Table 6. AB0463 is the antibody disclosed in patent document (WO 2020 / 191342 A1). AB0388 is an antibody disclosed in patent document (US 2022 / 0168440 A1). AB1502 is an antibody in which the N297A substitution mutation is added to the heavy chain constant region of AB0976. AB1503 is an antibody in which the heavy chain constant region of AB0976 is modified to the constant region of human IgG4 having the S228P substitution mutation.

[0700] Experimental Example 5. Evaluation of the recombinant protein antigen binding ability of anti-Claudin-6 antibody (ELISA)

[0701] Experimental Example 5.1. Evaluation of Antigen Binding Ability of 9 Types of Antibodies

[0702] To select antibodies that possess specific binding characteristics to human Claudin-6 while simultaneously not binding to human Claudin-9, the binding affinities for human Claudin-6 and Claudin-9 of nine anti-Claudin-6 antibodies with different framework domain amino acid sequences were evaluated using an ELISA-based solution binding test. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with Claudin-6 protein (Claudin-6 protein-VLP, Human; MedChemExpress, Catalog # HY-P77899) or Claudin-9 protein (Claudin-9 protein-His, Human; Sino biological, Catalog # 21185-HNAH) at a concentration of 0.25 μg / ml in 1X PBS solution at 4°C for 16 hours. Afterward, the plate was washed five times with 1X PBS containing 0.05% (v / v) tween 20, and then Claudin-6 was treated with 3% (v / v) BSA (bovine serum albumin) in 1X PBS and Claudin-9 with 1% (v / v) BSA in 1X PBS for 2 hours to block non-specific binding sites.

[0703] After washing the above plate five times with 1X PBS containing 0.05% (v / v) tween 20, anti-Claudin-6 or anti-Claudin-6 / 9 antibodies were treated on a 96-well microtiter plate with 1 / 4 serial dilutions in buffer to adjust concentrations from 100 nM to 0.02 pM and 12 points, and the binding ability was analyzed by ELISA as follows. Specifically, after incubating at 37°C for 1 hour, the plate was washed 5 times with PBS containing 0.05% (v / v) Tween 20, and then HRP-conjugated human Fc multiplexed antibody reagent (ThermoFisher, Catalog # 31413) was diluted to a ratio of 1:30,000 and added to the washed microtiter plate. The reaction was carried out at 37°C for 30 minutes to detect antibodies bound to the plate. After the reaction, color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440) for 3 to 6 minutes. The enzymatic reaction was stopped with 0.5 mol / L (1N) sulfuric acid, and absorbance was measured at 450 nm and 650 nm using a microplate reader (molecular device).

[0704] The results obtained above are shown in Fig. 8.

[0705] As shown in Figure 8, antibodies with a combination of a heavy chain variable region and a light chain variable region exhibited Claudin-6 binding ability similar to AB0976. Among the nine antibodies with a combination of a heavy chain variable region and a light chain variable region, AB0976 was confirmed to have the best specific binding ability to Claudin-6.

[0706] Experimental Example 5.2. Evaluation of the Specificity of the Antibody for Claudin-6

[0707] For AB0976, which showed the best binding ability in the above results, a comparative evaluation of ligand binding ability against human Claudin-6 and Claudin-9 was performed together with control antibodies AB0463 and AB0388. The specific method was carried out in the same manner as described in Experimental Example 5.1.

[0708] The results obtained above are shown in Fig. 9 and Table 7 below.

[0709] [Table 7] Evaluation Results of Recombinant Protein Antigen Binding Ability of Anti-Claudin-6 Antibody and Anti-Claudin-6 / 9 Antibody

[0710]

[0711]

[0712] As shown in Figure 9, the antibody AB0976 according to the present invention exhibited superior ligand (antigen) binding activity compared to the control antibody AB0463, and showed similar ligand binding activity to the anti-Claudin-6 / 9 antibody AB0388. However, unlike AB0388, no binding activity was observed for the Claudin-9 protein, confirming that AB0976 has specific ligand binding activity for the Claudin-6 protein.

[0713] Experimental Example 5.3. Evaluation of binding ability according to modification of the heavy chain constant region of the antibody

[0714] To verify whether binding ability is maintained even when the heavy chain constant region of AB0976 is altered, AB1502, which has the same sequence as AB0976 but contains the N297A mutation in the IgG1 Fc backbone, and AB1503, which has a completely different Fc backbone, IgG4, were produced and purified, respectively, and a comparative evaluation was performed. The specific method was carried out in the same manner as described in Experimental Example 5.1.

[0715] The experimental results are shown in Fig. 10.

[0716] As shown in Figure 10, AB1502 and AB1503 exhibited specific binding activity against the Claudin-6 antigen similar to AB0976, despite having different Fc sequences, while almost no binding activity was observed against the Claudin-9 antigen. These results imply that the Claudin-6 specific binding activity of AB0976 is dependent on the antibody's CDR sequence, suggesting that the antibody's specific binding characteristics can be maintained even if the antibody's constant region is altered.

[0717] Experimental Example 5.4. Evaluation of Antibody Binding Specificity

[0718] To verify the specificity of the antibody against Claudin family proteins, an evaluation was performed using Claudin-3 Protein-VLP, Human (MedChemExpress, HY-P700472) and Claudin-4 Protein-VLP, Human (MedChemExpress, HY-P700405). The specific method was carried out in the same manner as described in Experimental Example 5.1.

[0719] The above experimental results are shown in Fig. 11. As shown in Fig. 11, AB0976 did not show binding activity against Claudin-9 antigen, as well as Claudin-3 and Claudin-4 antigens, but showed specific ligand binding activity against Claudin-6 antigen.

[0720]

[0721] Experimental Example 6. Measurement of binding dissociation constant using SPR

[0722] The target protein binding dissociation constants of the antibody AB0976 of the present disclosure and the control antibodies AB0463 and AB0388 were measured using the Biacore™ 8K SPR system (Cytiva), a Surface Plasmon Resonance (SPR) measuring instrument. The antibodies, which are the ligands, were diluted to a level of 30 μg / mL in 10 mM Sodium Acetate, pH 4.5 immobilization buffer on a Series S Sensor Chip CM5 (Cytiva, 29149603) and flowed at a flow rate of 10 μL / min to fix the concentration at a level of 1,000 or 5,000 RU. Subsequently, the binding rate constant (Ka) was measured by flowing the target proteins (analytes), human Claudin-6 protein (Acrobiosystems, CL6-H5289) and human Claudin-9 protein (Acrobiosystems, CL9-H5586), at the concentrations specified in Table 11 for 90 seconds, and then the dissociation rate constant (Kd) was measured by flowing for 210 seconds. The binding rate constant, dissociation rate constant, and binding dissociation constant (K D The ) value was obtained by applying a 1:1 binding model using Biacore™ 8K evaluation software (cytiva).

[0723] The analysis results are as shown in Table 8 below.

[0724] [Table 8] Results of binding affinity analysis of anti-Claudin-6 antibody and anti-Claudin-6 / 9 antibody for target proteins

[0725]

[0726]

[0727] The affinity of AB0976, the anti-Claudin-6 antibody of the present disclosure, for human Claudin-6 is K D = 3.54×10 -9It was evaluated as M, which was confirmed to be superior to the affinity of the control antibody. In addition, AB0976 was found not to bind to human Claudin-9, confirming that it has a selective and specific binding affinity for human Claudin-6.

[0728]

[0729] Experimental Example 7. Evaluation of Antibody Binding Ability to Cell Lines (Flow Cytometry)

[0730] The cell lines used were Claudin-6 CHO Cell Line (BPS Bioscience, 78527-M), Claudin-9 CHO Cell Line (BPS Bioscience, 78687), PA-1 (ATCC, CRL-1572), SNU-119 (KCLB, 00119), OVCAR-3 (ATCC, HTB-161), OV-90 (ATCC, CRL-3585), BeWo (ATCC, CCL-98), and OUMS-23 (JCRB, JCRB1022). Cells for flow cytometry analysis were detached from culture flasks using trypsin-EDTA, and the number of viable cells was measured using trypan blue. The cells were centrifuged at 1200 rpm for 3 minutes, washed with 1X PBS, and 1 x 10⁶ cells were placed in each well of a 96 V-type plate. 5Cells were seeded. Cells were incubated with the primary antibody in FACS buffer (1X PBS containing 2% FBS) at 4°C for 1 hour. The primary antibody was serially diluted 1 / 4 with the buffer to adjust the concentration from 100 nM to 0.38 pM, 10 points. Cells treated with the primary antibody to the hIgG1 isotype control were used as a control. The primary antibodies AB0976, AB0463, and AB0388 were treated and evaluated and compared. After washing the cells twice with FACS buffer, they were treated with a 1:1,000 ratio Alexa Fluor 647-conjugated secondary antibody (goat-derived anti-human IgG Fcγ fragment specific antibody-Alexa Fluor 647, Jackson ImmunoResearch, 109-606-098) diluted with FACS buffer and incubated for 30 minutes at 4°C under dark conditions.

[0731] After washing the cells twice with FACS buffer, they were resuspended in 200 μL of cold FACS buffer, and the samples were analyzed using a BD LSRFortessa X-20 flow cytometer (BD Biosciences).

[0732] The results of the analysis of the cell binding affinity of the antibodies are shown in Figs. 12, 13, and Table 9 below.

[0733] [Table 9] Results of cell line binding ability evaluation of anti-Claudin-6 antibody and anti-Claudin-6 / 9 antibody

[0734]

[0735]

[0736] As a result of comparing the cellular binding affinities of the antibody AB0976 of the present disclosure with the control antibodies AB0463 and AB0388, AB0976 exhibited binding affinities equivalent to or superior to those of the control antibodies in various cell lines with different target expression levels. Furthermore, in stable-expression cell lines transduced with Claudin-6 and Claudin-9 genes, AB0976 demonstrated specific binding activity to Claudin-6 when compared to the anti-Claudin-6 / 9 antibody AB0388.

[0737] Due to these characteristics, AB0976 was evaluated as an antibody that secures stable and reproducible binding ability in various cell lines, while simultaneously possessing enhanced selectivity and specificity for Claudin-6.

[0738]

[0739] Experimental Example 8. Evaluation of antibody binding specificity against Claudin family protein stable expression cell lines (Flow cytometry)

[0740] To evaluate binding specificity to Claudin family proteins, cell lines stably expressing human Claudin-6, Claudin-9, Claudin-3, and Claudin-4 genes were constructed. Specifically, stable expression cell lines were established by transfecting human-derived HEK-293 cells and mouse-derived 3T3 fibroblasts with the said genes.

[0741] The established cells were treated with trypsin to prepare a cell suspension. Subsequently, antibodies B1 (anti-claudin-6 / 9 mouse IgG chimeric antibody of Daiichi Sankyo, US Publication No. 2022 / 0168440), SC105 (anti-Claudin-6 antibody of AbbVie Stemcentrx LLC, US Registration No. 10,053,511), and KM3907 (anti-Claudin-4 antibody of Kyowa Hakko Kirin Co., Ltd, EP Publication No. 2,138,576) were appropriately diluted in FACS buffer, added to the cells, and incubated for 1 hour under dark conditions at 4°C. After the reaction, cells were washed three times with FACS buffer to remove unconjugated antibodies, and then stained with a fluorescently labeled goat-derived anti-mouse IgG secondary antibody (Alexa Fluor 647 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, 109-605-088)) for 1 hour under dark conditions at 4°C. The stained cells were analyzed using an Agilent NovoCyte flow cytometer.

[0742] The established cells were treated with trypsin to prepare a cell suspension. Subsequently, antibodies B1, SC105, and KM3907 were appropriately diluted in FACS buffer, added to the cells, and incubated for 1 hour under dark conditions at 4°C. After the incubation, the cells were washed three times with FACS buffer to remove unconjugated antibodies, and then stained with a fluorescently labeled anti-human IgG secondary antibody for 1 hour under dark conditions at 4°C. The stained cells were analyzed using an Agilent NovoCyte flow cytometer.

[0743] The results obtained from the above experiment are shown in Fig. 14. As shown in Fig. 14, it was confirmed that B1 binds to both Claudin-6 and Claudin-9 in HEK293T and 3T3 cell lines that stably express the human Claudin-6 gene. SC105 specifically bound to Claudin-6, but its binding ability was lower compared to B1. Meanwhile, KM3907 did not bind to Claudin-6 and showed high binding ability to Claudin-4. Considering that AB0976 of the present disclosure showed similar levels of binding ability to B1 and Claudin-6 expressing cell lines, these results indirectly confirmed that AB0976 exhibits higher binding ability than SC105.

[0744] Accordingly, the antibody AB0976 according to the present invention simultaneously secures excellent binding specificity and binding ability for Claudin family proteins, and was evaluated as a differentiated antibody that combines high selectivity and affinity compared to various control antibodies.

[0745]

[0746] Experimental Example 9. Analysis of Internalization

[0747] Cells to be used for the internalization analysis were detached from culture plates using trypsin-EDTA, and the live cell count was determined by trypan blue treatment. The cell lines used were PA-1, SNU-119, OV-90, BeWo, and OUMS-23. Using the medium dedicated to each cell line, PA-1 was 0.1 x 10⁶ 6 cells / mL, for other cell lines 0.2x10 6The antibodies were diluted to a concentration of cells / mL, and 5,000 and 10,000 cells were seeded into 50 μL aliquots per well of a Nunc EDGE 96-well plate. The cells were incubated overnight at 37°C in a 5% CO2 incubator to allow for cell attachment. The antibody concentration was adjusted to 8 μg / mL (approx. 55 nM) by diluting it with the medium specific to each cell line. For the experiment to confirm the concentration-dependent internalization of AB0976, IncuCyte® FabFluor-pH Red was used for labeling, followed by 37°C for 15 minutes in the dark. The antibodies were evaluated and compared by treating AB0976, AB0463, and AB0388. For the experiment to confirm the concentration-dependent internalization of AB0976, the concentrations were adjusted from 100 nM to 3.13 nM at 6 points using 1 / 2 serial dilutions with buffer. AB0237 and a group treated only with medium (Cell only) were used as negative controls. Labeled antibodies were added at the same volume (50 μL) as the cells to achieve a final concentration of 4 μg / mL or 50 nM. Phase and Red signals were analyzed at 1-hour intervals for 48, 72, and 120 hours using incucyte imaging.

[0748] The results of antibody internalization analysis in various cell lines are shown in Figures 15 and 16. When comparing the antibody AB0976 according to the present invention with control antibodies AB0463 and AB0388, AB0976 exhibited superior internalization activity compared to the control antibodies in various cell lines with different target expression levels. That is, the fluorescence signal generated when the antibody enters the cell was observed to be faster and stronger in AB0976 compared to the control antibodies.

[0749] Additionally, the results of the concentration-dependent internalization analysis of AB0976 in the PA-1 cell line are shown in Fig. 17. As a result, it was confirmed that the internalization efficiency of the antibody AB0976 according to the present invention increased in proportion to the concentration. Furthermore, observations up to 120 hours showed that the fluorescence signal gradually became stronger as time progressed.

[0750] Therefore, the antibody AB0976 according to the present invention possesses excellent intracellular uptake capability and is expected to maximize the intracellular delivery efficiency of drugs through concentration-dependent and long-lasting internalization characteristics. Due to these characteristics, AB0976 may be particularly advantageous for development in a form combined with various substances (e.g., labels, nucleic acids, proteins, drugs, nanoparticles, etc.). Furthermore, AB0976 offers significantly improved performance in terms of internalization efficiency and delivery duration compared to existing antibodies, and may possess the advantage of enhancing therapeutic effects.

[0751]

[0752] Experimental Example 10. Evaluation of antibody binding affinity to human Claudin-6 antigen-expressing cell lines with mutations

[0753] To identify the epitope of AB0976 of the present disclosure, DNA containing a sequence encoding human Claudin-6 was first synthesized and inserted into plasmid DNA. Subsequently, using the plasmid DNA as a template, DNA for Claudin-6 expression was constructed by introducing mutations M29L, R145Q, or Q156L, respectively, using a site-directed mutagenesis kit (ENB, E0554S). These mutations correspond to regions L29, Q145, and L156, which correspond to the Claudin-9 sequence, and were introduced into corresponding sites on the Claudin-6 protein. Therefore, through analysis using the variants, it was possible to determine whether the epitope recognized by AB0976 depends on specific amino acid residues of Claudin-6.

[0754] ExpiCHO cells (Thermofisher, A29127) are 6.0 x 10 6 The cells were cultured at a concentration of cells / mL. Plasmid DNA was diluted in OptiPRO SFM (Thermofisher, 12309019), mixed with ExpiFectamine CHO reagent (Thermofisher, A29129), and reacted at room temperature for 5 minutes. After treating the cells with the transduction mixture, they were cultured in a shaking incubator at 37°C under 8% CO2 conditions. 18 to 22 hours after transduction, ExpiCHO Feed and Enhancer were added, and the cells were cultured for an additional 48 hours.

[0755] Afterward, harvest and count the cells, then transfer them to a 96-well plate at a volume of 2.0 x 10 5Cells were dispensed into cells / well. The cells were centrifuged at 1500 rpm for 3 minutes at 4°C to remove the supernatant, and then incubated with the primary and secondary antibodies, diluted in FACS buffer prepared with 1X PBS and 1% BSA, at 4°C for 1 hour each. Centrifugation and washing were repeated under the same conditions between each step. Finally, the cells were suspended in fixation buffer (BD, 554655), stored in the dark at 4°C, and then analyzed using a flow cytometer.

[0756] The acquired data were gated using FlowJo (v10.10.0) software, and the mean fluorescence intensity (MFI) of cells bound to anti-Claudin-6 antibody or anti-human Fc antibody was calculated. Using the calculated values, an MFI curve against log[antibody concentration] was constructed using GraphPad Prism (v10.3.1), and a non-linear regression analysis (log[agonist] vs. response - variable slope, 4-parameter model) was applied to determine the EC 50 The value was calculated.

[0757] As shown in Fig. 18, it was confirmed that the binding ability of the AB0976 antibody decreased in the M29L variant of human Claudin-6. This showed a distinct difference compared to the AB0463 antibody, in which a decrease in binding ability was observed only for the Q156L variant of Claudin-6. In other words, while the binding ability of AB0463 is dependent on the Q156 position of Claudin-6, it was confirmed that for AB0976, the M29 position, as well as the Q156 position, acts as an epitope affecting binding ability. Therefore, it was found that although the two antibodies both target Claudin-6, the epitope sites affecting binding ability differ. Furthermore, regarding the binding evaluation results for the Q156L variant, AB0976 showed no binding ability in Q156L, whereas AB0388 demonstrated binding ability even in the Q156L variant. This suggests that while AB0976 maintains the specificity of not binding to Claudin-9, AB0388 can cross-bind to Claudin-9.

[0758]

[0759] Experimental Example 11. In vivo monotherapy test of a monoantibody in a human-derived ovarian cancer cell line OVCAR-3 xenograft mouse model

[0760] To evaluate the in vivo monotherapy study of AB0976, a mouse tumor xenograft model using the human-derived ovarian cancer cell line OVCAR-3 was established, and the antibody was administered via tail vein. 5×10⁻¹⁰ 7-week-old BALB / c-Nude mice 6 OVCAR-3 was mixed with an equal volume of Corning® Matrigel® matrix and implanted via subcutaneous injection into the flank. The lengths of the long axis (L) and short axis (W) of the tumor were measured using a vernier caliper, and the final tumor volume was calculated using the following formula.

[0761] Tumor volume (mm³) = (0.5) x L x W 2

[0762] Tumor volume was observed at intervals of 3-4 days for 7 weeks after drug administration, and the measured tumor volumes for the administration of the test substance are shown in the table below.

[0763] Average tumor volume is 159 mm 3 When the target was reached, group separation was performed using a random sequential assignment method (n=8 in each group). AB0976 was administered as a single dose of 9 mg / kg (mpk), and as a control, an hIgG1 isotype control was administered as a single dose of 9 mpk.

[0764] The results are shown in Table 10.

[0765] [Table 10] Average tumor volume on day 42 of administration (mm²) 3 )

[0766]

[0767]

[0768] 1533.25 mm in the AB0976 9 mpk administration group 3 An average tumor volume was observed, and in the hIgG1 isotype control, 1543.676 mm 3 An average tumor volume was observed. Therefore, experimental results performed in an OVCAR-3 xenograft mouse model expressing human Claudin-6 confirmed that the direct anticancer effect of the AB0976 antibody is limited through antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This implies that the toxicity of the antibody alone to cells is low. Therefore, the antibody is unlikely to exhibit unexpected or uncontrollable toxicity even when utilized in combination with other substances.

[0769] Therefore, the fact that antitumor activity was not observed in in vivo experiments of the above antibody suggests that the antibody of the present invention actually possesses highly advantageous characteristics when utilized in a form combined with various substances.

[0770]

[0771] Experimental Example 12. Effects of ADC

[0772] Experimental Example 12.1. Preparation of AB0976-37728 ADC

[0773] AB0976 dissolved in PBS buffer (20 mM, pH 6) containing 5 mM EDTA (5 mg / ml, 0.17 μmol) was reduced by adding a 5 mM TCEP (Tris (2-carboxyethyl) phosphine) solution (0.34 ml, 1.70 μmol). The antibody / TCEP solution was incubated at 25°C for 2 hours. After reduction was complete, the reaction solution was cooled to 25°C. Subsequently, a solution of the camptothecin derivative-linker compound 37728 (P37728) dissolved in DMA (10 mg / ml, 3.06 μmol) was added to the reduced antibody solution. The camptothecin derivative-linker used has the following structure:

[0774]

[0775] The reaction solution was incubated at 25°C for 2 hours while conjugating the toxin-linker to the antibody reduced via thiol maleimide binding. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultrafilter to obtain AB0976-37728 ADC (7.97 mg / ml, 20 mg) dissolved in His (20 mM, pH 6) buffer. The biochemical characteristics of the resulting ADC were analyzed by measuring purity and aggregate content using size exclusion chromatography (SEC) and high-pressure liquid chromatography (HPLC), and by measuring drug loading (DAR) using hydrophobic interaction chromatography HPLC (HIC-HPLC). The average number of conjugated drug molecules per antibody molecule was 7.9.

[0776] Experimental Example 12.2. In vitro cytotoxicity of AB0976-37728 ADC

[0777] The in vitro cytotoxicity of AB0976-37728 ADC was tested in ovarian cancer cells (PA-1 and Ovcar-3), liver cancer cells (Huh-7 and HepG2), and 293T-CLDN6 stable cells (293T cells stably expressing human CLDN6 protein). All cell lines were cultured in appropriate culture media at 37°C in a humidified incubator environment with 5% CO2. Cells were plated onto 96-well plates. Cell inoculation numbers ranged from 500 cells / 100 µL / well to 6,000 cells / 100 µL / well. Cells were inoculated overnight at 37°C in a humidified atmosphere with 5% CO2. AB0976-37728 ADC and IgG-37728 ADC (negative control) were prepared from stock solution and diluted to appropriate working concentrations 24 hours after cell inoculation. Serial 10-fold dilutions were performed at 7 sites using culture medium. Final concentrations ranged from 1,000 nM to 0.001 nM. Cells were cultured with ADCs for 72 hours. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot#PL701) was added to each well at 37°C for 1–4 hours, and absorbance was measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices) and SoftMax Pro 5.4.1 software. Inhibition rate was calculated using the following formula: (Average absorbance of treated samples / Average absorbance of control samples) × 100. For all cell analyses, dose-response curves were generated using GraphPad Prism 7 3-parameter curve fitting.

[0778] Figures 19 and 20 show representative death curves of AB0976-37728 ADC and IgG1-37728 ADC in PA-1, Ovcar-3, Huh7, HepG2, and 293T-CLDN6 cells.

[0779]

[0780] Table 11 summarizes the CLDN6 expression levels in the tested cells and the IC50 of AB0976-37728. AB0976-37728 exhibited the strongest cytotoxicity in 293T-CLDN6 and PA-1 cells with high CLDN6 expression, showing an IC50 value of less than 1 nM. Additionally, it induced strong cytotoxicity (IC50 values ​​in the single-digit nM range) in OVCAR-3 cells with moderate CLDN6 expression, showed the weakest activity in HepG2 cells, and showed no activity in Huh7 cells with low CLDN6 expression.

[0781] [Table 11] ADC IC50

[0782]

[0783]

[0784] Experimental Example 12.3. Bystander killing effect of AB0976-37728 ADC

[0785] The ability of ADCs to kill antigen-negative cells surrounding antigen-positive cells is called the bystander effect. To determine whether AB0976-37728 induces the bystander effect, a flow cytometry-based apoptosis assay was performed. CLDN6-positive cells (293T-CLDN6) and CLDN6-negative cells (NCI-N87) in the exponential growth phase were harvested, counted, and suspended in culture media adjusted for cell density. Both types of cells were seeded into the same wells of 24-well cell culture plates at a ratio of 0.125:1 or 0.25:1 and incubated overnight at 37°C. Then, AB0976-37728 was added at a concentration of 10 nM per well. Cells were cultured for an additional 5 days at 37°C in the presence of the ADC. After the end of culture, cells were harvested, transferred to round-bottom 96-well plates, washed with PBS, and resuspended in DPBS. After adding Herceptin antibodies and incubating for 60 minutes, goat anti-human IgG H&L (FITC) antibodies were administered to differentiate 293T-CLDN6 cells from NCI-N87 cells based on HER2 expression. Flow cytometry was performed using a BD Fortessa flow cytometer, and the number of CLDN6-positive and CLDN6-negative cells was analyzed using FlowJo software. The bystander effect was evaluated based on the viability of antigen-negative cells (e.g., CLDN6-negative cells) and was calculated as the number of antigen-negative cells surviving after ADC treatment divided by the total number of antigen-negative cells used.

[0786] The number of analyzed cells is shown in Figure 21. As shown in Figure 21, AB0976-37728 induced apoptosis in CLDN6-positive 293T cells, but had no apoptotic effect on CLDN6-negative NCI-N87 cells alone. Under conditions where 293T cells and negative NCI-N87 cells were co-cultured in vitro, AB0976-37728 induced apoptotic effects in both cells. Since AB0976-37728 exhibits CLDN6-specific cytotoxicity, NCI-N87 cell death may be induced by the released payload, which suggests a bystander apoptotic effect of AB0976-37728.

[0787] Experimental Example 12.4. In vivo anti-tumor activity of AB0976-37728 ADC

[0788] The anti-tumor activity of AB0976-37728 was evaluated using a CLDN6-positive PA-1 cell xenograft mouse model. 5 million PA-1 cells were harvested and subcutaneously implanted into the right flank of 6–7 week old Balb / c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd). Tumor size was approximately 150–200 mm 3 When [the threshold] was reached, 5 mice were randomly divided into different groups. Each group included a vehicle control group, an AB0976-37728 2.5 mg / kg group, and an AB0976-37728 5 mg / kg group. The ADC was administered intravenously at the indicated dose. Tumor volume was measured twice weekly. Tumor volume was calculated using the following formula: ½ x L x W; where L = length and W = width.

[0789] The results are shown in Fig. 22. Fig. 22 shows the change in in vivo tumor volume after ADC administration. As a result of administering AB0976-37728 at 2.5 mg / kg or 5 mg / kg, tumor growth was significantly inhibited in the PA-1 xenograft model.

[0790] Experimental Example 12.5. Plasma stability of AB0976-37728 ADC

[0791] To evaluate the plasma stability of the ADC, AB0976-37728 (0.1 mg / ml) was incubated in blank human plasma at 37°C for up to 96 hours. Samples were collected at each time point, and the total antibody (naked antibody and conjugated antibody) and residual conjugated antibody (ADC) were measured using ELISA. Briefly, to measure total antibodies, human CLDN6 protein was coated onto a microplate to capture the AB0976 antibody. After removing unbound antibodies, the bound total AB0976 antibody was detected using a polyclonal antibody (goat anti-human IgG Fc specific antibody) conjugated with horseradish peroxidase (HRP). To measure only the AB0976-37728 ADC, a mouse anti-DXd antibody was added as a secondary antibody. The concentration of the AB0976-37728 ADC was measured using a goat anti-mouse IgG (H+L) antibody conjugated with HRP.

[0792] Figure 23 shows the percentage of total antibodies and AB0976-37728 ADCs remaining at each time point. The trend of AB0976-37728 was similar to the trend of total antibodies. These results suggest that AB0976-37728 ADCs are stable in human plasma.

Claims

1. An anti-claudin 6 antibody that binds to human claudin 6 protein or an antigen-binding fragment thereof, The above anti-Claudin 6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and The above heavy-chain variable region comprises a first heavy-chain complementarity determining region (HCDR1) of SEQ ID NO. 1; a second heavy-chain complementarity determining region (HCDR2) of SEQ ID NO. 2; and a third heavy-chain complementarity determining region (HCDR3) of SEQ ID NO. 3, and The above light-chain variable region includes the first light-chain complementarity determining region (LCDR1) of SEQ ID NO. 4; the second light-chain complementarity determining region (LCDR2) of SEQ ID NO. 5; and the third light-chain complementarity determining region (LCDR3) of SEQ ID NO.

6.

2. In Paragraph 1, The above anti-Claudin 6 antibody or its antigen-binding fragment comprises a heavy chain variable region having 95% or more sequence identity with SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22, and a light chain variable region having 95% or more sequence identity with SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25, and The above heavy chain variable region includes HCDR1 of SEQ ID NO. 1; HCDR2 of SEQ ID NO. 2; and HCDR3 of SEQ ID NO. 3, and The above light chain variable region comprises LCDR1 of SEQ ID NO. 4; LCDR2 of SEQ ID NO. 5; and LCDR3 of SEQ ID NO. 6, Anti-Claudin 6 antibody or its antigen-binding fragment.

3. In any one selected from paragraphs 1 to 2, The above heavy-chain variable region comprises a first heavy-chain framework region (Heavy-chain Framework region 1; HFR1) of SEQ ID NO. 7; a second heavy-chain framework region (Heavy-chain Framework region 2; HFR2) of SEQ ID NO. 8 or SEQ ID NO. 9; a third heavy-chain framework region (Heavy-chain Framework region 3; HFR3) of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12; and a fourth heavy-chain framework region (Heavy-chain Framework region 4; HFR4) of SEQ ID NO. 13, and The above light-chain variable region comprises a first light-chain framework region (Light-chain Framework region 1; LFR1) of SEQ ID NO. 14 or SEQ ID NO. 15; a second light-chain framework region (Light-chain Framework region 2; LFR2) of SEQ ID NO. 16 or SEQ ID NO. 17; a third light-chain framework region (Light-chain Framework region 3; LFR3) of SEQ ID NO. 18; and a fourth light-chain framework region (Light-chain Framework region 4; LFR4) of SEQ ID NO. 19, Anti-Claudin 6 antibody or its antigen-binding fragment.

4. In any one selected from paragraphs 1 to 3, The above anti-Claudin 6 antibody or its antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO. 20 and the light chain variable region of SEQ ID NO. 25, Anti-Claudin 6 antibody or its antigen-binding fragment.

5. In any one selected from paragraphs 1 to 4, The above anti-Claudin 6 antibody comprises the heavy chain constant region of SEQ ID NO. 26 and the light chain constant region of SEQ ID NO. 32, Anti-Claudin 6 antibody or its antigen-binding fragment.

6. In any one selected from paragraphs 1 to 5, The above anti-Claudin 6 antibody comprises the heavy chain of SEQ ID NO. 37 and the light chain of SEQ ID NO. 42, Anti-Claudin 6 antibody or its antigen-binding fragment.

7. In any one selected from paragraphs 1 through 6, The class of the above anti-Claudin 6 antibody is IgG, and the IgG is IgG1, IgG2, IgG3, or IgG4, Anti-Claudin 6 antibody or its antigen-binding fragment.

8. In any one selected from paragraphs 1 through 7, The above anti-Claudin 6 antibody is a humanized antibody, Anti-Claudin 6 antibody or its antigen-binding fragment.

9. In any one selected from paragraphs 1 through 8, The above anti-Claudin 6 antibody or its antigen-binding fragment is capable of binding to the human Claudin 6 protein of SEQ ID NO. 56, Anti-Claudin 6 antibody or its antigen-binding fragment.

10. In any one selected from paragraphs 1 through 9, The binding affinity of the above anti-Claudin 6 antibody or its antigen-binding fragment for human Claudin 6 protein is higher than the binding affinity for human CLDN3 protein, human CLDN4 protein, or human CLDN9 protein, Anti-Claudin 6 antibody or its antigen-binding fragment.

11. In any one selected from paragraphs 1 through 10, The above anti-Claudin 6 antibody or its antigen-binding fragment specifically binds only to human Claudin 6 protein among human Claudin 6 protein, human Claudin 3 protein, human Claudin 4 protein, and human Claudin 9 protein, Anti-Claudin 6 antibody or its antigen-binding fragment.

12. In any one selected from paragraphs 1 to 11, The above anti-Claudin 6 antibody or its antigen-binding fragment can be internalized in cells expressing human Claudin 6 protein, Anti-Claudin 6 antibody or its antigen-binding fragment.

13. In any one selected from paragraphs 1 through 12, The above antigen-binding fragment can bind to the human Claudin 6 protein, and The above antigen-binding fragment is any one selected from F(ab), F(ab'), F(ab')2, F(ab')2, single-chain variable fragment (scFv), and scFv-Fc. Anti-Claudin 6 antibody or its antigen-binding fragment.

14. A molecule that binds to a human CLDN6 protein, comprising an anti-CLDN6 antibody of any one of claims 1 to 13 or an antigen-binding fragment thereof.

15. An anti-Claudin 6 antibody or an antigen-binding fragment thereof of any one of claims 1 to 13; and an immunoconjugate comprising a drug.

16. A composition comprising the following: An anti-Claudin 6 antibody of any one of claims 1 to 13 or an antigen-binding fragment thereof; a molecule that binds to the human CLDN6 protein of claim 14; or an immunoconjugate of claim 15.

17. An isolated polynucleotide comprising a sequence encoding the heavy chain of any one of claims 1 to 13 of an anti-claudin 6 antibody.

18. An isolated polynucleotide comprising a sequence encoding the light chain of any one of claims 1 to 13 of an anti-claudin 6 antibody.

19. An expression vector comprising one or more of the isolated polynucleotides of claim 17 and the isolated polynucleotides of claim 18.

20. A cell comprising one or more of the isolated polynucleotide of claim 17, the isolated polynucleotide of claim 18, and the expression vector of claim 19.

21. A method for producing antibodies comprising the following: Culturing the cells of paragraph 20 under conditions that allow antibody formation.

Images