Humanized antibody specific to claudin 4 and use thereof

Abstract

The present invention relates to a humanized antibody specific for claudin 4 and use thereof and, more specifically, to: a humanized antibody specific for claudin 4 having enhanced binding affinity and specificity for claudin 4 and reduced immunogenicity; and use thereof for cancer treatment and diagnosis.

Description

Humanized antibody specific to Claudin 4 and uses thereof

[0001] This application claims priority to Korean Patent Application No. 10-2025-0051134 filed on April 18, 2025, and the entire specification thereof is a reference to this application. The present invention relates to a humanized antibody specific to claudin 4 and its uses, and more specifically, to a humanized antibody specific to claudin 4 with improved binding affinity and specificity to claudin 4 and reduced immunogenicity, and its use for cancer treatment and diagnosis.

[0002]

[0003] Claudin is a major essential membrane protein for tight junctions between cells, and there are 24 families in mammals; humans possess the claudin family, excluding claudin 13 found in mammals. The claudin family has a similar structure in which it penetrates the cell wall and is embedded, and most have a structure containing two extracellular loops.

[0004]

[0005] Claudin is known to play a role in controlling the flow of molecules between cells, but recent studies have reported that it is closely associated with the development of cancers such as colon cancer, gastric cancer, breast cancer, esophageal cancer, and ovarian cancer. In particular, it has been reported that Claudin 3 and Claudin 4 of the Claudin family are overexpressed in ovarian cancer, which is known to have a poor prognosis due to the lack of distinct symptoms and appropriate treatment methods other than surgery and chemotherapy.

[0006]

[0007] As such, the increase or decrease in Claudin expression, which has high specificity in cancer tissues, can be used as a predictive indicator for cancer development. Furthermore, as Claudin serves as a useful biomarker in the diagnosis and treatment of cancer, various groups are attempting to develop therapeutic agents targeting Claudin. In particular, the United States, Japan, and other countries are developing therapeutic antibodies targeting Claudin 3 and 4, which are known to be overexpressed in ovarian cancer, and a monoclonal antibody targeting both Claudin 3 and 4 simultaneously has also been reported.

[0008]

[0009] Humanized antibodies are antibodies derived from non-human species (e.g., mouse antibodies) whose amino acid sequences have been modified to increase similarity to antibody variants naturally occurring in humans. The "humanization" process is typically applied to monoclonal antibodies developed for administration to humans and is performed when the process of developing specific antibodies involves generation in non-human immune systems (e.g., mice or rats). The protein sequences of antibodies produced in this manner differ from naturally occurring antibodies in humans and, therefore, are immunogenic when administered to human patients. Humanized antibodies are considered different from chimeric antibodies, which possess large stretches of non-human proteins, even though they have protein sequences similar to human antibodies.

[0010]

[0011] Meanwhile, humanized antibodies can be manufactured without generating chimeric intermediates. Direct generation of humanized antibodies can be achieved by inserting an appropriate CDR coding segment (responsible for the desired binding characteristics) into a human antibody scaffold, a process known as "CDR (complementarity-determining region) grafting." Typically, the DNA coding for the antibody's CDR can be sequenced after an antibody with the desired characteristics has been produced in a mouse or rat (or another non-human animal). Once the exact sequence of the desired CDR is identified, this sequence is inserted into a construct containing DNA of the human antibody framework.

[0012]

[0013] However, it is well known that simply inserting the known antibody CDR sequence into a human antibody framework alters the CDR structure within the antibody necessary for binding to the antigen, resulting in an unexpected decrease in antibody binding affinity.

[0014]

[0015] To address this problem, there is a technical challenge to design humanized antibodies that exhibit stable binding affinity, specificity, stability, and reduced immunogenicity by identifying residues within the human antibody framework that play a potentially important role in restoring antibody binding efficiency and inducing reversal mutations in the murine sequence.

[0016]

[0017] Accordingly, in order to produce a humanized antibody that includes the CDR sequence of a rat-derived anti-claudin 4 antibody and has improved binding affinity and specificity for claudin 4, the applicant analyzed amino acid residues in the human antibody framework sequence that may affect binding affinity with claudin 4 and back-mutated them, thereby completing a humanized antibody that exhibits improved binding affinity and specificity compared to the parent antibody.

[0018]

[0019] Accordingly, the object of the present invention is to provide a humanized monoclonal antibody specific to human Claudin 4 or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region:

[0020] A heavy chain FR (framework region) 1 selected from the group consisting of SEQ ID NOs 22, 27 and 32; a heavy chain CDR (complementary determining region) 1 of SEQ ID NO. 16; a heavy chain FR2 selected from the group consisting of SEQ ID NOs 23, 28 and 33; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs 18, 24, 29 and 34; a heavy chain FR3 of SEQ ID NO. 25 or 30; a heavy chain CDR3 of SEQ ID NO. 20; and a heavy chain variable region comprising a heavy chain FR4 of SEQ ID NO. 26 or 31; and

[0021] A light chain variable region comprising: a light chain FR1 selected from the group consisting of SEQ ID NOs 42, 45, and 47; a light chain CDR1 of SEQ ID NO. 36; a light chain FR2 selected from the group consisting of SEQ ID NOs 43, 46, and 48; a light chain CDR2 of SEQ ID NO. 38; a light chain FR3 of SEQ ID NO. 39; a light chain CDR3 of SEQ ID NO. 40; and a light chain FR4 of SEQ ID NO. 44.

[0022]

[0023] Another object of the present invention is to provide a polynucleotide encoding the humanized monoclonal antibody (mAb) or its antigen-binding fragment, a vector comprising the same, and a host cell transfected with the vector.

[0024]

[0025] Another object of the present invention is to provide a composition for treating or diagnosing cancer comprising the humanized monoclonal antibody (mAb) or its antigen-binding fragment as an active ingredient.

[0026]

[0027] Another object of the present invention is to provide a composition for cancer treatment or diagnosis comprising the humanized monoclonal antibody or an antigen-binding fragment thereof.

[0028]

[0029] Another object of the present invention is to provide a composition for cancer treatment or diagnosis that essentially comprises the humanized monoclonal antibody or an antigen-binding fragment thereof.

[0030]

[0031] Another object of the present invention is to provide a use of the humanized monoclonal antibody or its antigen-binding fragment for preparing a composition for cancer treatment.

[0032]

[0033] Another object of the present invention is to provide a method for treating cancer comprising administering an effective amount of a composition containing the humanized monoclonal antibody or its antigen-binding fragment as an active ingredient to an individual in need thereof.

[0034]

[0035] Another object of the present invention is to provide a use of the humanized monoclonal antibody or its antigen-binding fragment for preparing a composition for cancer diagnosis.

[0036]

[0037] Another objective of the present invention is to provide a cancer diagnosis method comprising the following steps:

[0038] i) a step of administering to a subject the above-mentioned humanized monoclonal antibody or an immunoconjugate in which a label is conjugated to an antigen-binding fragment thereof;

[0039] ii) a step of the above immunoconjugate binding to a target site within the subject;

[0040] iii) a step of imaging and detecting a signal generated from the above immunoconjugate; and

[0041] iv) A step of diagnosing whether cancer has occurred in the target site based on the detected signal.

[0042]

[0043] To achieve the aforementioned objectives of the present invention, the present invention provides a humanized monoclonal antibody specific to human Claudin 4 or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region:

[0044] A heavy chain FR (framework region) 1 selected from the group consisting of SEQ ID NOs 22, 27 and 32; a heavy chain CDR (complementary determining region) 1 of SEQ ID NO. 16; a heavy chain FR2 selected from the group consisting of SEQ ID NOs 23, 28 and 33; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs 18, 24, 29 and 34; a heavy chain FR3 of SEQ ID NO. 25 or 30; a heavy chain CDR3 of SEQ ID NO. 20; and a heavy chain variable region comprising a heavy chain FR4 of SEQ ID NO. 26 or 31; and

[0045] A light chain variable region comprising: a light chain FR1 selected from the group consisting of SEQ ID NOs 42, 45, and 47; a light chain CDR1 of SEQ ID NO. 36; a light chain FR2 selected from the group consisting of SEQ ID NOs 43, 46, and 48; a light chain CDR2 of SEQ ID NO. 38; a light chain FR3 of SEQ ID NO. 39; a light chain CDR3 of SEQ ID NO. 40; and a light chain FR4 of SEQ ID NO. 44.

[0046] To achieve another objective of the present invention, the present invention provides a polynucleotide encoding the humanized monoclonal antibody (mAb) or an antigen-binding fragment thereof, a vector comprising the same, and a host cell transformed with the vector.

[0047]

[0048] To achieve another objective of the present invention, the present invention provides a composition for treating or diagnosing cancer comprising the humanized monoclonal antibody (mAb) or an antigen-binding fragment thereof as an active ingredient.

[0049]

[0050] To achieve another objective of the present invention, the present invention provides a composition for cancer treatment or diagnosis comprising the humanized monoclonal antibody or an antigen-binding fragment thereof.

[0051]

[0052] To achieve another objective of the present invention, the present invention provides a composition for cancer treatment or diagnosis that essentially comprises the humanized monoclonal antibody or an antigen-binding fragment thereof.

[0053]

[0054] To achieve another objective of the present invention, the present invention provides a use of the humanized monoclonal antibody or its antigen-binding fragment for preparing a composition for cancer treatment.

[0055]

[0056] To achieve another objective of the present invention, the present invention provides a method for treating cancer comprising administering an effective amount of a composition containing the humanized monoclonal antibody or its antigen-binding fragment as an active ingredient to an individual in need thereof.

[0057]

[0058] To achieve another objective of the present invention, the present invention provides a use of the humanized monoclonal antibody or its antigen-binding fragment for preparing a cancer diagnostic composition.

[0059]

[0060] To achieve another objective of the present invention, the present invention provides a cancer diagnosis method comprising the following steps:

[0061] i) a step of administering to a subject the above-mentioned humanized monoclonal antibody or an immunoconjugate in which a label is conjugated to an antigen-binding fragment thereof;

[0062] ii) a step of the above immunoconjugate binding to a target site within the subject;

[0063] iii) a step of imaging and detecting a signal generated from the above immunoconjugate; and

[0064] iv) A step of diagnosing whether cancer has occurred in the target site based on the detected signal.

[0065]

[0066] In this specification, the term "comprising" is used with the same meaning as "including" or "characterized by," and does not exclude additional components or steps of the method that are not specifically mentioned in the composition or method according to the present invention. Furthermore, the term "consisting of" means excluding additional elements, steps, or components that are not separately described. The term "essentially consisting of" means that, within the scope of the composition or method, it may include, in addition to the described materials or steps, materials or steps that do not substantially affect the basic characteristics thereof.

[0067]

[0068] The terms “anti-claudin 4 antibody,” “antibody recognizing claudin 4,” and “antibody against claudin 4” are used interchangeably herein and refer to an antibody that binds to the claudin 4 protein with sufficient affinity and specificity.

[0069]

[0070] In the present invention, the term "antigen" refers to a molecule or part of a molecule capable of inducing antibody formation and to which an antibody can bind. An antigen may have one or more than one epitope. The specific reaction mentioned above is intended to mean that the antigen will react in a highly selective manner with its corresponding antibody, rather than reacting with a number of antibodies that may be triggered by other antigens. The antigen according to the present invention is a Claudin 4 protein or a fragment thereof.

[0071]

[0072] In the present invention, the terms “antigenic determinant” or “epitope” refer to a region of an antigen molecule that reacts specifically with a specific antibody. A peptide sequence derived from an epitope may be used alone or with a carrier moiety to immunize animals by applying methods known in the art, and to produce additional polyclonal or monoclonal antibodies. The peptide isolated from the epitope may be used in a diagnostic method for detecting antibodies and may be utilized as a therapeutic agent when inhibition of the said antibody is required.

[0073]

[0074] Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulfide bonds, and two light chains, each connected to the heavy chains in a "Y" shape by disulfide bonds. Proteolytic degradation of the antibody generates Fv (variable fragment) and Fc (crystallizable fragment) domains. The Fab (antigen-binding fragment) contains a region where the polypeptide sequence is variable. The term F(ab')2 refers to two Fab' arms linked together by disulfide bonds. The central axis of the antibody is referred to as the Fc fragment. Each heavy chain has variable domains (VH) at one end, followed by multiple constant domains (CH). Each light chain has variable domains (VL) at one end and constant domains (CL) at the other end, the light chain variable domains are aligned with the heavy chain variable domains, and the light chain constant domains are aligned with the heavy chain first constant domain (CH1). The variable domains of each pair of light and heavy chains form antigen binding sites. The domains on the light and heavy chains have the same general structure, and each domain includes four framework regions connected by three hypervariable regions known as complementary determining regions (1-3), the sequences of which are relatively conserved. These domains contribute to the specificity and affinity of the antigen binding sites.The isotype of the heavy chain (gamma, alpha, delta, epsilon, or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain is one of the two isotypes (kappa, κ or lambda, λ) found in all antibody classes.

[0075]

[0076] The variable region of an immunoglobulin light or heavy chain consists of a "framework" region (FR) blocked by three hypervariable regions. Therefore, the term "hypervariable region" refers to the amino acid residues of the antibody that contribute to antigen binding. The hypervariable region includes amino acid residues from the "complementarity determining region" or "CDR" and / or amino acid residues from the "hypervariable loop." The "framework region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. Therefore, the "human framework region" is substantially identical to the framework region of naturally occurring human immunoglobulins (about 85% or more, usually 90 to 95% or more). The framework region of an antibody, which is a combined framework region of the constituent light and heavy chains, contributes to positioning and aligning the CDR to the appropriate location. The CDR essentially contributes to binding to the epitope of the antigen.

[0077]

[0078] The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full-length antibodies or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments of sufficient length to exhibit desired biological activity.

[0079]

[0080] The antibody according to the present invention is a molecule comprising at least an antigen-binding portion of the antibody. The antibody or antibodies according to the present invention include intact antibodies, e.g., polyclonal antibodies or monoclonal antibodies (mAbs), as well as their proteolytic fragments, e.g., Fab fragments (monovalent antibody fragments, or denoted as F(ab)) or F(ab')2 fragments (divalent antibody fragments). Single-chain antibodies are also included within the scope of the present invention.

[0081]

[0082] "Antibody fragment" generally comprises only a portion of an intact antibody that contains the antigen-binding site of the intact antibody and thereby retains the ability to bind to the antigen. Examples of antibody fragments included in this definition include: (i) a Fab fragment containing VL, CL, VH, and CH1 domains; (ii) a Fab' fragment which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment containing VH and CH1 domains; (iv) an Fd' fragment containing VH and CH1 domains and having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment containing VL and VH domains of a single arm of the antibody; (vi) a dAb fragment consisting of a VH domain (reference [Ward et al., Nature 1989, 341, 544-546]); (vii) an isolated CDR region; (viii) an F(ab')2 fragment, which is a divalent fragment containing two Fab' fragments connected by a disulfide bridge at the hinge region; (ix) a single-stranded antibody molecule (e.g., single-stranded Fv; scFv) (references [Bird et al., Science 1988, 242, 423-426]; and [Huston et al., PNAS (USA) 1988, 85, 5879-5883]); (x) A "diabody" comprising two antigen-binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (e.g., EP 404,097; WO 93 / 11161; and see reference [Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448]); (xi) A "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) forming a pair of antigen-binding sites together with a complementary light chain polypeptide (see reference [Zapata et al. Protein Eng.Includes , 1995, 8, 1057-1062]; and U.S. Patent No. 5,641,870).

[0083]

[0084] The single-strand antibody may be a single-strand complex polypeptide having antigen-binding ability and containing an amino acid sequence homologous or similar to the variable regions of the immunoglobulin light and heavy chains, i.e., a linked VH-VL or single-strand Fv (scFv).

[0085]

[0086] In the present invention, the term "monoclonal antibody (mAb)" means an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting said population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and are for a single antigen. Additionally, unlike polyclonal antibody preparations which typically contain different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. The modifier "monoclonal" should not be interpreted as requiring the production of the antibody by any specific method. Monoclonal antibodies (mAbs) may be obtained by methods known to those skilled in the art. For example, the monoclonal antibody intended for use according to the present invention may be produced by a hybridoma method described in literature known to the art, or by a recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from a phage antibody library using techniques known in the art, for example.

[0087]

[0088] The monoclonal antibody (mAb) of the present invention may be of any class of immunoglobulin including IgG, IgM, IgE, or IgA. The hybridoma producing the mAb may be cultured in vitro or in vivo. High-titer mAbs may be obtained by in vivo production in which cells from individual hybridomas are injected intraperitoneally into pristine-primed BALB / c mice to produce an ascites fluid containing a high concentration of the desired monoclonal antibody (mAb). An isotype mAb of IgM or IgG may be purified from the ascites fluid or from the culture supernatant using column chromatography methods widely known to those skilled in the art.

[0089]

[0090] In the present invention, the term "humanized antibody" means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any technique for producing human antibodies disclosed herein.

[0091]

[0092] In the present invention, the terms “antigen-binding fragment” or “molecule comprising an antigen-binding portion of an antibody” are intended to include any isotype intact immunoglobulin molecule produced by any animal cell line or microorganism, as well as Fab fragments, Fab’ fragments, F(ab’)2 fragments, variable portions of their heavy and / or light chains, Fab mini-antibodies, dimeric bispecific mini-antibodies, and single-chain antibodies comprising antigen-binding reactive fractions, but not limited to their antigen-binding reactive fractions, as well as any other type of molecule in which said antibody reactive fraction is physically inserted. The molecule may be provided by any known technique, including but not limited to enzymatic cleavage, peptide synthesis, or recombinant techniques.

[0093]

[0094] The present invention also provides a conservative amino acid variant of an antibody molecule according to the present invention. A variant according to the present invention that preserves the overall molecular structure of the encoded protein may also be prepared. Considering the characteristics of the individual amino acids constituting the disclosed protein product, a person skilled in the art will be able to recognize some reasonable substitutions. Amino acid substitutions, i.e., “conservative substitutions,” may be made based, for example, on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues involved. In the present invention, the term “antibody analog” means one antibody derived from another antibody by one or more conservative amino acid substitutions.

[0095]

[0096] In the present invention, the term "antibody variant" refers to any molecule comprising the antibody of the present invention. For example, a fusion protein in which an antibody or its antigen-binding fragment is connected to another chemical entity is considered an antibody variant.

[0097]

[0098] In the present invention, the term "non-fully humanized monoclonal antibody" means a monoclonal antibody comprising a heavy chain and / or light chain variable domain in which the amino acid sequence located on or immediately adjacent to the CDR is not fully humanized, that is, not identical to any known homology or corresponding sequence taken from a natural human antibody.

[0099]

[0100] In the present invention, the term "amino acid residue mutation" means the substitution, insertion, or deletion of a single amino acid residue. In the present invention, the term "amino acid residue back mutation" means the substitution of a single amino acid residue found in a human antibody framework with a corresponding amino acid residue found in a murine antibody framework.

[0101]

[0102] The present invention will be described in detail below.

[0103]

[0104] The present invention provides a humanized monoclonal antibody specific to human Claudin 4 or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region:

[0105] A heavy chain FR (framework region) 1 selected from the group consisting of SEQ ID NOs 22, 27 and 32; a heavy chain CDR (complementary determining region) 1 of SEQ ID NO. 16; a heavy chain FR2 selected from the group consisting of SEQ ID NOs 23, 28 and 33; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs 18, 24, 29 and 34; a heavy chain FR3 of SEQ ID NO. 25 or 30; a heavy chain CDR3 of SEQ ID NO. 20; and a heavy chain variable region comprising a heavy chain FR4 of SEQ ID NO. 26 or 31; and

[0106] A light chain variable region comprising: a light chain FR1 selected from the group consisting of SEQ ID NOs 42, 45, and 47; a light chain CDR1 of SEQ ID NO. 36; a light chain FR2 selected from the group consisting of SEQ ID NOs 43, 46, and 48; a light chain CDR2 of SEQ ID NO. 38; a light chain FR3 of SEQ ID NO. 39; a light chain CDR3 of SEQ ID NO. 40; and a light chain FR4 of SEQ ID NO. 44.

[0107]

[0108] The present invention has produced many humanized antibodies by combining specific sets of CDR sequences and human framework (FR) sequences and introducing specific mutations into these sequences to generate antibodies with modified variable regions and improved characteristics. Specifically, the newly designed humanized variable regions described in the present invention preserve important residues for maintaining the conformation and binding affinity of the antibody, while simultaneously reducing the incidence of potential T-cell epitopes, thereby minimizing the risk of adverse immune responses to the antibody. The antibodies disclosed in the present invention were designed based on factors including homology, T-cell epitopes, key residues, and predicted structures. Consequently, the humanized antibodies disclosed in the present invention exhibited improved binding affinity and specificity compared to parent antibodies.

[0109]

[0110] In one aspect of the present invention, the humanized antibody may comprise a heavy chain immunoglobulin constant domain (Fc domain) selected from the group consisting of α, γ, μ, δ, or ε human immunoglobulin heavy chain constant regions. Specifically, the humanized antibody may comprise a heavy chain immunoglobulin constant domain selected from the group consisting of a human IgG1 constant domain, a human IgG2 constant domain, a human IgG3 constant domain, a human IgG4 constant domain, a human IgM constant domain, a human IgE constant domain, and a human IgA constant domain. In one embodiment, the human IgG4 constant domain is a mutant form stable in solution and has little to no complement activation activity. In another embodiment, the heavy chain immunoglobulin constant domain may be a human IgG4 constant domain in which Ser at position 241 (Kabat numbering) is mutated to Pro.

[0111]

[0112] In another aspect of the present invention, the immunoglobulin light chain constant region domain may be selected from the group consisting of a constant region of a κ or λ human immunoglobulin light chain. Preferably, the immunoglobulin light chain constant region domain may be a constant region of a κ human immunoglobulin light chain.

[0113]

[0114]

[0115] In another aspect of the present invention, the isotype of the human Fc domain may be characterized as IgG1, IgG2, IgG3, or IgG4.

[0116]

[0117] In one aspect of the present invention, the humanized monoclonal antibody (mAb) or its antigen-binding fragment may be characterized by comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs 2 to 7; and a light chain variable region sequence selected from the group consisting of SEQ ID NOs 9 to 11.

[0118]

[0119] In one aspect of the present invention, the humanized monoclonal antibody (mAb) or its antigen-binding fragment may be characterized by comprising the following sequence combination:

[0120] (1) The heavy chain variable region sequence of SEQ ID NO. 2 and the light chain variable region sequence of SEQ ID NO. 9;

[0121] (2) The heavy chain variable region sequence of SEQ ID NO. 3 and the light chain variable region sequence of SEQ ID NO. 9;

[0122] (3) heavy chain variable region sequence of SEQ ID NO. 4 and light chain variable region sequence of SEQ ID NO. 9;

[0123] (4) heavy chain variable region sequence of SEQ ID NO. 5 and light chain variable region sequence of SEQ ID NO. 9;

[0124] (5) The heavy chain variable region sequence of SEQ ID NO. 6 and the light chain variable region sequence of SEQ ID NO. 9;

[0125] (6) The heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO. 9;

[0126] (7) The heavy chain variable region sequence of SEQ ID NO. 2 and the light chain variable region sequence of SEQ ID NO. 10;

[0127] (8) heavy chain variable region sequence of SEQ ID NO. 4 and light chain variable region sequence of SEQ ID NO. 10;

[0128] (9) The heavy chain variable region sequence of SEQ ID NO. 5 and the light chain variable region sequence of SEQ ID NO. 10;

[0129] (10) heavy chain variable region sequence of sequence number 6 and light chain variable region sequence of sequence number 10;

[0130] (11) The heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO. 10;

[0131] (12) The heavy chain variable region sequence of sequence number 2 and the light chain variable region sequence of sequence number 11;

[0132] (13) The heavy chain variable region sequence of SEQ ID NO. 4 and the light chain variable region sequence of SEQ ID NO. 11;

[0133] (14) The heavy chain variable region sequence of SEQ ID NO. 5 and the light chain variable region sequence of SEQ ID NO. 11;

[0134] (15) The heavy chain variable region sequence of SEQ ID NO. 6 and the light chain variable region sequence of SEQ ID NO. 11; or

[0135] (16) The heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO. 11.

[0136]

[0137] In one embodiment of the present invention, the antigen-binding fragment may be selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2 and diabodies, but is not limited thereto.

[0138]

[0139] In one aspect of the present invention, the humanized monoclonal antibody (mAb) or its antigen-binding fragment may be characterized by being conjugated to one or more conjugate moietys.

[0140]

[0141] According to one embodiment of the present invention, it was confirmed that the humanized antibody of the present invention exhibits significantly superior internalization into cells expressing claudin 4 compared to the parent antibody. Therefore, when one or more conjugate moieties are conjugated to the humanized antibody according to the present invention, it may be useful because the internalization of these substances into cells can be induced with higher efficiency than with the parent antibody.

[0142]

[0143] The above conjugate moiety is a non-protein or digestible moiety that can be attached to an antibody. Various conjugate moietys may be connected to the antibody or its antigen-binding fragment provided herein. The conjugate moiety may be connected to the antibody or its antigen-binding fragment by, in particular, covalent bonding, affinity bonding, intercalation, coordination bonding, complexation, association, blending, or addition, among other methods.

[0144]

[0145] In certain embodiments, an antibody or its antigen-binding fragment is connected to one or more conjugates via a linker. In certain embodiments, the linker is a hydrazine linker, a disulfide linker, a difunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker. In certain embodiments, the linker is a lysosome-cleavable dipeptide, e.g., valine-citrulline (vc).

[0146]

[0147] The conjugate moiety may be a therapeutic agent (e.g., cytotoxic agent), a radioisotope, a detectable label (e.g., lanthanide, luminescent label, fluorescent label, or enzyme-substrate label), a pharmacokinetic modification moiety, or a purification moiety (e.g., magnetic beads or nanoparticles).

[0148]

[0149] Examples of detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansil, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luceriferase, glucoamylase, lysozyme, saccharide oxidase, β-D-galactosidase), radioisotopes, luminescent labels, chromophore moiety, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0150]

[0151] Examples of radioactive isotopes include 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re,188 Re, 153 Sm, 212 Bi, 32 It may include P and other lanthanides. Radiolabeled antibodies are useful for receptor target imaging experiments.

[0152]

[0153] In certain embodiments, the pharmacokinetic modification moiety may be a removal modifier that helps increase the half-life of the antibody. Exemplary examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, etc. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules.

[0154]

[0155] In certain embodiments, the conjugate moiety may be a purified moiety, such as magnetic beads or nanoparticles.

[0156]

[0157] In certain embodiments, the conjugate moiety comprises a cytotoxic agent, in which case the conjugate may be an antibody-drug conjugate (ADC).

[0158]

[0159] ADCs can be useful, for example, for the local delivery of cytotoxic agents in cancer treatment. This allows for the targeted delivery of cytotoxic agents to tumors and enables intracellular accumulation within them, which is particularly useful in cases where systemic administration of the aforementioned non-conjugated cytotoxic agent could result in unacceptable levels of toxicity to normal cells as well as the tumor cells to be eliminated.

[0160]

[0161] "Cytotoxic agent" may be any agent that is harmful to cancer cells or can damage or kill cancer cells. In certain embodiments, the cytotoxic agent is optionally a chemotherapy agent (e.g., growth inhibitor, DNA alkylating agent, topoisomerase inhibitor, tubulin binder, or other anticancer agent), a toxin, or a highly reactive radioisotope.

[0162]

[0163] Examples of cytotoxic agents include macromolecular bacterial toxins and phytotoxins, e.g., diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine, abrine, modesin, alpha-sarcine, Aleurites fordii protein, diantin protein, Phytolacca americana protein (PARI, PAPII, and PAP-S), momordica charantia inhibitor, cursin, crotin, saponaria officinalis inhibitor, gelonin, restrictocin, phenomycin, enomycin, and trichothecene. These macromolecular toxins may be conjugated to the antibodies provided herein or their antigen-binding fragments using methods known in the art.

[0164]

[0165] Cytotoxic agents also include, for example, small molecule toxins such as geldanamycin and chemotherapy drugs, metansinoids, caliceamycin, taxol, cytocalacin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, vindecin, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and their analogs, and anmetanotics (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), Alkylating agents (e.g., mechlorethamine, thioepah chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)) and antimitotic agents (e.g., vincristine and vinblastine), caliceamycin, metansinoids, dolastatin, auristatin (e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin It may be F(MMAF: Monomethyl auristatin F)), trichothecene, and CC1065, and derivatives thereof having cytotoxic activity. The toxin may be conjugated to the antibody or its antigen-binding fragment according to the present invention using methods known in the art.

[0166]

[0167] Cytotoxic agents can also be highly radioactive isotopes. Examples include At 211 , I 131 , I 125 , Y 90 , Re186 , Sm 153 , Bi 212 , P 32 , Pb 212 It includes radioisotopes of Lu. Methods for conjugating radioisotopes to antibodies are known in the art and are carried out, for example, through suitable ligand reagents. The ligand reagent has a chelating ligand capable of binding to, chelating, or otherwise complexing the radioisotope metal, which also has a functional group reactive to the cysteine ​​thiol of the antibody or antigen-binding fragment. Exemplary chelating ligands include DOTA, DOTP, DOTMA, DTPA, and TETA.

[0168]

[0169] In certain embodiments, the antibody is attached to the conjugate moiety through a linker, for example, a hydrazine linker, a disulfide linker, a difunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[0170]

[0171] Exemplary difunctional linkers include, for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl subverate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., It includes 1,5-difluoro-2,4-dinitrobenzene.

[0172]

[0173] In certain embodiments, the linker is cleavable under specific physiological conditions, thereby facilitating the release of a cytotoxic agent from the cell. For example, the linker may be an acid-unstable linker, a peptidase-sensitive linker, a photo-unstable linker, a dimethyl linker, or a disulfide-containing linker. In some embodiments, the linker may include amino acid residues, e.g., a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide. The amino acid residues within the linker may be naturally occurring or unnaturally occurring amino acid residues. Examples of the linker include valine-citrulline (ve or val-cit), valine-citrulline (ve or val-cit), alanine-phenylalanine (af or ala-phe), glycine-valine-citrulline (gly-yal-cit), glycine-glycine-glycine (gly-gly-gly), and valine-citrulline-p-aminobenzyloxycaronyl ("vc-PAB"). The amino acid linker component can be designed and optimized for its selectivity to enzymatic cleavage by specific enzymes, for example, tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0174]

[0175] The present invention also provides a polynucleotide encoding the humanized monoclonal antibody (mAb) or an antigen-binding fragment thereof, a vector comprising said polynucleotide, and a host cell transfected with said vector.

[0176]

[0177] In the present invention, "polynucleotide" may be described as an oligonucleotide or a nucleic acid, and includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of said DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring eucleotide analogs), and hybrids thereof. The polynucleotide may be single-stranded or double-stranded. The polynucleotide refers to a base sequence encoding an antibody consisting of a heavy chain and a light chain having a CDR configuration specific to human CLDN 4 protein, or a VH and VL configuration.

[0178]

[0179] The polynucleotide encoding the antibody of the present invention or its antigen-binding fragment can be obtained by methods well known in the art. For example, based on a DNA sequence or corresponding amino acid sequence encoding part or all of the heavy chain and light chain of the antibody, it can be synthesized using oligonucleotide synthesis techniques well known in the art, such as the polymerase chain reaction (PCR) method.

[0180]

[0181] The "vector" of the present invention is used for the purpose of replication or expression of the polynucleotide of the present invention for the recombinant production of the antibody of the present invention or its antigen-binding fragment, and generally comprises one or more of a signal sequence, a replication origin, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably may be a vector comprising the polynucleotide of the present invention operably linked to a regulatory sequence, e.g., a promoter.

[0182]

[0183] A plasmid, a type of vector, refers to a linear or circular double-helix DNA molecule to which external polynucleotide fragments can be attached. Another form of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), into which additional DNA fragments can be introduced into the viral genome. Certain vectors can undergo autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors, including those of bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell genome by introduction into the host cell and thereby replicated together with the host genome.

[0184]

[0185] In the present invention, the term "vector" may be understood as having the same meaning as "expression vector," which is a form of vector capable of expressing the polynucleotide. A polynucleotide sequence is "operably linked" to a regulatory sequence where the regulatory sequence influences the expression (e.g., level, timing, or position of expression) of the polynucleotide sequence. The regulatory sequence is a sequence that influences the expression (e.g., level, timing, or position of expression) of the nucleic acid to which it is operablely linked. The regulatory sequence may exert its influence, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). The regulatory sequence includes promoters, enhancers, and other expression regulatory elements.

[0186]

[0187] The cell line of the present invention is not particularly limited in type, provided that it is a cell that can be used to express a polynucleotide encoding an antibody or a fragment thereof included in the expression vector of the present invention. The cell (host cell) transformed with the expression vector according to the present invention may be a prokaryote (e.g., Escherichia coli), a eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells), or a hybridoma derived therefrom. Preferably, it may be a cell derived from a mammal, including humans.

[0188]

[0189] Prokaryotes suitable for this purpose are Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, e.g., Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella; Bacillus belonging to the genus Bacilli (e.g., B. subtilis and B. licheniformis); Includes Pseudomonas (e.g., P. aeruginosa) and Streptomyces. The cells of the present invention are not particularly limited as long as they are capable of expressing the vector of the present invention, but preferably may be E. coli.

[0190]

[0191] As the cell of the present invention, the eukaryotic organism most commonly used is Saccharomyces cerevisiae. However, many other genera, species, and strains, but not limited thereto, such as *Schizosaccharomyces pombe*, *Cluiveromyces* hosts, such as *K. lactis*, *K. fragilis* (ATCC 12,424), *K. bulgaricus* (ATCC 16,045), *K. wickeramii* (ATCC 24,178), *K. waltii* (ATCC 56,500), *K. drosophilarum* (ATCC 36,906), and *K. K. thermotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. Niger (A. niger) is available.

[0192]

[0193] The term "transformation" above is used with the same or similar meaning as "transfection" or "transduction." It refers to a modification of the genotype of a host cell caused by the introduction of an exogenous polynucleotide (a polynucleotide encoding the antibody or fragment thereof of the present invention), and signifies the introduction of the exogenous polynucleotide into the host cell regardless of the method used for the transformation. The exogenous polynucleotide introduced into the host cell may be maintained within the host cell's genome or may remain unintegrated, and the present invention encompasses both.

[0194]

[0195] A recombinant vector capable of expressing the antibody or fragment thereof of the present invention can be transformed by introducing it into a cell to produce the antibody or fragment thereof by methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and known methods for introducing nucleic acids into a cell.

[0196]

[0197] Additionally, the cell of the present invention is a cultured cell that can be transformed or transfected with the polynucleotide of the present invention or a vector containing it, and which can be subsequently expressed within the host cell. A recombinant cell refers to a cell that has been transformed or transfected with the polynucleotide to be expressed. The cell of the present invention may also be a cell that contains the polynucleotide of the present invention but does not express it to a desired level unless a regulatory sequence is introduced into the cell to be operably linked to the polynucleotide.

[0198]

[0199] The cells of the present invention can be cultured in various media. Commercially available media, such as Ham's F1O (Sigma-Aldrich Co., St. Louis, MO), Minimal Essential Medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich Co.), are suitable for culturing cells. Hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or equivalent energy sources may be added to the media if necessary.

[0200]

[0201] The present invention provides a method for producing a humanized antibody or a fragment thereof that binds to a Claudin 4 protein, comprising the steps of: culturing the cell under conditions in which a polynucleotide is expressed to produce a polypeptide comprising a light chain and a heavy chain variable region; and recovering the polypeptide from the cell or the culture medium in which the polypeptide is cultured.

[0202] The cell of the production method in the present invention is as described above and comprises a polynucleotide encoding the antibody of the present invention. The polypeptide of the production method may be the antibody of the present invention or a fragment thereof itself, or it may have an additional amino acid sequence other than the antibody of the present invention or a fragment thereof attached thereto.

[0203]

[0204] In this case, the antibody of the present invention or its fragment can be removed using methods well known to a person skilled in the art. The culture medium composition and culture conditions may vary depending on the type of cell, and these can be appropriately selected and adjusted by a person skilled in the art.

[0205]

[0206] The above antibody molecules may accumulate in the cytoplasm of the cell, be secreted from the cell, or be targeted to the periplasm or extracellular medium (supernatant) by an appropriate signal sequence, and targeting to the periplasm or extracellular medium is preferable. In addition, it is preferable to refold the produced antibody molecules using methods well known to those skilled in the art and to make them conform to a functional form. The recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cell, and this can be appropriately selected and controlled by a person with ordinary knowledge in the art.

[0207]

[0208] The polypeptide may be produced within the cell, in the surrounding cytoplasmic space, or secreted directly into the medium. If the polypeptide is produced within the cell, the cell may be destroyed as a first step to release the protein. Particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from this expression system is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit protein degradation, a protease inhibitor, for example PMSF, may be included in any preceding step, and an antibiotic may be included to prevent the growth of accidental contaminants. The antibody produced from the cell may be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and the antibody of the present invention may preferably be purified via affinity chromatography.

[0209]

[0210] The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer comprising the humanized monoclonal antibody (mAb) or its antigen-binding fragment as an active ingredient.

[0211]

[0212] In the present invention, the cancer may preferably be a cancer expressing claudin 4, and may be, for example, breast cancer, ovarian cancer, stomach cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, or ureteral cancer.

[0213]

[0214] The term "treatment" in the present invention comprehensively refers to improving symptoms caused by the cancer, and may include curing, substantially preventing, or improving the condition of the cancer, and includes, but is not limited to, alleviating, curing, or preventing one or most symptoms resulting from the cancer.

[0215]

[0216] In the present invention, the content of the composition is not significantly limited depending on the purpose or aspect of use, and may, for example, be 0.01 to 99 weight%, preferably 0.5 to 50 weight%, more preferably 1 to 30 weight% based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient. The pharmaceutical composition of the present invention may contain 0.1 to 99.9 weight% of a humanized monoclonal antibody or an antigen-binding fragment thereof prepared by the method of the present invention, and may contain 99.9% to 0.1 weight% of a carrier.

[0217]

[0218] The pharmaceutical composition according to the present invention may comprise only the antibody or a fragment thereof according to the present invention, or may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may additionally comprise excipients or diluents. In the above, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness. The carrier includes all types of solvents, dispersion media, water vapor oils or oil vapor water emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

[0219]

[0220] Pharmaceutically acceptable carriers may additionally include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Additionally, they may include various drug delivery materials used for oral administration of peptide preparations. Furthermore, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose and glycol, etc., and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may additionally include lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may be referenced from the following literature.

[0221]

[0222] The composition of the present invention may be administered to mammals, including humans, by any method. For example, it may be administered orally or parenterally. Parenteral administration methods may include, but are not limited to, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration.

[0223]

[0224] The pharmaceutical composition of the present invention can be formulated into an oral or parenteral administration formulation according to the administration route described above. In the case of an oral administration formulation, the composition of the present invention can be formulated into a powder, granule, tablet, pill, coated tablet, capsule, liquid, gel, syrup, slurry, suspension, etc., using methods known in the art. For example, an oral formulation can be obtained by combining an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and processing it into a granular mixture to obtain a tablet or coated tablet. Examples of suitable excipients may include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants. Furthermore, the pharmaceutical composition of the present invention may additionally include anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives. In the case of parenteral formulations, they can be formulated in the form of injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are generally known in all pharmaceutical chemistry.

[0225]

[0226] The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or administered via a fractionated treatment protocol involving multiple doses administered over a long period. The content of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg per kg of patient body weight per day, most preferably 0.1 μg to 500 mg. However, since the effective dose for a patient is determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, a person of ordinary knowledge in the art would be able to determine an appropriate effective dose of the composition of the present invention in light of these factors. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0227]

[0228] The term "effective amount" of the present invention refers to an amount that, when administered to an individual, produces an effect of improving, treating, detecting, diagnosing, or suppressing or reducing cancer or said disease. The term "individual" may be an animal, preferably a mammal, particularly including humans, and may also be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient requiring said effect.

[0229]

[0230] The present invention also provides a cancer diagnostic composition comprising the humanized monoclonal antibody (mAb) or its antigen-binding fragment as an active ingredient.

[0231]

[0232] As described above, the antibody can specifically bind to Claudin 4 of cancer cells. Therefore, when a composition containing the antibody is treated to cancer cells expressing Claudin 4, the antibody binds to the Claudin 4 antigen of the cancer cells to form an antigen-antibody complex, thereby allowing for the separation of cancer cells that bind to the antibody and normal cells that do not bind to it.

[0233]

[0234] The present invention also provides an immunoconjugate in which a therapeutic agent or a label is conjugated to a humanized monoclonal antibody or an antigen-binding fragment thereof.

[0235]

[0236] In the present invention, the term "immunoconjugate" refers to an antibody or an antigen-binding fragment thereof conjugated to an additional moiety. The antibody of the present invention may be conjugated to various moietys, for example, to improve the therapeutic / diagnostic properties of the antibody, to facilitate detection, or for imaging or therapeutic purposes. For example, the antibody of the present invention may be conjugated to a therapeutic agent, a detectable label, and / or a transport moiety.

[0237]

[0238] The antibody of the present invention or its antigen-binding fragment may be conjugated with a therapeutic agent (e.g., nucleotide, peptide, protein, cytokine, amino acid, lipid, carbohydrate, small molecule compound, radioactive isotope, and combinations thereof), and in this case, the immunoconjugate may also be called an antibody-drug conjugate (ADC), an antibody-cytokine fusion protein (ACFP), etc.

[0239] The above-mentioned immunoconjugate can deliver a conjugated therapeutic agent to a cell (e.g., a tumor cell) through specific binding to an antigen on a target cell. In this case, the immunoconjugate may enter the interior of the target cell to exert a cytotoxic effect of the conjugated therapeutic agent, or it may bind to the surface of the target cell so that the conjugated therapeutic agent interacts with the adjacent environment (e.g., adjacent cells, immune cells, etc.) to exert enhanced pharmacological activity.

[0240] In one aspect of the present invention, the therapeutic agent is a cytotoxic agent. In a specific embodiment, the therapeutic agent may be selected from the group consisting of alkylating agents, mitotic inhibitors, anticancer antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof. Examples of the alkylating agents include, but are not limited to, nitrogen mustard (e.g., mechlorethamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethyleneimine (e.g., thiotepa, etc.), sulfate esters and polyols (e.g., busulfan, dibromomannitol), nitrosourea (e.g., camustine, lomustine, etc.), platinum-based antitumor agents (e.g., cisplatin, oxaliplatin, carboplatin, etc.). Examples of the above-mentioned mitotic inhibitors include, but are not limited to, metansinoids (e.g., metansin, metansinol, C-3 ester of metansinol), taxanes (e.g., docetaxel, paclitaxel or nanoparticle paclitaxel), and vinca alkaloids (e.g., vindecin sulfate, vincristine, vinblastine or vinorelbine). Examples of the above-mentioned anticancer antibiotics include, but are not limited to, actinomycin, anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), caliceamicin, and duocarmycin. Examples of the above anmetatars include, but are not limited to, folic acid antagonists (e.g., methotrexate, etc.), pyrimidine antagonists (e.g., 5-fluorouracil, fluoxuridine, cytarabine, capecitabine, gemcitabine, etc.), purine antagonists (e.g., 6-mercaptopurine, 6-thioguanine, etc.), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, pentostatin, etc.).Examples of the above-mentioned topoisomerase inhibitors include, but are not limited to, camtothecin and its derivatives (e.g., irinotecan, topotecan, etc.), amsacrin, daunomycin, adriamycin, epipodophyllotoxin, ellipticin, epirubicin, etoposide, razoxane, tenifoside, etc. Examples of the above-mentioned tyrosine kinase inhibitors include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, restaurtinib, nilotinib, semaxanib, sunitinib, vandetanib, etc. Examples of the above-mentioned radionuclides include, 131 I, 111 In, 90 Y, 177 Lu, etc. are included but not limited thereto. In certain embodiments, the therapeutic agent may be selected from the group consisting of platinum-based antineoplastics, anthracycline antibiotics, taxane compounds, nucleoside analogs, camtothecin compounds and analogs or homologues thereof and any combination thereof.

[0241]

[0242] The antibody or its antigen-binding fragment of the present invention may be bound to a detectable label to provide measurability, for example, to facilitate quantification or imaging. The labeled antibody may be used in various analyses using various labels, particularly in immunoassays. Preferred labels may include, but are not limited to, radioisotopes, enzymes, coenzymes, fluorescent substances, chemiluminescent substances, chromogenic substances, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes (e.g., fluorescent dyes, tandem dyes), etc.

[0243]

[0244] The antibody or its antigen-binding fragment of the present invention may be conjugated to a transport moiety. A transport moiety typically refers to a substance that mediates the transport of the antibody to a target, for example, a specific part of the (human) body or cell.

[0245]

[0246] In the present invention, the immunoconjugate comprises various dual-functional protein coupling materials, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), dual-functional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., It can be a "cleavable linker" that can be conjugated using 1,5-difluoro-2,4-dinitrobenzene and facilitates the release of therapeutic agents within the cell. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers may be used, but are not limited thereto.

[0247]

[0248] The present invention provides the use of the humanized monoclonal antibody or its antigen-binding fragment for preparing a composition for cancer treatment.

[0249]

[0250] The present invention provides a method for treating cancer comprising administering an effective amount of a composition containing the humanized monoclonal antibody or its antigen-binding fragment as an active ingredient to an individual in need thereof.

[0251]

[0252] The present invention provides a use of the humanized monoclonal antibody or its antigen-binding fragment for preparing a cancer diagnostic composition.

[0253]

[0254] The present invention provides a cancer diagnosis method comprising the following steps:

[0255] i) a step of administering to a subject the above-mentioned humanized monoclonal antibody or an immunoconjugate in which a label is conjugated to an antigen-binding fragment thereof;

[0256] ii) a step of the above immunoconjugate binding to a target site within the subject;

[0257] iii) a step of imaging and detecting a signal generated from the above immunoconjugate; and

[0258] iv) A step of diagnosing whether cancer has occurred in the target site based on the detected signal.

[0259]

[0260] Examples of the above labels may include (e.g., fluorescein, rhodamine, dansil, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luceriferase, glucoamylase, lysozyme, saccharide oxidase, β-D-galactosidase), radioisotopes, luminescent labels, chromophore moiety, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0261]

[0262] Examples of radioactive isotopes include 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y,90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 It may include P and other lanthanides. Radiolabeled antibodies are useful for receptor target imaging experiments.

[0263]

[0264] As is known to those skilled in the art, the imaging of the above-mentioned subject may include imaging or monitoring techniques recognized in the art, such as magnetic resonance imaging, computerized tomography (CT scan), positron tomography (PET scan), radiography, ultrasound, etc.

[0265]

[0266] Diagnosing whether cancer has developed in the target site based on the detected signal of the present invention means evaluating the presence or level of claudin 4 in a neoplasm, tumor, or cancer by a detectably labeled humanized monoclonal antibody or an antigen-binding fragment thereof according to a method known in the relevant art. More specifically, it may include quantitatively or qualitatively analyzing the intensity, distribution, or pattern of the signal obtained from the immunoconjugate and evaluating the presence or expression level of human claudin 4 in the target site by comparing it with a reference value, a background signal, or a control.

[0267]

[0268] In the step of diagnosing whether cancer has occurred, if cancer is diagnosed to have occurred based on the presence and expression level of human claudin 4 at the target site, a step of treating the cancer may be additionally included. In the treatment step, a conventionally used cancer treatment method may be used and may include administering the composition of the present invention or an anticancer agent.

[0269]

[0270] The above anticancer agent may be selected from a group consisting of chemotherapy agents, targeted anticancer agents, oncolytic viruses, antibody therapeutic agents, immunotherapy agents (e.g., immune checkpoint inhibitors), and combinations thereof.

[0271]

[0272] The humanized antibody according to the present invention exhibits improved binding strength and specificity compared to the parent antibody, and has a significantly higher internalization rate into cells expressing claudin 4, so it can be very usefully utilized in the development of therapeutic agents and diagnostic agents targeting cells expressing claudin 4.

[0273]

[0274] Figures 1a to 1c show the results of analyzing the degree of humanization through the Humaness score of selected humanized Claudin 4 antibodies.

[0275] Figure 2 shows the results of analyzing the binding patterns of humanized claudin 4 antibodies to human claudin 4 and mouse claudin 4.

[0276] Figures 3a to 3o and Figure 4 show the results of confirming the cross-reactivity of humanized claudin 4 antibodies against other claudins.

[0277] Figure 5 shows the results of measuring the antigen-antibody binding strength between Claudin 4 and humanized Claudin 4 antibodies using BLI equipment.

[0278] Figures 6a to 6c show the results of measuring the binding affinity between a humanized anti-claudin 4 antibody and human claudin 4 (hCLDN4) using a MDA-MB-453 cell line expressing human claudin 4 by flow cytometry.

[0279] Figures 7, 8a, and 8b show the results of evaluating antibody-dependent cytotoxicity (ADCC) against MDA-MD-453 using the anti-Claudin 4 antibody and the NK92-MI-CD16a cell line expressing human CD16a.

[0280] Figures 9a to 9g and Figure 10 show the results of evaluating the cell internalization efficiency of each humanized anti-Claudin 4 antibody by performing a ZAP assay on the non-small cell lung cancer cell line SNU-1327 or the triple-negative breast cancer cell line MDA-MB-453.

[0281] Figure 11 shows the results of evaluating the degree of internalization of humanized anti-claudin 4 antibody in MDA-MB-453 cells using flow cytometry.

[0282] Figure 12 is a diagram showing the experimental schedule for evaluating the anticancer activity of humanized anti-claudin 4 antibody in mice xenografted with MDA-MB-453 cell lines.

[0283] Figures 13a to 13d show the results of evaluating the anticancer activity of humanized anti-claudin 4 antibodies in mice xenografted with MDA-MB-453 cell lines.

[0284]

[0285] The present invention will be explained in detail below through the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention.

[0286]

[0287] Example 1: Production of Humanized Anti-Claudin 4 Antibody

[0288] Based on the light and heavy chain sequences of the publicly known chimeric anti-human claudin 4 monoclonal antibody (Chimeric anti-hCLDN4 mAb, hereinafter c4Ab1 or parent antibody), a human germ cell-derived framework sequence with the most similar sequence was selected and utilized for antibody design. A humanization sequence was designed by considering antigen-antibody binding affinity, VH / VL pairing, Vernier zone-related amino acid residues, and various factors that may affect the structural stability and binding affinity of the antibody. The lengths of the framework sequences and CDR sequences of c4Ab1 (parent antibody) and the selected human germ cell-derived sequence were confirmed using Kabat, IMGT, and Chothia numbering. The binding structure between the humanized anti-claudin 4 antibody and claudin 4 was predicted through reported epitope region-based prediction and docking simulation. Based on the corresponding antigen-antibody binding structure prediction data, amino acid residues expected to affect the binding affinity with human CLDN4 were selected from the framework sequence of c4Ab1 (parent antibody), excluding the CDR region, through in silico alanine scanning of the humanized anti-Claudin 4 antibody. Alternatively, amino acid residues affecting antibody structure formation and those inducing energy value instability were selected through in silico structural analysis of the humanized antibody sequence obtained by CDR grafting using the framework sequence of the human germline sequence and the CDR sequence of c4Ab1 (parent antibody). Back-mutations were introduced into the humanized anti-Claudin 4 antibody sequence using the amino acid residues selected from the c4Ab1 (parent antibody) sequence and the amino acid residues selected from the humanized antibody sequence obtained by CDR grafting.Additionally, the FR (framework region) 3 portion of the VL sequence of the humanized anti-claudin 4 antibody with the above back-mutation was replaced with the FR3 sequence of c4Ab1 (parent antibody), and consequently, a humanized antibody sequence was also designed with 8 to 10 amino acids back-mutated to the amino acids of c4Ab1 (parent antibody) (Tables 1 to 3).

[0289]

[0290] [Table 1]

[0291]

[0292]

[0293] [Table 2]

[0294]

[0295]

[0296] [Table 3]

[0297]

[0298]

[0299] [Table 4]

[0300]

[0301] The designed humanized anti-Claudin 4 antibody and c4Ab1 (parent antibody) were obtained using the ExpiCHO-S cell line (Thermo Fisher Scientific, Waltham, MA, USA) as the host cell. Specifically, antibody-producing cell lines were produced by transiently transfecting the ExpiCHO-S cell line according to the manufacturer's protocol using the genetic information of each antibody, the ExpiFectamine™ CHO Transfection Kit (Thermo Fisher Scientific), and OptiPRO™ SFM (Thermo Fisher Scientific). The produced cell lines were cultured using ExpiCHO Expression Medium (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0302] The simple binding affinity of each antibody to the cell line culture medium and the antigen (hCLDN3) was measured. Specifically, the antigen-antibody binding affinities of c4Ab1 (parent antibody) and the designed humanized antibody were primarily compared using an AHC (Anti-hIgG capture) Biosensor (Sartorius) and BLI equipment according to the manufacturer's protocol (Table 5).

[0303]

[0304] [Table 5]

[0305]

[0306] Among the designed humanized antibody sequences, six heavy chain sequences (H21a, H21b, H21-m1, H24, H24a, H24m-1) and three light chain sequences (L17-2, L20-2, L21-2) were finally selected by additionally considering the Humanness score (Figs. 1a to 1c), and were used in subsequent examples.

[0307]

[0308] Example 2: Confirmation of the humanness score of the humanized anti-claudin 4 antibody

[0309] The humanness scores of the light and heavy chain sequences of the finally selected humanized anti-Claudin 4 antibodies were confirmed using T20 and Z-score results. A public database was used for Z-score analysis, and predicted results were verified using a specific program for T20 score analysis. Results showed improved humanness scores in the humanized antibodies compared to c4Ab1 (parent antibody), and humanness scores similar to those of Trastuzumab and Omalizumab, which are generally well-known humanized antibodies, were obtained (Figs. 1a to 1c).

[0310]

[0311] Example 3: Verification of cross-reactivity

[0312] Cross-reactivity with other claudin proteins (human claudin: hCLDN 1, hCLDN 3, hCLDN 4, hCLDN 5, hCLDN 6, hCLDN 8, hCLDN 9, hCLDN 17; murine claudin: mCLDN 3, mCLDN 4) was evaluated through flow cytometry for several types of humanized anti-claudin 4 antibodies prepared by combining the heavy chain sequence and light chain sequence selected in Example 1.

[0313] Each claudin protein was artificially overexpressed in HEK293 cells (Korean Cell Line Bank, Cat. No. 21573) and utilized for cross-reactivity analysis. Specifically, cDNA encoding each claudin protein was cloned into pcDNA3.1(+) (Invitrogen), and the cloned products were transfected into HEK293 cells using Opti-MEM (gibco, #31985-062) and Lipofectamine 2000 (invitrogen, #11668-019), respectively. Subsequently, the cells were treated with neomycin to perform initial selection of the transformed cells, and additionally, cell populations with high expression levels of each claudin protein were obtained and cultured using BD FACSAria III (BD Biosciences, San Jose, CA, USA). Every day, we used a Celigo (Nexcelom Bioscience, USA) instrument to verify whether the cells were derived from a single cell, and finally obtained HEK293 cell lines that stably express each claudin.

[0314] After culturing each of the above cell lines and untransformed HEK293 cells to be used as a control to a sufficient number, each cell was harvested and 3 x 10 6 cells / mL (100 µl) were dispensed. Subsequently, 10 µg / mL of the humanized anti-Claudin 4 antibody prepared in Example 1 and c4Ab1 (parent antibody) were each administered to each dispensed cell. o After treatment at C for 1 hour, the body was washed three times repeatedly with PBS / 1% FBS buffer. Subsequently, 4 doses of 1:100 diluted anti-human IgG-FITC antibody (Jackson Immunoresearch Laboratories) were added. oTreatment was performed in C for 45 minutes. After washing three times with PBS / 1% FBS buffer, the cross-reactivity of the humanized anti-claudin 4 antibody was analyzed using a flow cytometer (BD FACS Calibur).

[0315] In the case of c4Ab1 (parent antibody), some cross-reactivity with other claudins besides claudin 4 was observed; however, in the newly designed humanized anti-claudin 4 antibody after the humanization process described herein, this cross-reactivity disappeared, and it was confirmed that it specifically binds only to claudin 4. In particular, it was confirmed that the humanized anti-claudin 4 antibody of this specification significantly reduces binding to murine claudin 4 (mCLDN4) and specifically binds very strongly to human claudin 4 (hCLDN4) (Figs. 2 to 4).

[0316]

[0317] Example 4: Evaluation of binding strength (BLI & Flow cytometry)

[0318] The binding affinity between the humanized anti-claudin 4 antibody of this specification and the antigen (hCLDN4) was measured using a BLI instrument. Specifically, each humanized anti-claudin 4 antibody (5 μg / ml) prepared in Example 1 using the Human Fc capture method was immobilized as a ligand on a biosensor, and experiments were performed using human recombinant claudin 4 protein (Origen, Cat. No. TP300490) as the analyte. Various concentration ranges were established by serially diluting the analyte in ½ increments starting from an initial concentration of 500 nM. 10x KB was used as the buffer for the experiment, and both the binding and dissociation steps were carried out for 300 seconds. The results were measured using an Octet QK384 (ForteBio) according to the manufacturer's protocol. The measurement results are shown in Figure 5.

[0319] The binding affinity between the humanized anti-claudin 4 antibody of this specification and the antigen (hCLDN4) was measured by flow cytometry. Specifically, each antibody was applied to the MDA-MB-453 (Korean Cell Line Bank, Cat. No. 30131) cell line expressing human claudin 4 at various concentration ranges (concentration ranges set from a final antibody concentration of 200 nM at cell treatment to below 4). o After treatment in C for 1 hour, the cells were washed three times repeatedly with PBS / 1% FBS buffer. Subsequently, each cell was treated with 4 doses of anti-human IgG-FITC antibody (Jackson Immunoresearch Laboratories) diluted 1:100. o After treatment in C for 1 hour, the samples were washed three times with PBS / 1% FBS buffer. Finally, the binding affinity between each antibody described herein and the antigen (hCLDN4) was analyzed using a flow cytometer (BD FACSCalibur). (Figs. 6a to 6c)

[0320]

[0321] Example 5: Confirmation of anticancer activity of anti-Claudin 4 antibody in Claudin 4-positive triple-negative breast cancer cell lines

[0322] Anticancer activity (antibody-dependent cell-mediated cytotoxicity (ADCC)) was confirmed using an anti-claudin 4 antibody (c4Ab1 (parent antibody) and the antibody of Example 1, with Sacituzumab, an anti-TROP-2 antibody, used as a control) in MDA-MD-453 (Korean Cell Line Bank, Cat. No. 30131) and an NK92-MI-CD16a cell line expressing human CD16a.

[0323] MDA-MB-453 2 X 10 6After dispensing cells / mL (100 µl) into each well of a 96-well plate, anti-Claudin-4 antibodies at various concentration ranges (10 µg / mL to 0.001 µg / mL) were administered the following day at room temperature for 30 minutes. Subsequently, 4 x 10⁶ cells were added to each well. 6 After treating NK92-MI-CD16a cells at cells / mL (25ul) and culturing for 4 hours, the anticancer effect of the antibody was confirmed through the LDH (lactate dehydrogenase) measurement method using EZ-LDH (DoGenBio, Cat# DG-LDH1000).

[0324]

[0325] As can be seen in Figures 7, 8a, and 8b, it was confirmed that the anticancer activity of the humanized anti-claudin 4 antibody in MDA-MB-453 was similar to that of c4Ab1 (parent antibody).

[0326]

[0327] Example 6: Evaluation of Cell Internalization of Humanized Anti-Claudin 4 Antibody Using ZAP Assay

[0328] The cellular internalization efficiency of each humanized anti-Claudin 4 antibody was evaluated by performing a ZAP assay using an anti-Claudin 4 antibody and 'Hum-ZAP' (ADVANCED TARGETING SYSTEM, Cat# IT-22), an anti-human antibody conjugated with saporin that induces apoptosis through ribosome inhibition, on SNU-1327 (non-small cell lung cancer) or MDA-MB-453 (triple-negative breast cancer) cell lines. SNU-1327 or MDA-MB-453 cell lines were plated in 96-well plates at a density of 2.5 x 10 per well 4The cells were dispensed at a concentration of 100 μL / mL. To evaluate the cellular internalization efficiency of the humanized anti-Claudin 4 antibody across various concentration ranges (set so that the antibody concentration at the time of cell treatment was a maximum of 300 nM and below), each antibody was diluted with a pre-prepared 18 nM Hum-ZAP reagent, and the diluted antibody-reagent mixture was reacted at room temperature for 15 minutes to obtain an antibody-reagent complex. Subsequently, the culture medium for each cell was removed, and 75 μL of fresh culture medium was added. Then, 25 μL of the prepared antibody-reagent complex was added to each cell and cultured for 72 hours, after which the cellular internalization of the humanized anti-Claudin 4 antibody was quantitatively evaluated using the WST-1 assay with EZ-Cytox (DoGenBio, Cat# EZ-3000).

[0329]

[0330] As can be seen in FIGS. 9a to 9g and FIG. 10, the cellular internalization efficiency of the humanized anti-claudin 4 antibody of this specification was significantly improved compared to c4Ab1 (parent antibody). Furthermore, each cell line is a Trop-2 positive cell line, which is a cell that simultaneously expresses claudin 4 and Trop-2. When comparing the cellular internalization efficiency of the anti-Trop2 antibody sacituzumab, a component of TRODELVY (sacituzumab govitecan-hziy), and the humanized anti-claudin 4 antibody of this specification in each cell line, it was confirmed that the humanized anti-claudin 4 antibody of this specification showed superior performance.

[0331]

[0332] These results suggest that the humanized antibody according to the present invention may be suitable for targeted therapeutic applications requiring effective cellular uptake, such as antibody-drug conjugates (ADCs).

[0333]

[0334] Furthermore, although increased cellular internalization of antibodies generally leads to a decrease in ADCC effect, the humanized antibodies according to the present invention exhibited ADCC activity equivalent to or greater than that of the parent antibody. This balance between cellular internalization and ADCC activity indicates the potential for the humanized antibodies according to the present invention to be utilized in various therapeutic applications.

[0335]

[0336] Example 7: Evaluation of Cell Internalization of Humanized Anti-Claudin 4 Antibody Using Flow Cytometry

[0337] After culturing the MDA-MB-453 cells, the cells were harvested at a rate of 5 x 10 5 They were dispensed one by one. To each dispensed cell, 10 µg / mL of humanized Claudin 4 antibody was administered under different conditions (4 o C, 1 hour; 37 o They were treated with (C, 24 hours). Afterwards, they were washed three times repeatedly with PBS / 1% FBS buffer. Subsequently, each cell was treated with 4 doses of anti-human IgG-FITC antibody (Jackson Immunoresearch Laboratories) diluted 1:100. o After treatment in C for 1 hour, the cells were washed three times with PBS / 1% FBS buffer. Finally, the cellular internalization of each antibody was analyzed using a flow cytometer (BD FACSCalibur). (Fig. 11)

[0338] The degree of cellular internalization based on the measured data was calculated using the following formula.

[0339] Relative MFI = hCLDN4 MFI / Control MFI

[0340] Internalization (%) = { ( 1hr Relative MFI - 24hr Relative MFI ) / ( 1hr Relative MFI )}

[0341]

[0342] Example 8: Evaluation of the efficacy of humanized anti-claudin 4 antibody in an in vivo environment

[0343] The efficacy of the humanized anti-claudin 4 antibody of this specification was evaluated in a xenograft mouse model using cancer cell lines. Specifically, human triple-negative breast cancer cell lines, specifically MDA-MB-453, were cultured under standard conditions, harvested, washed with DPBS, and resuspended in 50% Matrigel. Subsequently, 5 x 10^6 cells were injected subcutaneously into the right flank of each experimental mouse (NOG mouse, female, 6 to 8 weeks old, KoaTech). After the tumor size reached approximately 100 mm³, the antibody of this specification, the experimental substance, was injected. The antibody was injected intraperitoneally (IP) into each mouse at a dose of 10 mg / kg, and sequentially 2 x 10 6 Canine NK92-MI-CD16a cells (suspended in 100 μL DPBS) were injected via the tail vein (intravenous; IV). Antibody administration was performed a total of 5 times at intervals of 3 to 4 days. Tumor size was measured twice a week using a digital vernier caliper, and tumor volume was calculated according to the following formula.

[0344] [Tumor Volume Calculation Formula]

[0345] Tumor Volume (mm³) = Diameter (mm) × Area (mm²) × 0.5

[0346]

[0347] Based on the measured tumor volume, the Tumor Growth Inhibition (%TGI) and the ratio of the final tumor volume of the treatment group to the control group (tumor volume over control volume, %T / C) were calculated for each individual. The formula is as follows.

[0348] [%TGI Calculation Formula]

[0349] %TGI = [1 - (T n -T0) / (C n -C0)] x 100

[0350] = [1 - (Average increase in tumor volume of treatment group / Increase in tumor volume of control group)]×100

[0351]

[0352] [%T / C Calculation Formula]

[0353] %T / C = T n / C n

[0354] = (Tumor volume of treatment group / Average tumor volume of control group) × 100

[0355] (T : Treatment group, C : Control group, T n : Tumor volume on the final measurement date of the treatment group, C n : Tumor volume of the control group on the final measurement date, T0: Tumor volume of the treatment group on the initial measurement date, C0: Tumor volume of the control group on the initial measurement date)

[0356]

[0357] The daily tumor volume, body weight, %TGI, and %T / C of each individual are shown in Fig. 12 and Figs. 13a to 13d.

[0358]

[0359] Statistical significance

[0360] Statistical analysis was performed using one-way and two-way ANOVA to compare differences between treatment groups. Where statistical significance was confirmed, Tukey-Kramer post-hoc tests were applied to confirm differences between specific groups. Data were expressed as mean ± standard deviation (SD), and a P-value less than 0.05 was considered statistically significant. Significance levels were indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001.

[0361]

[0362]

[0363] The humanized antibody according to the present invention exhibits enhanced binding affinity and specificity for human claudin 4 compared to the parent antibody, and also has a significantly higher internalization rate into cells expressing claudin 4, so it can be very usefully utilized in the development of therapeutic agents and diagnostic agents targeting cells expressing claudin 4, and thus has very high industrial applicability.

Claims

1. A humanized monoclonal antibody specific to human Claudin 4 comprising the following heavy chain variable region and light chain variable region, or an antigen-binding fragment thereof: A heavy chain FR (framework region) 1 selected from the group consisting of SEQ ID NOs 22, 27 and 32; a heavy chain CDR (complementary determining region) 1 of SEQ ID NO. 16; a heavy chain FR2 selected from the group consisting of SEQ ID NOs 23, 28 and 33; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs 18, 24, 29 and 34; a heavy chain FR3 of SEQ ID NO. 25 or 30; a heavy chain CDR3 of SEQ ID NO. 20; and a heavy chain variable region comprising a heavy chain FR4 of SEQ ID NO. 26 or 31; and A light chain variable region comprising: a light chain FR1 selected from the group consisting of SEQ ID NOs 42, 45, and 47; a light chain CDR1 of SEQ ID NO. 36; a light chain FR2 selected from the group consisting of SEQ ID NOs 43, 46, and 48; a light chain CDR2 of SEQ ID NO. 38; a light chain FR3 of SEQ ID NO. 39; a light chain CDR3 of SEQ ID NO. 40; and a light chain FR4 of SEQ ID NO.

44.

2. The humanized monoclonal antibody or its antigen-binding fragment, characterized in that, in claim 1, it comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs 2 to 7; and a light chain variable region sequence selected from the group consisting of SEQ ID NOs 9 to 11.

3. The humanized monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that the antibody comprises a human Fc domain.

4. A humanized monoclonal antibody or its antigen-binding fragment characterized in that, in paragraph 3, the isotype of the human Fc domain is IgG1, IgG2, IgG3, or IgG4.

5. In Paragraph 1, (1) The heavy chain variable region sequence of SEQ ID NO. 2 and the light chain variable region sequence of SEQ ID NO. 9; (2) The heavy chain variable region sequence of SEQ ID NO. 3 and the light chain variable region sequence of SEQ ID NO. 9; (3) heavy chain variable region sequence of SEQ ID NO. 4 and light chain variable region sequence of SEQ ID NO. 9; (4) heavy chain variable region sequence of SEQ ID NO. 5 and light chain variable region sequence of SEQ ID NO. 9; (5) The heavy chain variable region sequence of SEQ ID NO. 6 and the light chain variable region sequence of SEQ ID NO. 9; (6) The heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO. 9; (7) The heavy chain variable region sequence of SEQ ID NO. 2 and the light chain variable region sequence of SEQ ID NO. 10; (8) heavy chain variable region sequence of SEQ ID NO. 4 and light chain variable region sequence of SEQ ID NO. 10; (9) The heavy chain variable region sequence of SEQ ID NO. 5 and the light chain variable region sequence of SEQ ID NO. 10; (10) heavy chain variable region sequence of sequence number 6 and light chain variable region sequence of sequence number 10; (11) The heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO. 10; (12) The heavy chain variable region sequence of sequence number 2 and the light chain variable region sequence of sequence number 11; (13) The heavy chain variable region sequence of SEQ ID NO. 4 and the light chain variable region sequence of SEQ ID NO. 11; (14) The heavy chain variable region sequence of SEQ ID NO. 5 and the light chain variable region sequence of SEQ ID NO. 11; (15) The heavy chain variable region sequence of SEQ ID NO. 6 and the light chain variable region sequence of SEQ ID NO. 11; or (16) A humanized monoclonal antibody or an antigen-binding fragment thereof characterized by comprising the heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO.

11.

6. A humanized monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2 and Diabadi.

7. The humanized monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that the humanized monoclonal antibody or its antigen-binding fragment is conjugated to one or more conjugate moietys.

8. A polynucleotide encoding a humanized monoclonal antibody according to any one of claims 1 to 7 or an antigen-binding fragment thereof.

9. A vector comprising the polynucleotide of claim 8.

10. A host cell transfected with the vector of claim 9.

11. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a humanized monoclonal antibody according to any one of claims 1 to 7 or an antigen-binding fragment thereof.

12. A pharmaceutical composition according to claim 10, characterized in that the cancer is breast cancer, ovarian cancer, stomach cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, or ureteral cancer.

13. A cancer diagnostic composition comprising, as an active ingredient, a humanized monoclonal antibody according to any one of claims 1 to 7 or an antigen-binding fragment thereof.

14. An immunoconjugate in which a therapeutic agent or a label is conjugated to a humanized monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

15. An immunoconjugate according to claim 14, characterized in that the therapeutic agent is selected from the group consisting of nucleotides, peptides, proteins, cytokines, amino acids, lipids, carbohydrates, low-molecular-weight compounds, radioactive isotopes, and combinations thereof.

16. An immunoconjugate according to claim 14, characterized in that the label is selected from the group consisting of radioactive isotopes, fluorescent dyes, enzymes, and combinations thereof.

17. Use of a humanized monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for preparing a composition for treating cancer.

18. A method for treating cancer comprising administering an effective amount of a composition comprising, as an active ingredient, a humanized monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 to an individual in need thereof.

19. Use of a humanized monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for preparing a composition for cancer diagnosis. 20.i) A step of administering to a subject a humanized monoclonal antibody according to any one of claims 1 to 7 or an immunoconjugate in which a label is conjugated to an antigen-binding fragment thereof; ii) a step of the above immunoconjugate binding to a target site within the subject; iii) a step of imaging and detecting a signal generated from the above immunoconjugate; and iv) A cancer diagnosis method comprising the step of diagnosing whether cancer has occurred in the target site based on the detected signal.

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