CN117597362A - Antibodies against claudin-6 and uses thereof - Google Patents

Abstract

The present disclosure provides antibodies and antibody fragments thereof that bind to human CLDN 6. The antibodies of the present disclosure can be used in antibody-based immunotherapeutic approaches to direct a cytotoxic response against or to target CLDN6 expressing cancers for disruption by antibody drug conjugates.

Description

Antibodies against tight junction protein-6 and uses thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This PCT application claims priority to U.S. application serial number 63/240,399, filed on September 3, 2021, entitled “Antibodies against tight junction protein-6 and uses thereof” and U.S. application serial number 63/155,304, filed on March 2, 2021, entitled “Antibodies against tight junction protein-6 and uses thereof”, the entire contents of which are incorporated herein by reference.

Sequence Listing

This application contains a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on February 28, 2022 is named "122863-5006-WO_ST25_Sequence_Listing.TXT" and is 34,000 bytes in size.

Technical Field

The present disclosure belongs to the field of immunotherapy, and relates to antibodies and fragments thereof that bind to human tight junction protein-6 (Claudin-6) (CLDN6), polynucleotide sequences encoding these antibodies, and cells that produce them. The present disclosure also relates to therapeutic compositions comprising these antibodies, and methods of using them for cancer detection, prognosis, and antibody-based immunotherapy.

Background Art

The tight junction protein (CLDN) family consists of 27 members, showing different expression patterns in a cell and tissue type selective manner. Tight junction proteins are integral membrane proteins located in the tight junctions (tight junctions, TJs) of epithelial cells and endothelial cells. CLDNs interact with each other in the same cell (cis interaction) and adjacent cells (trans interaction), thereby forming TJs with tissue-specific barrier functions. Each cell type expresses more than one member of the tight junction protein family. In normal physiology, tight junction proteins interact with a variety of proteins and are closely involved in signal transduction from tight junctions and to tight junctions (Lal-Nag, M and Morin, P.J., Genome Biol 10: 235, 2009).

CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (ELI and EL2). The extracellular loops of tight junction proteins from adjacent cells interact with each other to seal the cell sheet and regulate paracellular transport between the luminal and basolateral spaces. The tight junction protein structure is highly conserved among different family members. CLDN6 contains 220 amino acids and is 23 kDa in size, showing a typical protein structure of tight junction proteins.

Unlike most widely expressed tight junction proteins, CLDN6 is characterized by selective expression (Hewitt et al., BMC Cancer, 6: 186, 2006). CLDN6 is an oncofetal tight junction molecule expressed in various types of embryonic epithelial cells. Disorders of tight junctions and dysregulation of tight junction molecules are common hallmarks of cancer cells and are often associated with malignant transformation. CLDN6 expression is abnormally activated in a variety of cancer types, including gastric cancer, lung cancer and ovarian adenocarcinoma, endometrial cancer and embryonic carcinoma, childhood brain tumors (e.g., atypical teratoma/rhabdoid tumor) and germ cell tumors (Hassimoto et al., J Pharmacol Exp Ther 368: 179-186, 2019; Kojima et al., Cancers 2020, 12, 2748). Therefore, CLDN6 has been identified as a tumor-associated antigen. As a tumor-associated antigen, CLDN6 can be classified as a differentiation antigen because it is expressed in the early stages of epidermal morphogenesis, where it is essential for epidermal differentiation and barrier formation. The unique expression pattern of CLDN6 in cancer but not in adult normal tissues, coupled with its accessibility to antibodies on the cell surface, makes CLDN6 a promising target for diagnostic and immunotherapeutic approaches in multiple cancer types.

There is a high degree of sequence conservation between CLDN6 and other tight junction proteins. The high homology between CLDN6 and other tight junction proteins (e.g., CLDN9, CLDN4, and CLDN3) makes it difficult to provide CLDN6 antibodies with properties suitable for therapeutic use (such as specificity, affinity, and safety).

Antibody-based immunotherapies targeting claudin-6 may modulate the activity of CLDN6 and/or direct a cytotoxic response against cancers expressing CLDN6. Therefore, there is a need for antibodies against CLDN6.

Summary of the invention

The present disclosure addresses the above needs by providing anti-CLDN6 antibodies and fragments that can bind to tight junction protein-6 present on cancer cells. These antibodies and fragments thereof are characterized by a unique set of CDR sequences, specificity for CLDN6, and are useful for cancer detection, prognosis, and immunotherapy. More specifically, the present disclosure relates to antibodies that bind to human CLDN6, and their use for detecting the presence of tumor cells and/or tumor stem cells expressing CLDN-6, modulating CLDN6-mediated activity of cells localized in the tumor microenvironment, or directing a cytotoxic response against cancers expressing CLDN6.

According to some embodiments, the antibody or antibody fragment comprises a set of six complementarity determining region (CDR) sequences selected from three CDRs of the heavy chain (HC) variable region (selected from SEQ ID NOs: 1, 3, 23, 24, 26, 28 and 30) and three light chain CDRs of the light chain (LC) variable region (selected from SEQ ID NOs: 2, 4, 25, 27, 29 and 31), or an analog or derivative thereof having at least 90% sequence identity with the identified antibody or fragment sequence.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 6, and CDR3: SEQ ID NO: 7; and/or a light chain variable region comprising CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 9, and CDR3: SEQ ID NO: 10.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, and CDR3: SEQ ID NO: 13; and/or a light chain variable region comprising CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, and CDR3: SEQ ID NO: 16.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34; and/or a light chain variable region comprising CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40; and/or a light chain variable region comprising CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 42, and CDR3: SEQ ID NO: 43.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 44, and CDR3: SEQ ID NO: 40; and/or a light chain variable region comprising CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 45, and CDR3: SEQ ID NO: 43.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 46, CDR2: SEQ ID NO: 47, and CDR3: SEQ ID NO: 48; and/or a light chain variable region comprising CDR1: SEQ ID NO: 49, CDR2: SEQ ID NO: 50, and CDR3: SEQ ID NO: 51.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from SEQ ID NO: 1, 3, 23, 24, 26, 28, and 30.

In other embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable light chain sequence selected from SEQ ID NO: 2, 4, 25, 27, 29 and 31.

In other embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 23, 24, 26, 28 and 30 and a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 25, 27, 29 and 31.

In some embodiments, the anti-CLDN6 antibody or antibody fragment comprises a variable heavy chain and a variable light chain sequence selected from the following combinations:

(a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2;

(b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

(c) a variable heavy chain sequence comprising SEQ ID NO: 23 and a variable light chain sequence comprising SEQ ID NO: 2;

(d) a variable heavy chain sequence comprising SEQ ID NO: 24 and a variable light chain sequence comprising SEQ ID NO: 25;

(e) a variable heavy chain sequence comprising SEQ ID NO: 26 and a variable light chain sequence comprising SEQ ID NO: 27;

(f) a variable heavy chain sequence comprising SEQ ID NO: 28 and a variable light chain sequence comprising SEQ ID NO: 29; and

(g) a variable heavy chain sequence comprising SEQ ID NO: 30 and a variable light chain sequence comprising SEQ ID NO: 31.

In some embodiments, an immunoconjugate is provided, comprising an antibody that binds to CLDN6 covalently linked to a cytotoxic agent, wherein the antibody comprises a variable heavy chain and a variable light chain sequence selected from the group consisting of:

(a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; and

(b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

(c) a variable heavy chain sequence comprising SEQ ID NO: 23 and a variable light chain sequence comprising SEQ ID NO: 2;

(d) a variable heavy chain sequence comprising SEQ ID NO: 24 and a variable light chain sequence comprising SEQ ID NO: 25;

(e) a variable heavy chain sequence comprising SEQ ID NO: 26 and a variable light chain sequence comprising SEQ ID NO: 27;

(f) a variable heavy chain sequence comprising SEQ ID NO: 28 and a variable light chain sequence comprising SEQ ID NO: 29; and

(g) a variable heavy chain sequence comprising SEQ ID NO: 30 and a variable light chain sequence comprising SEQ ID NO: 31.

In some embodiments, an immunoconjugate is provided, comprising an antibody that binds to Claudin-6 covalently linked to a cytotoxic agent, wherein the antibody comprises (a) a heavy chain variable region comprising CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 6, and CDR3: SEQ ID NO: 7; and/or a light chain variable region comprising CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 9, and CDR3: SEQ ID NO: 10; (b) a heavy chain variable region comprising CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, and CDR3: SEQ ID NO: 13; and/or a light chain variable region comprising CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, and CDR3: SEQ ID NO: 16; (c) a heavy chain variable region comprising CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34; and/or a light chain variable region comprising CDR1: SEQ ID NO: 35 NO:35, CDR2: SEQ ID NO:36 and CDR3: SEQ ID NO:37; (d) a heavy chain variable region comprising CDR1: SEQ ID NO:38, CDR2: SEQ ID NO:39 and CDR3: SEQ ID NO:40; and/or a light chain variable region comprising CDR1: SEQ ID NO:41, CDR2: SEQ ID NO:42 and CDR3: SEQ ID NO:43; (e) a heavy chain variable region comprising CDR1: SEQ ID NO:38, CDR2: SEQ ID NO:44 and CDR3: SEQ ID NO:40; and/or a light chain variable region comprising CDR1: SEQ ID NO:41, CDR2: SEQ ID NO:45 and CDR3: SEQ ID NO:43; and (f) a heavy chain variable region comprising CDR1: SEQ ID NO:46, CDR2: SEQ ID NO:47 and CDR3: SEQ ID NO:48. NO:48; and/or a light chain variable region comprising CDR1: SEQ ID NO:49, CDR2: SEQ ID NO:50 and CDR3: SEQ ID NO:51.

In some embodiments, the anti-CLDN6 antibodies and antibody fragments thereof comprise one or more heavy chain variable region CDRs disclosed in Table 1 and/or one or more light chain variable region CDRs disclosed in Table 2.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof exhibits one or more of the following structural and functional characteristics, alone or in combination: (a) binds to cells expressing human CLDN6 on their cell surface; (b) selectively binds to Claudin-6 CHO-K1 cells, with a signal (e.g., MFI) that is about 20 to 25 times greater than the binding activity of an isotype control antibody to CLDN6 expressed on Claudin-6-CHO-K1 cells, or 20 to 30 times greater than the binding activity of an isotype control antibody to CLDN6 expressed on Claudin-6-HEK293 cells, and selectively binds to Claudin-9 HEK293 cells, with a signal that is only 16 times greater than the binding activity of the isotype control antibody; (c) binds equally to cells expressing Claudin-6 (Claudin-6 CHO-K1 cells) and Claudin-9 (HEK293 cells), Its signal is about 25 to 60 times the binding activity of the isotype control antibody; (d) weakly binds to cells expressing CLDN3 and CLDN4 or does not bind at all; (e) binds to NEC8 cells that endogenously express Claudin-6, but does not bind to NEC8 cells in which the Claudin-6 gene has been knocked out; (f) optionally cross-reacts with mouse CLDN6; (g) after binding, it is efficiently internalized from the surface of cells positive for Claudin-6, inducing endocytosis-mediated cytotoxicity in NEC8 cells that endogenously express Claudin-6; and (h) exhibits one or more immune effector functions against cells carrying CLDN6 in its native conformation, wherein the one or more immune effector functions are selected from antibody-dependent cell-mediated cytotoxicity (ADCC), T cell-dependent cytotoxicity (TDCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP).

In some embodiments, the anti-CLDN6 antibody specifically binds to human cells expressing endogenous levels of claudin-6 and host cells engineered to overexpress claudin-6, without exhibiting binding to claudin-3 or claudin-4. In one embodiment, the anti-CLDN6 antibody binds to CLDN6 endogenously expressed on human testicular embryonic carcinoma (NEC8 cells). In some embodiments, the anti-CLDN6 antibody binds to CLDN6 endogenously expressed on human ovarian cancer (OV90 cells). In some embodiments, the CLDN6-specific antibody or antibody fragment binds to human claudin-6 with an affinity of less than 100 nM. In other embodiments, the CLDN6/9-specific antibody or antibody fragment selectively binds to human CLDN6 and human CLDN9.

In some embodiments, the antibody is capable of binding to CLDN6 associated with the surface of a cell expressing CLDN6. Preferably, the cell expressing CLDN6 is an intact cell, in particular a non-permeabilized cell, and the CLDN6 protein bound by the antibody is associated with the cell surface in a native (e.g., non-denatured) conformation. In a specific embodiment, the antibody is substantially unable to: bind to CLDN9 associated with the surface of a cell expressing CLDN9; or bind to CLDN4 associated with the surface of a cell expressing CLDN4 and/or bind to CLDN3 associated with the surface of a cell expressing CLDN3.

In one embodiment, the anti-CLDN6 antibody or antibody fragment selectively binds to CLDN6 relative to CLDN9, and does not bind to tight junction protein-3 (CLDN3), tight junction protein-4 (CLDN4). In another embodiment, the anti-CLDN6 antibody or antibody fragment binds to both CLDN6 and CLDN 9 equally (e.g., without preference for either of the two CLDNs). In some embodiments, the antibody exhibits an EC ₅₀ of less than about 50 nM (e.g., less than about 75 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM) in a FACS-based assay using any of the host cells engineered to overexpress CLDN6 (e.g., 293T-CLDN6 or CHO-CLDN6).

In some embodiments, the antibody or fragment thereof exhibits one or more immune effector functions against cells carrying CLDN6 in its native conformation, wherein the one or more immune effector functions are preferably selected from antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), induction of apoptosis and inhibition of proliferation, preferably, the effector function is ADCC and/or CDC.

In some embodiments, the antibody or fragment thereof can be linked to one or more therapeutic effector moieties (e.g., radiolabels, cytotoxins, therapeutic enzymes, agents that induce apoptosis, etc.) to provide targeted cytotoxicity, e.g., killing tumor cells. In some embodiments, the anti-CLDN6 antibody specifically binds to human CLDN6 present on the surface of tumor cells, effectively inducing internalization of CLDN6 or directing cell-mediated killing of tumor cells.

In some embodiments, the anti-CLDN6 antibody is incorporated into an immunoconjugate comprising an anti-CLDN6 antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes (i.e., radioconjugates).

In some embodiments, the anti-CLDN6 antibody or antibody fragment may comprise a substitution or modification of the constant region (i.e., Fc region), including but not limited to amino acid residue substitutions, mutations and/or modifications, which produces a compound having one or more enhanced ADCC, CDC, ADCP, TDCC antibody-mediated effector functions.

In some embodiments, the anti-CLDN6 antibody or antibody fragment comprises six (6) CDRs or variants thereof derived from the VH or VL domain of a single anti-CLDN6 antibody disclosed herein. For example, the binding agent may comprise all six CDR regions of an anti-CLDN6 antibody (referred to as "NR.N6.Abl"). In a representative example, the antibody or antibody fragment thereof may comprise the amino acid sequences of SEQ ID NOs: 5-7 and SEQ ID NOs: 8-10, which represent CDR1, CDR2 and CDR3 of the variable heavy chain region and CDR1, CDR2 and CDR3 of the variable light chain region of an anti-human CLDN6 antibody (referred to herein as "NR.N6.Abl").

Any anti-CLDN6 antibody disclosed herein may be a fully human, chimeric, CDR-grafted, humanized or recombinant antibody, or a fragment thereof. In an alternative embodiment, the disclosed anti-CLDN6 antibodies or antibody fragments may be developed for use in various alternative formats, including bispecific or multispecific formats.

In some embodiments, the CLDN6 antibody is a full-length antibody. In some embodiments, the antibody is an antibody fragment. In further embodiments, the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab') ₂ , Fd, Fv, scFv and scFv-Fc fragments, single-chain antibodies, minibodies, and diabodies.

The present disclosure also provides nucleic acids encoding any of the anti-CLDN6 antibodies disclosed herein. In related embodiments, the present disclosure provides vectors comprising one or more nucleic acids encoding the anti-CLDN6 antibodies disclosed herein, or host cells comprising the vectors.

CLDN6 antibodies and antibody fragments thereof can be used to treat cancer. Cancer cells expressing CLDN6 are suitable targets for CLDN6-targeted treatments (such as treatments using antibodies against CLDN6). Such methods for treating or cancer may include administering a composition or formulation comprising a CLDN6 antibody or an antibody fragment thereof to a subject in need thereof.

For example, a CLDN6 antibody or antibody fragment thereof can be administered alone (e.g., as a monotherapy), or in combination with other immunotherapeutic agents and/or chemotherapies. In one embodiment, a CLDN6 antibody or fragment is used to prepare an ADC suitable for mediating killing of cancer cells expressing CLDN6. In an alternative embodiment, a CLDN6 antibody is used to engineer a recombinant antibody designed to kill tumor cells by enhanced ADCC, ADCP, TDCC or CDD.

The present disclosure also provides a method for treating cancer, comprising administering a pharmaceutical composition comprising an ADC to a subject in need thereof, wherein the ADC comprises an anti-CLDN6 antibody or antibody fragment disclosed herein coupled to a payload. In some embodiments, the method comprises administering a pharmaceutical composition comprising an anti-CLDN ADC to a subject in need thereof, wherein the cancer is selected from uterine cancer, testicular cancer, ovarian cancer, and lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

When read in conjunction with the accompanying drawings, the foregoing summary of the invention and the specific embodiments of the present disclosure below will be better understood. For the purpose of illustrating the present disclosure, shown in the drawings are currently preferred embodiments. However, it should be understood that the present disclosure is not limited to the precise arrangements, embodiments and means shown.

Figure 1 provides the amino acid sequences of the VH and VL domains of human anti-Claudin-6 antibodies and their respective CDR sequences (Kabat numbering). Sequence identifiers are provided, and the CDRs are underlined in the variable domain sequences.

Figures 2A and 2B show the binding of anti-CLDN6 antibodies and isotype control antibodies to Claudin-6-CHO-K1 transfected cells and untransfected parental CHO-K1 cells by FACS.

Figures 3A and 3B show the binding of anti-CLDN6 antibodies and isotype control antibodies to Claudin-9-HEK293 transfected cells by FACS.

FIG. 4A and FIG. 4B show the binding of anti-claudin antibodies to Claudin-3-CHO-K1 transfected cells by FACS.

FIG. 5A and FIG. 5B show the binding of anti-claudin antibodies to Claudin-4-CHO-K1 transfected cells by FACS.

FIG. 6A and FIG. 6B show the binding of anti-CLDN6 antibodies to NEC8 tumor cells endogenously expressing human CLDN6 by FACS.

Figures 7A and 7B show the binding of anti-CDN6 antibodies to OV90 tumor cells endogenously expressing human CLDN6 by FACS.

FIG. 8A and FIG. 8B show the binding of anti-CLDN antibodies to MCF7 cells endogenously expressing Claudin-3 (CLDN3) and Claudin-4 (CLDN4) by FACS.

Figure 9 shows the binding of rabbit polyclonal anti-CLDN9 antibodies to human tumor cell lines NEC8, OV90 and MCF7 and Claudin-9-HEK293 transfected cells by FACS.

Figures 10A, 10B and 10C show dose response curves of the anti-claudin-6 antibodies NR.N6.Ab1 and NR.N6.Ab2 of the present disclosure binding to cell lines overexpressing claudin-6 by FACS. HEK293 cells overexpressing claudin-6 (Figure 10A); HEK293 cells overexpressing claudin-9 (Figure 10B); and CHO cells overexpressing claudin-6 (Figure 10C).

Figures 11A and 11B show the antibody-dependent cellular cytotoxicity (ADCC) mediated by NR.N6.Abl and NR.N6.Ab2 against NEC8 cells (Figure 11A) and OV90 cells (Figure 11B) that endogenously express human CLDN6.

Figures 12A, 12B and 12C show the antibody-mediated endocytosis induced by anti-claudin-6 antibodies NR.N6.Ab1 and NR.N6.Ab2 in NEC8 tumor cells (Figure 12A); OV90 tumor cells endogenously expressing human CLDN6 (Figure 12B) and HEK293 cells overexpressing claudin-6 (Figure 12C).

Figures 13A, 13B, 13C and 13D show the binding of anti-CLDN6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5, NR.N6.Ab6 and isotype control antibodies to HEK293 cells overexpressing Claudin-6 and HEK293 cells overexpressing Claudin-9 by FACS.

Figures 14A, 14B, 14C, 14D and 14E show the binding of anti-CLDN6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.4.N6.Ab5, NR.N6.Ab6, NR.NR6.Ab1 and isotype control antibodies to NEC8 cells endogenously expressing Claudin-6 and NEC8 cells in which the Claudin-6 gene was knocked out (NEC8 Claudin-6KO) by FACS.

Figures 15A, 15B, 15C, 15D, 15E and 15F show the binding of anti-CLDN6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 and two positive controls MAB4620 and MAB4219 to CHO-K1 cells overexpressing Claudin-6, CHO-K1 cells overexpressing Claudin-3 and CHO-K1 cells overexpressing Claudin-4 by FACS.

Figures 16A, 16B, 16C and 16D show dose response curves of anti-CLDN6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 of the present disclosure to HEK293 cells overexpressing Claudin-6 and HEK293 cells overexpressing Claudin-9 by FACS.

Figures 17A, 17B and 17C show dose-response curves of the anti-CLDN6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 of the present disclosure with NEC8 cells endogenously expressing Claudin-6 and NEC8 cells in which the Claudin-6 gene was knocked out (NEC8 Claudin-6 KO) by FACS.

Figures 18A, 18B, 18C and 18D show dose response curves of the anti-CLDN6 antibodies NR.N6.Ab1, variants of NR.N6.Ab1N73D and isotype controls of the present disclosure with HEK293 cells overexpressing Claudin-6, HEK293 cells overexpressing Claudin-9, NEC8 cells endogenously expressing Claudin-6 and NEC8 Claudin-6 KO cells by FACS.

Figures 19A and 19B show the antibody-dependent cellular cytotoxicity (ADCC) mediated by NR.N6.Ab1, NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 against NEC8 cells endogenously expressing human CLDN6 (Figure 19A) and NEC8 cells with claudin-6 knocked out (Figure 19B).

Figures 20A and 20B show the internalization activity of NR.N6.PC1, NR.N6.Ab1, NR.N6.Ab3, NR.N6.Ab4, and NR.N6.Ab5 on NEC8 and NEC8 cells in which Claudin-6 was knocked out.

Figures 21A and 21B show the antibody-mediated endocytosis induced by anti-claudin-6 antibodies NR.N6.Ab1, NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 in NEC8 tumor cells endogenously expressing human CLDN6 (Figure 21A) and NEC8 cells with claudin-6 knocked out (Figure 21B).

DETAILED DESCRIPTION

In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present disclosure belongs.

Throughout this disclosure, the following abbreviations will be used:

mAb or Mab or MAb—monoclonal antibody.

CDR—complementarity determining region in an immunoglobulin variable region.

VH or VH—immunoglobulin heavy chain variable region.

VL or VL—immunoglobulin light chain variable region.

FR—antibody framework region, the immunoglobulin variable region excluding the CDR region.

The term "claudin-6" or "CLDN6" (used interchangeably herein) preferably relates to human CLDN6, in particular to a protein comprising the amino acid sequence of SEQ ID NO: 1 according to the sequence listing, or a variant of said amino acid sequence. The term "CLDN6" includes any CLDN6 variant, such as post-translationally modified variants and conformational variants. The amino acid sequences of human, cynomolgus monkey and murine CLDN6 are provided in the NCBI reference sequences: NP_067018.2 (human) (SEQ ID NO: 17), XP_005591080.1 (cynomolgus monkey) (SEQ ID NO: 18) and NP_061247.1 (mouse) (SEQ ID NO: 19). The orthologs of CLDN6 in cynomolgus monkey and mouse have >99% and ~88% identity with the human protein, respectively.

The term "CLDN9" relates to human CLDN9, in particular to a protein comprising the amino acid sequence according to SEQ ID NO: 20 (NCBI reference sequence NP_066192.1), or a variant of said amino acid sequence. Human CLDN6 and human CLDN9 proteins have 71.8% identity.

The term "CLDN4" relates to human CLDN4, in particular to a protein comprising the amino acid sequence according to SEQ ID NO: 21 (NCBI reference sequence NP_001296.1), or a variant of said amino acid sequence. Human CLDN6 and human CLDN4 proteins have 59.1% identity.

The term "CLDN3" relates to human CLDN3, in particular to a protein comprising the amino acid sequence according to SEQ ID NO: 22 (NCBI reference sequence NP_001297.1), or a variant of said amino acid sequence. Human CLDN6 and human CLDN3 proteins have an identity of 56.7%.

The term "percent identity" is intended to indicate the percentage of identical amino acid residues between the two sequences obtained after optimal alignment of the two sequences to be compared, which percentage is purely statistical, the differences between the two sequences being randomly distributed and distributed throughout their length. Sequence comparisons between two amino acid sequences are usually performed by comparing these sequences after optimal alignment, the comparison being performed by segments or by a "comparison window" in order to identify and compare local regions of sequence similarity. In addition to manual alignment, optimal alignment for comparing sequences can also be produced by the local homology algorithm of Smith and Waterman (1981, Ads App. Math. 2, 482), the local homology algorithm of Neddiernan and Wunsch (1970, J. Mol. Biol. 48, 443), the similarity search method of Pearson and Lipman (1988, Proc. Natl Acad. Sci. USA 85, 2444), or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI).

As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies).

As used herein, the term "cross-reactivity" refers to the ability of the anti-human CLDN6 antibodies described herein to bind to CLDN6 from different species. For example, the antibodies described herein may also bind to CLDN6 from another species (eg, or rat or mouse CLDN6).

Exemplary antibodies such as IgG comprise two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The hypervariable region generally comprises the following amino acid residues: about amino acid residues 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and about 31-35B (HCDR1; "H" for heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health, 1994. of Health, Bethesda, Md. (1991) and/or these residues form a hypervariable loop (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, for example, each antibody constituting the population is identical and/or binds to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or produced during the production process of monoclonal antibody preparations (these variants are usually present in small amounts). Contrary to polyclonal antibody preparations that typically contain different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any method. For example, the monoclonal antibody used according to the present disclosure can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and other exemplary methods of these methods and preparation of monoclonal antibodies are described herein.

The term "chimeric" antibody refers to a recombinant antibody in which a portion of the heavy chain and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (as long as they exhibit the desired biological activity). In addition, complementary determining regions (CDRs) can be grafted to change certain properties of the antibody molecule, including affinity or specificity. Typically, variable domains are obtained from antibodies ("parent antibodies") of experimental animals (such as rodents), while constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibodies can direct effector functions in human subjects and are less likely to cause adverse immune responses than the parent (e.g., mouse) antibodies from which the chimeric antibodies are derived.

The term "humanized antibody" refers to an antibody that is engineered to include one or more human framework regions and non-human (e.g., mouse, rat or hamster) complementary determining regions (CDRs) in the variable region of the heavy chain and/or light chain. In certain embodiments, except for the CDR regions, the humanized antibody comprises a completely human sequence. Compared to non-humanized antibodies, humanized antibodies are generally less immunogenic to humans and therefore may provide therapeutic benefits in certain cases. Those skilled in the art will understand humanized antibodies and will also understand suitable techniques for their production. See, e.g., Hwang, W.Y.K., et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833-37; and U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; and Selick et al., WO 90/07861, the entire contents of each of which are incorporated herein by reference.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and/or an antibody that has been prepared using any technique known to those skilled in the art for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising residues that bind to non-human antigens. Human antibodies can be produced using various techniques known in the art, including methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147 (I): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic challenge, but in which the endogenous loci have been disabled, e.g., immunized HuMab mice (see, e.g., Nils Lonber et al., 1994, Nature 368:856-859, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918, and WO 01/09187 for HuMab mice), xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE ^™ technology), or Trianni mice (see, e.g., WO 2013/063391, WO 2017/035252, and WO 2017/136734).

The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "antigen binding domain" of an antibody (or simply "antibody binding domain") or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen complex. Examples of binding fragments included in the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity determining regions (CDRs) and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker.

The "variable domain" (V domain) of an antibody mediates binding and imparts antigenic specificity to a particular antibody. However, variability is not evenly distributed in the 110 amino acid spans of the variable domain. In fact, the V region is composed of a relatively constant extension of 15-30 amino acids called a framework region (FR) and an extremely variable shorter region of 9-12 amino acids each length of which is referred to herein as a "hypervariable region" or CDR that separates the framework region. As will be appreciated by those skilled in the art, the accurate numbering of CDRs and placement in different numbering systems may be different. However, it should be understood that the disclosure of variable heavy chain sequences and/or variable light chain sequences includes the disclosure of related CDRs. Therefore, the disclosure of each variable heavy chain region is the disclosure of vhCDR (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is the disclosure of vlCDR (e.g., vlCDR1, vlCDR2, and vlCDR3).

As used herein, the term "complementarity determining region" or "CDR" refers to a short polypeptide sequence within the variable region of the heavy and light chain polypeptides that is primarily responsible for mediating specific antigen recognition. There are three CDRs (referred to as CDR1, CDR2, and CDR3) in each VL and each VH. Unless otherwise indicated herein, CDRs and framework regions are annotated according to the Kabat numbering scheme (Kabat E.A., et al., 1991, Sequences of proteins of Immunological interest, NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, Md.).

In other embodiments, the CDRs of an antibody may be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745, which is incorporated herein by reference in its entirety, or according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7:132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27:209-212, the entire contents of each of which are incorporated herein by reference. See, e.g., Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), the entire contents of which are incorporated herein by reference. In other embodiments, the CDRs of an antibody may be identified according to the AbM numbering scheme, which refers to the AbM hypervariable regions, represents a compromise between the Kabat CDRs and the Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), the entire contents of which are incorporated herein by reference.

"Framework" or "framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4.

"Human common framework" is a framework that represents the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences comes from a subgroup of variable domain sequences. Typically, the sequence subgroup is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup Ill as described above by Kabat et al.

The "hinge region" is generally defined as stretching from 216-238 (EU numbering) or 226-251 (Kabat numbering) of human IgGl. The hinge can be further divided into three distinct regions, the upper hinge, the middle hinge (eg, core), and the lower hinge.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab) ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment (the name reflects the ability to crystallize readily). The Fab fragment consists of an intact light (L) chain as well as the variable region domain (VH) of the heavy (H) chain and the first constant domain (CH1) of one heavy chain. Pepsin treatment of an antibody produces a single large F(ab) ₂ fragment that roughly corresponds to two disulfide-linked Fab fragments that have divalent antigen-binding activity and are still able to cross-link antigen. Fab fragments differ from Fab' fragments in that there are several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH refers herein to Fab' in which the cysteine residues of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments originally were produced as pairs of Fab' fragments having hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Fv" consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight non-covalent association. Six hypervariable loops (3 loops each in the H chain and L chain) are generated from the folding of these two domains, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide also comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the structure required for antigen binding. For a review of sFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "antigen binding domain" of an antibody (or simply "antibody binding domain") or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen complex. Examples of binding fragments included in the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity determining regions (CDRs) and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker.

The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH-VL unit has polyepitopic specificity (i.e., capable of binding to two different epitopes on one biomolecule, or each epitope on different biomolecules). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, bispecific diabodies, and triabodies. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets.

"Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different targets. However, in contrast to bispecific antibodies, bispecific antibodies have two antigen-binding arms with the same amino acid sequence, and each Fab arm is capable of recognizing two antigens. Dual specificity allows antibodies to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, a multispecific antibody in the form of IgG1 binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Single specificity" refers to the ability to bind only one epitope. Multispecific antibodies can have a structure similar to that of a complete immunoglobulin molecule and include an Fc region, such as an IgG Fc region. Such structures include, but are not limited to, IgG-Fv, IgG-(scFv) ₂ , DVD-Ig, (scFv) ₂ -(scFv) ₂ -Fc, and (scFv) ₂ -Fc-(scFv) _2. In the case of IgG-(scFv) ₂ , the scFv can be linked to the N-terminus or C-terminus of the heavy or light chain.

As used herein, the term "bispecific antibody" refers to a monoclonal antibody, typically a human or humanized antibody, that has binding specificities for at least two different antigens. In the present disclosure, one of the binding specificities may be for CLDN6 and the other may be for any other antigen, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacteria-derived protein or a bacterial surface protein, etc.

As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises a VH and a VL domain connected by a linker that is too short (e.g., a linker consisting of five amino acids) to allow intramolecular binding of the VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain, thereby forming a homodimeric structure. Thus, the term "tribody" refers to a trivalent antibody comprising three peptide chains, each peptide chain comprising a VH domain and a VL domain connected by a linker that is too short (e.g., a linker consisting of 1-2 amino acids) to allow intramolecular binding of the VH and VL domains within the same peptide chain.

When used to describe various antibodies disclosed herein, the term "isolated antibody" refers to an antibody that has been identified and separated and/or recovered from a cell or cell culture expressing it. An isolated antibody or antibody fragment may include a variant of an antibody or antibody fragment having one or more post-translational modifications (e.g., C-terminal lysine shearing) that occur during production, purification, and/or storage of the antibody or antibody fragment. Contaminant components in its natural environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other protein solutes or non-protein solutes. In some embodiments, the isolated antibody is purified to a purity greater than 95% or 99%, measured by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC) methods. For a review of antibody purity assessment methods, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007). In preferred embodiments, the antibody is purified: (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.

With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target refers to binding that is measurably distinct from non-specific interactions. For example, specific binding can be measured by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target can be expressed, for example, as a molecule having a Kd for the target of 10-4 M or less, or 10-5 M or less, or 10-6 M or less, or 10-7 M or less, or 10-8 M or less, or 10-9 M or less, or 10-10 M or less, or 10-11 M or less or 10-12 M or less, or a Kd in the range of 10-4 M to 10-6 M, or 10-6 M to 10-10 M, or 10-7 M to 10-9 M. Those skilled in the art will appreciate that affinity and KD values are inversely related. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specifically binds" refers to wherein the molecule binds to CLDN6 or an epitope of CLDN6 without substantially binding to any other polypeptide or polypeptide epitope.

As used herein, the term "binding to CLDN6" refers to the ability of an antibody or antigen-binding fragment to recognize and bind to endogenous human CLDN6 when endogenous human CLDN6 is present on the surface of normal or malignant cells, or on the surface of recombinant host cells engineered to overexpress human CLDN6.

The term "affinity" as used herein refers to the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined as 1/Kd. Methods for determining mAb affinity can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., ed., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993) and Muller, Meth. Enzymol. 92: 589-601 (1983), all of which are incorporated herein by reference. One standard method well known in the art for determining mAb affinity is to use surface plasmon resonance (SPR) screening (eg, by analysis using a BIAcore ^™ SPR analysis instrument).

"Epitope" is a term of art, which refers to one or more sites of interaction between an antibody and its antigen. Description is found in (Janeway, C, Jr., P.Travers et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8, New York, Garland Publishing, Inc.): "Antibodies can usually only recognize a small portion of the surface of a macromolecule (such as a protein) ... [certain epitopes] may be composed of amino acids from different parts of the [antigen] polypeptide chain that are gathered together by protein folding. This type of antigenic determinant is referred to as a conformational epitope or a discontinuous epitope, because the structure identified is composed of segments of proteins that are discontinuous in the antigen amino acid sequence but gathered together in a three-dimensional structure. On the contrary, the epitope composed of a single segment of a polypeptide chain is referred to as a continuous epitope or a linear epitope" (Janeway, C.Jr., P.Travers et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8. New York, Garland Publishing, Inc.).

As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka), expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. Preferred methods for determining the KD of an antibody include biolayer interferometry (BLI) analysis (preferably using a Fortebio Octet RED device), surface plasmon resonance (preferably using a biosensor system (e.g., surface plasmon resonance system)), or flow cytometry and Scatchard analysis.

" _EC50 " with respect to an agent and a specific activity (e.g., binding to a cell, inhibiting enzyme activity, activating or inhibiting an immune cell) refers to the effective concentration of an agent that produces 50% of its maximal response or effect with respect to that activity. " _EC100 " with respect to an agent and a specific activity refers to the effective concentration of an agent that produces its substantially maximal response with respect to that activity.

As used herein, the term "antibody-drug conjugate" (ADC) refers to an immunoconjugate consisting of a recombinant monoclonal antibody covalently linked to a cytotoxic agent (called a payload) via a synthetic linker. Immunoconjugates (antibody-drug conjugates, ADCs) are a class of highly effective antibody-based cancer therapeutics. ADCs consist of a recombinant monoclonal antibody covalently linked to a cytotoxic agent (called a payload) via a synthetic linker. ADCs combine the specificity of monoclonal antibodies with the potency of small molecule chemotherapeutic drugs, facilitating the targeted delivery of highly cytotoxic small molecule drug moieties directly to tumor cells.

As used herein, the term "endocytosis" refers to the process by which eukaryotic cells internalize plasma membrane fragments, cell surface receptors, and components from the extracellular fluid. Endocytosis mechanisms include receptor-mediated endocytosis. The term "receptor-mediated endocytosis" refers to a biological mechanism by which a ligand, upon binding to its target, triggers membrane invagination and contraction, internalization and transport into the cytosol, or translocation into an appropriate intracellular compartment.

The term "bystander effect" refers to target cell-mediated killing of healthy cells adjacent to tumor cells targeted by an antibody drug conjugate. The bystander effect is usually caused by the ability of hydrophobic cytotoxic drugs to diffuse out of antigen-positive target cells and into adjacent antigen-negative healthy cells. The presence or absence of the bystander effect can be attributed to aspects of the linker and coupling chemistry used to generate the immunoconjugate.

The term "effector function" is derived from the interaction of the antibody Fc region with certain Fc receptors, including but not necessarily limited to C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), T cell-dependent cytotoxicity (TCDD) and down-regulation of cell surface receptors. Such effector functions generally require the Fc region to be combined with an antigen binding domain (e.g., an antibody variable domain).

As used herein, the terms "antibody-based immunotherapy" and "immunotherapy" are used to refer generally to any form of therapy that relies on the targeting specificity of an anti-CLDN6 antibody, a bispecific molecule comprising an anti-CLDN6 antibody or its antibody fragment or CDR, an antigen binding domain or fusion protein to mediate a direct or indirect effect on cells expressing CLDN6. The term refers to treatment methods including the use of naked antibodies, bispecific antibodies (including T cell engagement, NK cell engagement and other immune cell/effector cell engagement forms), antibody drug conjugates, cell therapy using T cells (CAR-T) or NK cells (CAR-NK) engineered to contain anti-CLDN6 chimeric antigen receptors and oncolytic viruses containing CLDN6-specific binding agents, and gene therapy by delivering the antigen binding sequence of an anti-CLDN6 antibody and expressing the corresponding antibody fragment in vivo.

Tight junction protein family

Claudins are integral membrane proteins that comprise the major structural proteins of tight junctions, which are the apical-most cell-cell adhesion junctions in polarized cell types such as those found in epithelial or endothelial cell sheets.

The tight junction protein family of human proteins consists of at least 24 members ranging in size from 22-34 kDa. All tight junction proteins have a four-span membrane topology, in which both protein ends are located in the intracellular face of the membrane, forming two extracellular (EC) loops, EC1 and EC2. Typically, EC1 is about 50-60 amino acids in size, and EC2 is smaller than EC1, typically containing about 25 amino acids. The EC loop mediates head-to-head homophilicity, and for certain combinations of tight junction proteins, the EC loop mediates heterophilic interactions, resulting in the formation of tight junctions.

Claudin-6

Tight junction protein-6 (CLDN6) is usually expressed in humans as a 220 amino acid precursor protein; the first 21 amino acids constitute a signal peptide. The amino acid sequence of the CLDN6 precursor protein is publicly available on the National Center for Biotechnology Information (NCBI) website as NCBI reference sequence NP_067018.2, and is provided herein as SEQ ID NO: 17.

CLDN6 is highly expressed in germ cell tumors, including seminoma, embryonal carcinoma and yolk sac tumor, as well as certain gastric adenocarcinomas, lung adenocarcinomas, ovarian adenocarcinomas and endometrial carcinomas (Ushiku T, et al., Histopathology 61(6):1043–1056, 2012, Hewitt KJ, Agarwal R, Morin PJ. The claudin gene family: expression innormal and neoplastic tissues. BMC Cancer 2006; 6; 186; Micke, P. et al. (2014) Aberrantly activated Claudin-6 and 18.2 as potential therapy targets in non-small-cell lung cancer. Int. J. Cancer 135, 2206–2214; Lal-Nag, M. et al. (2012) Claudin-6: a novel receptor for CPE-mediated cytotoxicity in ovarian cancer. Oncogenesis 1, e33; Ben-David, U. et al. (2013) Immunologic and chemical targeting of the tight junction protein Claudin-6eliminates tumorigenic human pluripotent stem cells. Nat. Commun. 4, 1992).

The human CLDN6 protein is very closely related to the human CLDN9 protein sequence in the extracellular domain (ECD), with >98% identity in ECD1 and >91% identity in ECD2. Human CLDN4 is also closely related to human CLDN6 in the ECD sequence, with >84% identity in ECD1 and >78% identity in ECD2. The discovery of monoclonal antibodies (MAbs) against CLDN6 is plagued by high homology to endogenously expressed tight junction protein-9 (CLDN9), which differs from CLDN6 in the extracellular domain by only 3 amino acids (2 in ECD1 and 1 in ECD2). The deduced cynomolgus monkey protein ECD sequences of CLDN4, CLDN6, and CLDN9 proteins are 100% identical to the respective human ECD sequences. Therefore, the disclosed anti-human CLDN6 antibodies and fragments are expected to cross-react with cynomolgus monkey CLDN6 (data not shown). In addition, the claudin-6 gene is highly conserved among different species; for example, the human and mouse genes show 88% homology at the DNA and protein levels.

Targeting CLDN6 for cancer therapy

In the past few years, it has become increasingly clear that tight junctions play a role in the proliferation, transformation, and metastasis of cancer cells. Dysregulation of tight junction proteins can lead to the destruction of tight junctions in epithelial cells, which in turn leads to loss of cell polarity and impaired epithelial integrity. Overexpression of CLDN6 in tumor cells may be related to dysregulation of tight junction protein localization due to tumor cell dedifferentiation, or to the need for rapidly growing cancer tissue to effectively absorb nutrients within abnormally vascularized tumor masses (Morin PJ., Cancer Res. 1; 65 (21): 9603-6, 2005). Reduced cell-cell adhesion and increased cancer cell migration are considered to be the main events of epithelial cell to mesenchymal transition (EMT), which is an important step in cancer progression and metastasis.

Anti-CLDN6 Antibodies

The disclosed anti-CLDN6 antibodies (NR.N6.Ab1, NR.N6.Ab2, NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6) selectively bind to human CLDN6 or human CLDN6/9. These antibodies and fragments thereof are characterized by a unique set of CDR sequences for CLDN6 and can be used as monotherapy or in combination with other anticancer agents for cancer immunotherapy.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof exhibits one or more of the following structural and functional characteristics, alone or in combination: (a) binds to cells expressing human CLDN6 on their cell surface; (b) selectively binds to Claudin-6 CHO-K1 cells, with a signal (e.g., MFI) that is about 20 to 25 times the binding activity of an isotype control, or 20 to 30 times the binding activity of an isotype control antibody to CLDN6 expressed on Claudin-6-HEK293 cells; (c) binds equally to cells expressing Claudin-6 (Claudin-6 CHO-K1 cells) and Claudin-9 (HEK293 cells), with a signal that is about 25 to 60 times the binding activity of an isotype control antibody; (d) binds to cells expressing CLDN3, CLD3, or CHO-K1 cells; N4 cells weakly bind or not bind at all; (e) bind to NEC8 cells endogenously expressing claudin-6, but not to NEC8 cells in which the claudin-6 gene has been knocked out; (f) optionally cross-react with mouse CLDN6; (g) after binding, it is efficiently internalized from the surface of cells positive for claudin-6, inducing endocytosis-mediated cytotoxicity in NEC8 cells endogenously expressing claudin-6; and (h) exhibits one or more immune effector functions against cells carrying CLDN6 in its native conformation, wherein the one or more immune effector functions are selected from antibody-dependent cell-mediated cytotoxicity (ADCC), T cell-dependent cytotoxicity (TDCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP).

In one embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1. For example, the anti-CLDN6 antibody or antibody fragment thereof may comprise a set of CDRs corresponding to those in one of the anti-CLDN6 antibodies disclosed in Table 1 (e.g., the CDRs of NR.N6.Ab1).

In another embodiment, the anti-CLDN6 antibody comprises a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2. For example, the anti-CLDN6 antibody or antibody fragment thereof may comprise a set of CDRs corresponding to those in one of the anti-CLDN6 antibodies disclosed in Table 2 (e.g., the CDRs of NR.N6.Ab2).

In an alternative embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a VH having a set of CDRs disclosed in Table 1 (HCDR1, HCDR2 and HCDR3) and a VL having a set of CDRs disclosed in Table 2 (LCDR1, LCDR2 and LCDR3).

In one embodiment, the antibody can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody that specifically binds to human CLDN6, or an antigen-binding portion thereof. In one embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises all six CDR regions of the NR.N6.Ab1 or NR.N6.Ab2 antibody that forms the chimeric or humanized antibody.

Table 1: CDR sequences of variable heavy chains of anti-CLDN6 antibodies

Anti-CLDN6 Ab HCDR1 HCDR2 HCDR3 NR.N6.Ab1 SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:7 NR.N6.Ab2 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 NR.N6.Ab3 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:34 NR.N6.Ab4 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40 NR.N6.Ab5 SEQ ID NO:38 SEQ ID NO:44 SEQ ID NO:40 NR.N6.Ab6 SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:48

Table 2: CDR sequences of anti-CLDN6 variable light chains

Anti-CLDN6 Ab LCDR1 LCDR2 LCDR3 NR.N6.Ab1 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:10 NR.N6.Ab2 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 NR.N6.Ab3 SEQ ID NO:35 SEQ ID NO:36 SEQ ID NO:37 NR.N6.Ab4 SEQ ID NO:41 SEQ ID NO:42 SEQ ID NO:43 NR.N6.Ab5 SEQ ID NO:41 SEQ ID NO:45 SEQ ID NO:43 NR.N6.Ab6 SEQ ID NO:49 SEQ ID NO:50 SEQ ID NO:51

In one embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a VH having a set of complementarity determining regions (CDR1, CDR2 and CDR3) selected from:

(i) CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 6, CDR3: SEQ ID NO: 7;

(ii) CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, CDR3: SEQ ID NO: 13;

(iii) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, CDR3: SEQ ID NO: 34;

(iv) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, CDR3: SEQ ID NO: 40;

(v) CDR1:SEQ ID NO:38, CDR2:SEQ ID NO:44, CDR3:SEQ ID NO:40; and

(vi) CDR1: SEQ ID NO: 46, CDR2: SEQ ID NO: 47, CDR3: SEQ ID NO: 48.

In one embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a VL having a set of complementarity determining regions (CDR1, CDR2 and CDR3) selected from:

(i) CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 9, CDR3: SEQ ID NO: 10;

(ii) CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, CDR3: SEQ ID NO: 16

(iii) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, CDR3: SEQ ID NO: 37;

(iv) CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 42, CDR3: SEQ ID NO: 43;

(v) CDR1:SEQ ID NO:41, CDR2:SEQ ID NO:45, CDR3:SEQ ID NO:43; and

(vi) CDR1: SEQ ID NO: 49, CDR2: SEQ ID NO: 50, CDR3: SEQ ID NO: 51.

In another embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises:

(a) a VH having a set of complementarity determining regions (CDR1, CDR2 and CDR3) selected from the group consisting of:

(i) CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 6, CDR3: SEQ ID NO: 7; and

(ii) CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, CDR3: SEQ ID NO: 13;

(iii) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, CDR3: SEQ ID NO: 34;

(iv) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, CDR3: SEQ ID NO: 40;

(v) CDR1:SEQ ID NO:38, CDR2:SEQ ID NO:44, CDR3:SEQ ID NO:40; and

(vi) CDR1:SEQ ID NO:46, CDR2:SEQ ID NO:47, CDR3:SEQ ID NO:48; and

(b) a VL having a set of complementarity determining regions (CDR1, CDR2 and CDR3) selected from:

(i) CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 9, CDR3: SEQ ID NO: 10;

(ii) CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, CDR3: SEQ ID NO: 16;

(iii) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, CDR3: SEQ ID NO: 37;

(iv) CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 42, CDR3: SEQ ID NO: 43;

(v) CDR1:SEQ ID NO:41, CDR2:SEQ ID NO:45, CDR3:SEQ ID NO:43; and

(vi) CDR1: SEQ ID NO: 49, CDR2: SEQ ID NO: 50, CDR3: SEQ ID NO: 51.

In one embodiment, the antibody comprises a combination of a VH and a VL having a set of complementary determining regions (CDR1, CDR2 and CDR3) selected from:

(i) VH: CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 6, CDR3: SEQ ID NO: 7, VL: CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 9, CDR3: SEQ ID NO:10，

ii) VH: CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, CDR3: SEQ ID NO: 13, VL: CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, CDR3: SEQ ID NO:16、

iii) VH: CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, CDR3: SEQ ID NO: 34, VL: CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, CDR3: SEQ ID NO:37，

iv) VH: CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, CDR3: SEQ ID NO: 40, VL: CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 42, CDR3: SEQ ID NO:43，

v) VH: CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 44, CDR3: SEQ ID NO: 40, VL: CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 45, CDR3: SEQ ID NO:43, and

vi) VH: CDR1: SEQ ID NO: 46, CDR2: SEQ ID NO: 47, CDR3: SEQ ID NO: 48, VL: CDR1: SEQ ID NO: 49, CDR2: SEQ ID NO: 50, CDR3: SEQ ID NO:51.

In one embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 23, 24, 26, 28 and 30, and/or a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 25, 27, 29 and 31.

In one embodiment, the anti-CLDN6 antibody or its antibody fragment comprises a pair of variable heavy chain and variable light chain sequences selected from the following combinations: a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4; a variable heavy chain sequence comprising SEQ ID NO: 23 and a variable light chain sequence comprising SEQ ID NO: 2; a variable heavy chain sequence comprising SEQ ID NO: 24 and a variable light chain sequence comprising SEQ ID NO: 25; a variable heavy chain sequence comprising SEQ ID NO: 26 and a variable light chain sequence comprising SEQ ID NO: 27; a variable heavy chain sequence comprising SEQ ID NO: 28 and a variable light chain sequence comprising SEQ ID NO: 29; and a variable heavy chain sequence comprising SEQ ID NO: 30 and a variable light chain sequence comprising SEQ ID NO: 31. The skilled artisan will further appreciate that variable light chains and variable heavy chains can be independently selected or mixed and matched to prepare anti-CLDN6 antibodies comprising combinations of variable heavy chains and variable light chains other than the pairs identified above.

In an alternative embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a pair of variable heavy chain and variable light chain sequences selected from the following combinations: a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 1 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 2; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 3 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 4; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 23 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 2; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 24 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 25; NO: 26 has a variable heavy chain sequence with 90%, 95% or 99% identity and a variable light chain sequence with 90%, 95% or 99% identity to SEQ ID NO: 27; a variable heavy chain sequence with 90%, 95% or 99% identity to SEQ ID NO: 28 and a variable light chain sequence with 90%, 95% or 99% identity to SEQ ID NO: 29; and a variable heavy chain sequence with 90%, 95% or 99% identity to SEQ ID NO: 30 and a variable light chain sequence with 90%, 95% or 99% identity to SEQ ID NO: 31. The skilled person will further understand that the variable light chain and the variable heavy chain can be independently selected or mixed and matched to prepare an anti-CLDN6 antibody comprising a combination of variable heavy chains and variable light chains other than the pairs identified above.

In some embodiments, the antibody is a full-length antibody. In other embodiments, the antibody is an antibody fragment, including, for example, an antibody fragment selected from the group consisting of: Fab, Fab', F(ab) ₂ , Fv, domain antibody (dAb) and complementarity determining region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, and polypeptides containing at least a sufficient immunoglobulin portion to selectively bind CLDN6 to the polypeptide.

In some embodiments, the variable region domain of the anti-CLDN6 antibody disclosed herein can be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or a fragment thereof. Similarly, the VL domain can be linked to a CK domain or a fragment thereof. In this way, for example, the antibody can be a Fab fragment, in which the antigen binding domain comprises the relevant VH and VL domains, the C-termini of which are covalently linked to the CH1 and CK domains, respectively. The CH1 domain can have other amino acid extensions, for example, to provide a hinge region or a portion of a hinge region domain as present in a Fab fragment, or to provide other domains, such as antibody CH2 and CH3 domains.

Thus, in one embodiment, the antibody fragment comprises at least one CDR as described herein. As described herein, the antibody fragment may comprise at least two, three, four, five or six CDRs. The antibody fragment may further comprise at least one variable region domain of an antibody as described herein. The variable region domain may be of any size or amino acid composition and will generally comprise at least one CDR sequence responsible for binding to human anti-CLDN6, such as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 described herein, and which is adjacent to or in frame with one or more framework sequences.

In some embodiments, the anti-CLDN6 antibody is a monoclonal antibody. In some embodiments, the anti-CLDN6 antibody is a human antibody. In an alternative embodiment, the anti-CLDN6 antibody is a murine antibody. In some embodiments, the anti-CLDN6 antibody is a chimeric antibody, a bispecific antibody, or a humanized antibody.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises one or more conservative amino acid substitutions. One skilled in the art will recognize that a conservative amino acid substitution is a substitution of one amino acid with another amino acid having similar structural or chemical properties (e.g., similar side chains). Exemplary conservative substitutions are described in the art, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Publication Company, 4th Edition (1987).

"Conservative modification" refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions refer to amino acids being replaced by amino acid residues with similar side chains. Families of amino acid residues having similar side chains are well defined and include amino acids having the following side chains: acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). In addition, any native residue in the polypeptide may also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1998) Acta Physiol Scand Suppl 643:55-67; Sasaki et al. (1998) Adv Biophys 35:1-24). Amino acid substitutions of the antibodies of the present disclosure may be performed by known methods, such as by PCR mutagenesis (U.S. Pat. No. 4,683,195).

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable heavy chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30. In other embodiments, the anti-CLDN6 antibody or antibody fragment thereof retains the binding and/or functional activity of an anti-CLDN6 antibody or antibody fragment thereof comprising the variable heavy chain sequence in SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30. In other further embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises the variable heavy chain sequence of SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30 and has one or more conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In yet further embodiments, one or more conservative amino acid substitutions fall within one or more framework regions of SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30 (based on the numbering system of Kabat).

In a specific embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the anti-CLDN6 heavy chain variable region sequence shown in SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30, comprises one or more conservative amino acid substitutions in the framework region (based on the numbering system of Kabat), and retains the binding and/or functional activity of an anti-CLDN6 antibody or antibody fragment thereof comprising a variable heavy chain sequence as shown in SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30 and a variable light chain sequence as shown in SEQ ID NO: 2, 4, 25, 27, 29 or 31.

In some embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable light chain sequence having an amino acid sequence having at least about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 25, 27, 29 or 31. In other embodiments, the anti-CLDN6 antibody or antibody fragment thereof retains the binding and/or functional activity of the anti-CLDN6 antibody or antibody fragment thereof comprising the variable light chain sequence of SEQ ID NO: 2, 4, 25, 27, 29 or 31. In other further embodiments, the anti-CLDN6 antibody or antibody fragment thereof comprises the variable light chain sequence of SEQ ID NO: 2, 4, 25, 27, 29 or 31 and has one or more conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the light chain variable sequence. In yet further embodiments, one or more conservative amino acid substitutions fall within one or more framework regions of SEQ ID NO: 2, 4, 25, 27, 29 or 31 (based on the numbering system of Kabat).

In a specific embodiment, the anti-CLDN6 antibody or antibody fragment thereof comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the anti-CLDN6 light chain variable region sequence shown in SEQ ID NO: 2, 4, 25, 27, 29 or 31, comprises one or more conservative amino acid substitutions (based on the numbering system of Kabat) in the framework region, and retains the binding and/or functional activity of an anti-CLDN6 antibody or antibody fragment thereof comprising a variable heavy chain sequence as shown in SEQ ID NO: 1, 3, 23, 24, 26, 28 or 30 and a variable light chain sequence as shown in SEQ ID NO: 2, 4, 25, 27, 29 or 31.

The therapeutic value of the antibodies of the present disclosure can be enhanced by coupling with cytotoxic drugs or agents to improve their effectiveness and efficacy. In some embodiments, the antibody is an antibody-drug conjugate (ADC), which comprises a CLDN6-specific antibody coupled to a cytotoxic effector (such as a radioisotope, a drug, or a cytotoxin).

The anti-CLDN6 antibodies of the present disclosure can also be used to develop antibody-based immunotherapeutics or cell therapies (such as CAR-T therapies) that rely on selective binding of CLDN6 or CLDN6/9 to direct patient effector cells (e.g., T cells or NK cells) to tumors, which contain bispecific T cell engaging antibodies, or bispecific molecules that redirect NK cells.

In an exemplary aspect, the anti-CLN6 antibody or fragment thereof of the present disclosure can be incorporated into an antigen binding protein in the form of a bispecific antibody capable of binding to two different antigens. More than fifty forms of bispecific antigen binding proteins are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7):838-847(2015); Zhang et al., Exp Hematol Oncol 6:12(2017); Spiess et al., Mol Immunol.; 67(2Pt A):95-106(2015). In an exemplary aspect, the anti-CDLN6 antigen binding protein component of the bispecific antibody is a full-length antibody. In an alternative embodiment, the bispecific antigen binding protein comprises an anti-CLDN6 scFv comprising the LC and HC variable regions of any of the antibodies of the present disclosure.

In various aspects, the antigen binding fragment is based on the heavy chain variable region, and in other aspects, the antigen binding fragment is based on the light chain variable region. In exemplary aspects, the antigen binding fragment comprises at least part of both the HC variable region and the LC variable region. In exemplary aspects, the bispecific antigen binding protein comprises at least one (if not two) of the LC or HC variable regions of the CLDN6 antibody of the present disclosure, and at least one (if not two) of the LC and HC variable regions of the second antibody specific for the second antigen. In an exemplary example, the bispecific antigen binding protein comprises an scFV containing the LC or HC variable regions of the CLDN6 antibody of the present disclosure, and the LC and HC variable regions of the second antibody specific for the second antigen.

In an exemplary embodiment, the antigen binding protein is bispecific, which binds CLDN6 and a second antigen. In an exemplary example, the second antigen is a cell surface protein expressed by T cells. In exemplary aspects, the cell surface protein is a component of a T cell receptor (TCR), such as CD3. In an exemplary example, the second antigen is a co-stimulatory molecule that assists T cell activation, for example, CD40 or 4-1BB (CD137). In an alternative exemplary example, the second antigen is an immune checkpoint molecule (e.g., a protein involved in an immune checkpoint pathway), selected from B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA or SIGLEC7. Optionally, the immune checkpoint molecule is PD-1, LAG3, TIM3 or CTLA4.

The anti-CLDN6 antibodies or antigen-binding portions described herein, bispecific molecules or fusion proteins comprising a CLDN6 binding agent can be used for antibody-based treatment of diseases associated with cells expressing CLDN6. For example, the antibodies can be used to treat solid tumor cancer diseases associated with cells expressing CLDN6, such as breast cancer, lung cancer, ovarian cancer, testicular cancer, pancreatic cancer, gastric cancer, gallbladder cancer, and urothelial cancer.

In various embodiments, the anti-CLDN6 antibodies provided herein may comprise substitutions or modifications of the constant region (i.e., Fc region), including but not limited to amino acid residue substitutions, mutations and/or modifications, the resulting compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered binding of Fc ligands to Fc receptors (FcRs), enhanced or reduced ADCC, CDC, ADCP, TDCC, altered glycosylation and/or disulfide bonds and improved binding specificity. For example, by altering the amino acid residues involved in the interaction between the Fc domain and the Fc receptor (e.g., FcγRI, FcγRIIA and B, FcγRIII and FcRn), antibodies or antibody fragments with improved Fc effector functions can be generated, which can lead to increased cytotoxicity.

A key step in activating cytotoxic cells is the binding of mAbs to FcγRIIIa (CD16A) on immune effector cells, and the strength of this interaction is determined by the antibody isotype, the glycosylation pattern of the antibody Fc region, and FcγRIIIa polymorphism. Many publications have reported findings demonstrating the role of FcγR-mediated effector functions in antibody-based cancer therapies in clinical studies. Findings have shown an association between clinical responses (e.g., antibody efficacy) and the specific allotype that activates human FcγRs. Patients carrying the 158F allele have been reported to exhibit diminished clinical responses to certain therapeutic antibodies, including trastuzumab, rituximab, cetuximab, infliximab, and ipilimumab, as well as other therapeutic antibodies that utilize ADCC as a primary mechanism of action. Antibodies engineered to improve FcgR binding properties have been reported to drive superior anti-tumor responses and confer greater clinical benefit.

The discovery of activating and inhibitory FcγRs has led to translational research efforts focused on designing “fit-for-purpose” therapeutic antibodies based on FcγR binding activity, characterized by an activation/inhibition (A:I) ratio engineered to activate immune effector cells to perform specific functions. Cancer immunotherapy using monoclonal antibodies (mAbs) can promote the elimination of tumor cells through multiple mechanisms, including ADCC, ADCP, and/or CDC activity. Indeed, the therapeutic activity of several approved mAbs depends on the binding of the Fcγ region to low-affinity Fcγ receptors expressed on effector cells.

Several publications report the successful use of protein engineering strategies to design variant human IgG1 Fc domains (CH regions) with optimized FcgR binding properties and activation/inhibition (A:I) ratios suitable for optimizing cell-mediated effector functions. Particular efforts have focused on increasing the affinity of the Fc domain for the low-affinity receptor FcγIIIa. Several mutations within the Fc domain have been identified that directly or indirectly enhance binding to Fc receptors, thereby significantly enhancing cellular cytotoxicity (Lazar, G.A. PNAS 103:4005-4010 (2006), Shields, R.L. et al., J. Biol. Chem. 276:6591-6604 (2001) Stewart, R. et al., Protein Engineering Design and Selection 24:671-678 (2011) (Richards, J.O. et al., Mol. Cancer Ther. 7:2517-2575 (2008)).

CLDN6 binding

The anti-CLDN6 antibodies or antibody fragments thereof provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding strength of the antigen binding protein to CLDN6 can be described by its affinity, which is a measure of the strength of the interaction between the binding site of the antigen binding protein and the epitope. In various aspects, the affinity of the antigen binding protein is measured or ranked using a flow cytometry or fluorescence activated cell sorting (FACS)-based assay. Flow cytometry-based binding assays are known in the art. See, e.g., Cedeno-Arias et al., Sci Pharm 79(3):569-581(2011); Rathanaswami et al., Analytical Biochem 373:52-60(2008); and Geuijen et al., J Immunol Methods 302(1-2):68-77(2005). Selectivity can be based on the KD exhibited by the antigen binding protein for CLDN6 or a CLDN family member, wherein the KD can be determined by techniques known in the art, e.g., surface plasmon resonance, FACS-based affinity assays.

In various aspects, the relative affinity of the CLDN6 antibody is determined by a FACS-based assay, in which different concentrations of the CLDN6 antibody are incubated with cells expressing CLDN6 and the emitted fluorescence (which is a direct measure of antibody-antigen binding) is measured. A fluorescence curve is plotted for each dose or concentration. The maximum value is the lowest concentration at which the fluorescence reaches a plateau or reaches a maximum value (i.e., when binding saturation occurs). Half of the maximum value is considered the EC ₅₀ or IC ₅₀ , and the antibody with the lowest EC ₅₀ / IC ₅₀ is considered to have the highest affinity relative to other antibodies tested in the same manner.

In one aspect, the cell is genetically engineered to overexpress CLDN6. For example, the cell is a HEK293T or CHO cell engineered to express CLDN6. In an alternative aspect, the cell is an established human tumor cell line that endogenously expresses CLDN6. In various aspects, the cell is a cell from a human cell line (e.g., an ovarian cell line, an endometrial cell line, a germ cell tumor cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc.).

In one embodiment, the anti-CLDN6 antibody or antibody fragment of the present disclosure selectively binds to CLDN6 over CLDN9, and does not bind to claudin-3 (CLDN3) or claudin-4 (CLDN4). In an alternative embodiment, the anti-CLDN6 antibody or antibody fragment binds to both CLDN6 and CLDN 9 equally (e.g., with no preference for either of the two CLDNs), and does not bind to claudin-3 (CLDN3) or claudin-4 (CLDN4).

CLDN6 internalization and dose-dependent cytotoxicity

Preclinical characterization of the safety and antitumor activity of IMAB027-vcMMAE, an ADC comprising the anti-claudin-6 antibody-drug antibody IMAb027, included studies evaluating: internalization of IMAB027 in various CLDN6+ human ovarian cancer (OC) and testicular cancer (TC) cell lines; binding characteristics (by FACS) and cell viability assessed by XTT metabolic assay in cell culture as well as IMAB027–vcMMAE-mediated cytotoxic effects (direct and indirect bystander effects) (Türeci, et al., AACR; Cancer Res 2018;78(13 Suppl):Abstract #1778).

Türeci et al. reported that IMAB027 ACD robustly binds to and is internalized by cell lines expressing CLDN6, and can reduce the viability of CLDN6+ OC and TC cells by up to 100%, with EC50 values in the ng/mL range. In addition, after conjugation, IMAB027-vcMMAE retains the ability of IMAB027 to induce CLDN6+ cell death through antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. In monoculture, cell lines that do not express CLDN6 were not affected by IMAB027-vcMMAE; however, in cocultures of CLDN6+ and CLDN6-negative cells, IMAB027-vcMMAE exerted a bystander effect, causing the death of cocultured CLDN6-negative cells in addition to the death of the targeted CLDN6+ cells. Therefore, monoclonal antibodies specific for CLDN6 are known to mediate induced and efficient CLDN6 internalization and can be used to deliver cytotoxic agents to tumor cells expressing CLDN6.

Based on the in vitro evaluation of maximum binding capacity, EC ₅₀ , cell surface internalization and cytotoxicity, the suitability of the anti-CLDN6 antibodies of the present disclosure for use as ADC-based targeting antibodies for treating cancer can be evaluated. Therefore, the anti-CLDN6 antibodies of the present disclosure are suitable for use as ADC-based targeting antibodies for developing internalization site-specific ADCs for use in antibody-based immunotherapy methods for treating cancer.

The disclosed antibodies specific for CLDN6 are capable of mediating induced and efficient CLDN6 internalization, and in the presence of a secondary antibody coupled to the ADC, a dose-dependent cytotoxicity was generated. In the HEK293 cell line overexpressing CLDN6, the observed EC ₅₀ for cell killing ranged from 1.73 nM to 2.19 nM, and in the cancer cell line NEC8, the EC ₅₀ for cell killing ranged from 0.1 nM to 0.2 nM. In the cancer cell line OV90, the EC ₅₀ for cell killing ranged from 1.08 nM to 2.32 nM.

CLDN6 ADCC

Due to binding to CLDN6 expressed on the surface of target cells, the antibodies of the present disclosure can mediate target cell killing by one or more mechanisms of action (such as delivery of cytotoxic agents, or by directing ADCC-, CDC- or TDCC-mediated lysis). In one embodiment, the target cell is a primary or metastatic cancer cell.

In some aspects, the ability of the disclosed anti-CLDN6 antibodies to mediate killing (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), T-cell-dependent cytotoxicity (TDCC), and/or inhibition of cell proliferation) and/or phagocytosis of cells expressing CLDN6 can be assessed.

The anti-CLDN6 antibodies disclosed herein are also capable of directing ADCC against target cells expressing CLDN6 (whether endogenously expressed or engineered to overexpress human CLDN6 in host cells). In the cancer cell line NEC8, the EC ₅₀ range of ADCC activity was 0.40 nM to 9.83 nM. In the cancer cell line OV90, the EC ₅₀ range of ADCC activity was 0.3 nM to 0.75 nM.

Antibody-based immunotherapy

The goal of antibody-based immunotherapy using antibodies targeting tumor antigens is to eliminate cancer cells without damaging normal tissues. Therefore, the efficacy and safety of antibody-based immunotherapy in oncology depend largely on the intended mechanism of action, the relevant effector functions of the immune system, and the nature of the tumor-specific or tumor-associated target antigen.

The antibodies of the present disclosure can also be used to target payloads (e.g., radioisotopes, drugs, or toxins) to directly kill tumor cells, or can be used in conjunction with traditional chemotherapeutic agents to attack tumors through complementary mechanisms of action, which may include anti-tumor immune responses that are compromised due to the cytotoxic side effects of chemotherapeutic drugs on immune effector cells.

Antibody-drug conjugates (ADCs) are a class of highly effective antibody-based cancer therapeutics. ADCs consist of recombinant monoclonal antibodies covalently linked to cytotoxic agents (referred to as payloads) via synthetic joints. ADCs combine the specificity of monoclonal antibodies with the efficacy of small molecule chemotherapeutics, facilitating the direct targeted delivery of highly cytotoxic small molecule drug moieties to tumor cells. The targeting properties of ADCs allow for increased drug efficacy and limited systemic exposure. These characteristics together provide ADCs with less side effects and a wider therapeutic window of desired features (Peters et al., Biosci Rep, 35 (4): e00225, 2015).

Cell surface antigens suitable for use as ADC targets have two important properties: (i) high expression levels in target cells, limited or no expression in normal tissues, and (ii) efficient internalization in response to antibody binding. CLDN6 is overexpressed in a variety of cancers, including endometrial cancer, ovarian cancer, testicular cancer, and lung cancer (NSCLC). There is evidence that a large portion of the expressed CLDN protein remains associated with the cell surface of tumorigenesis, allowing localization and internalization of the antibodies or ADCs of the present disclosure.

As used herein, an "internalizing" antibody is an antibody that is taken up by a cell (along with any cytotoxins) after binding to a relevant antigen or receptor. For therapeutic applications, internalization preferably occurs in a subject in need thereof. The amount of ADC internalized can be sufficient to kill cells expressing the antigen, especially cancer stem cells expressing the antigen. Depending on the efficacy of the cytotoxin or ADC as a whole, in some cases, cellular uptake of a single antibody molecule is sufficient to kill the target cell bound to the antibody. For example, the efficacy of certain drugs is so high that the internalization of several molecules of toxins coupled to the antibody is sufficient to kill tumor cells. Whether an antibody is internalized after binding to a mammalian cell can be determined by various art-recognized assays, including those described in the examples below.

The generation of antibody-drug conjugates can be achieved by any technology known to those skilled in the art using any suitable payload drug, synthetic joints and coupling chemistry. Those skilled in the art will understand ADC and will also appreciate that developing ADC requires evaluation of several factors, including target antigen biology, antibody specificity, cytotoxicity and mechanism of action of payload drugs, stability and cutting of joints, sites of joint attachment, and the level of ADC heterogeneity produced by coupling chemistry. For the number of cytotoxic molecules attached to each antibody, heterogeneity can lead to the production of such a drug product, which contains non-effective substances (specie) (no drug payload) and each antibody has a substance with more than 4 drug parts (high load), which has the potential to be cleared more quickly and cause toxicity. In addition, the presence of non-potent substances (non-potentspecie) (antibodies without cytotoxic payloads) can reduce effectiveness by competing for the combination with the ADC target antigen. Therefore, it is desirable to produce an ADC drug product with a homogeneous mixture of antibodies, characterized in that it is consistent with drug: antibody ratio (DAR).

Most ADC candidates currently in clinical evaluation incorporate one of the three major classes of drugs (i.e., maytansinoids, auristatins, and PBD dimers) as the cytotoxic payload; however, other classes of payloads are also being used, such as calicheamicin (for gemtuzumab ozogamicin and inotuzumab ozogamicin) and sirolimus (for sirolimus). In general, cytotoxic drugs can be used as microtubule inhibitors (auritasins and maytansine alkaloids), or as DNA structure disruptors, including duocarmycin (DNA alkylation), calicheamicin (DNA double-strand cleavage), camptothecin analogs (topoisomerase inhibitors) (such as SN-38 and exatecan) or pyrrolobenzodiazepine (PBD) dimers (DNA chain crosslinking) (Shim et al.).

One of the key functions of the linker is to maintain the stability of the ADC in the blood circulation while allowing the release of toxins after being internalized by the target cells. Important parameters to be considered in the process of identifying a suitable linker include the cleavability of the linker and the details of the coupling chemistry (i.e., the location and nature of the connection). Broadly speaking, linkers are divided into two categories: cleavable and non-cleavable. Cleavable linkers take advantage of the difference between normal physiological conditions in the bloodstream and the intracellular conditions present in the cytoplasm of cancer cells (Peters et al., Biosci Rep, 35 (4): e00225, 2015). After the ADC antigen complex is internalized, changes in the microenvironment trigger the cleavage of the linker, releasing the cytotoxic payload, effectively targeting the toxicity to cancer cells expressing the target antigen. Broadly speaking, there are three types of cleavable linkers: hydrazine, disulfide and peptide linkers. In contrast, non-cleavable linkers rely only on the process of lysosomal degradation after the ADC antigen is internalized. After internalization of the ADC-antigen complex, proteases within the lysosome degrade the protein structure of the antibody, generating a single amino acid (usually cysteine or lysine) attached to the linker, and the cytotoxic agent released into the cytoplasm as an active drug. It is well known that linker chemistry is an important determinant of the specificity, potency, activity, and safety of ADCs.

Those skilled in the art will recognize that there are many techniques for chemically modifying proteins that are suitable for coupling linker payloads to TSA- or TAA-specific antibodies. Those skilled in the art will recognize that different coupling chemistries will provide different levels of control over the number and sites of drug attachment, potentially affecting the pharmacokinetics, toxicity, and therapeutic window of the resulting anti-CLDN6 ADC. Antibody-drug conjugates can be prepared by conjugating a drug to an antibody according to conventional techniques. Techniques for coupling therapeutic moieties to antibodies are well known to those skilled in the art, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (Second Edition), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506, (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

Those skilled in the art will appreciate that in addition to conventional conjugation techniques (involving conjugation to surface-exposed lysine or cysteine residues present in the antibody, either as a result of the natural amino acid sequence composition), there are many other site-specific drug conjugation methods that can be used to prepare anti-CLDN6 specific immunoconjugates. Site-specific conjugation chemistry methods aim to produce relatively homogeneous ADC products without changing the binding affinity of the antibody. In general, three strategies are mainly used for site-specific conjugation of antibodies: the use of engineered cysteines, the incorporation of non-natural amino acids, and enzyme conjugation, which uses reactive sites of antibodies designed to react specifically with bacterial enzymes (e.g., transglutaminases, glycosyltransferases, classases, or formylglycine generating enzymes) to generate post-translationally modified proteins in a site-specific manner. Techniques for site-specifically conjugating therapeutic moieties to antibodies are well known to those skilled in the art and include, but are not limited to, methods disclosed in U.S. Patent Nos. 7,723,485; 8,937,161; 9,000,130; 9,884,127; 9,717,803; 10,639,291; 10,357,472, U.S. Patent Application Publication No. 2015/0283259; US2017/0362334; US2018/0140714; and International Publication Nos. WO2013/092983; WO2013/092998; WO2014/072482; WO2014/202773; WO2014/202775; WO2015/155753; WO2015/191883; WO2016/102632; WO2017/059158; WO 2018/140590 and WO 2018/185526.

In an alternative embodiment, the anti-CLDN6 antibodies or antibody fragments of the present disclosure may interact with effector cells of the immune system, preferably through ADCC, TDCC, CDC or ADCP interactions (Kubota, T. et al. (2009) Cancer Sci. 100(9), 1566-1572; Nazarian et al., J. Bio. Scre., 2015, 20(4) 519-527).

In the context of the present disclosure, the term "immune effector function" includes any function mediated by components of the immune system that results in tumor growth inhibition and/or tumor development inhibition (including inhibition of tumor dissemination and metastasis). Preferably, the immune effector function results in killing of cancer cells. Preferably, the immune effector function in the context of the present disclosure is an antibody-mediated effector function. These functions include complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells carrying tumor-associated antigens.

Antibody-dependent cell-mediated cytotoxicity (ADCC) describes the cell killing ability of effector cells, which preferably requires the use of antibody-labeled target cells. Effector cells can include B cells, T cells, killer cells, NK cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells and/or basophils; More specifically, effector cells are T cells or NK cells. In some aspects, ADCC occurs when antibodies bind to antigens on tumor cells and antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells. Several Fc receptor families have been identified, and specific cell populations characteristically express limited Fc receptors. ADCC can be regarded as a mechanism that directly induces instant tumor destruction to varying degrees, resulting in antigen presentation and induction of tumor-directed T cell responses. Preferably, the in vivo induction of ADCC will result in tumor-directed T cell responses and host-derived antibody responses.

Complement-dependent cytotoxicity (CDC) is another method of cell killing that can be directed by antibodies. IgM is the most efficient isotype for complement activation, but IgG1 and IgG3 are also very efficient in directing CDC via the classical complement activation pathway.

Alternatively, the anti-CLDN6 antibodies of the present disclosure provided herein can be used for adaptive immune gene therapy to treat tumors. In one embodiment, the antibodies of the present disclosure (e.g., ScFv fragments) can be used to produce chimeric antigen receptors (CARs). "CAR" is a fusion protein consisting of an ECD, a transmembrane domain, and at least one intracellular domain, and the ECD comprises an anti-CLDN antibody of the present disclosure or an immunoreactive fragment thereof (e.g., ScFv fragment). In one embodiment, T cells, natural killer cells, or dendritic cells that have been genetically engineered to express CAR can be introduced into a subject with cancer to stimulate the subject's immune system to specifically target tumor cells expressing CLDN6.

Methods of producing antibodies

Anti-CLDN6 antibodies or antibody fragments thereof can be prepared by any method known in the art. For example, a soluble recombinant claudin-6 (CLDN6) protein, or a fragment of a CLDN6 peptide coupled to a carrier protein, can be used to immunize a recipient. Any suitable immunization method can be used. Such methods may include adjuvants, other immunostimulants, repeated booster immunizations, and the use of one or more immunization routes.

Any suitable source of human CLDN6 can be used as an immunogen to generate non-human or human anti-CLDN6 antibodies in the compositions and methods disclosed herein.

Different forms of CLDN6 antigens can be used to elicit an immune response for identifying biologically active anti-CLDN6 antibodies. Thus, the eliciting CLDN6 antigen can be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancers. In some aspects, the eliciting antigen is an isolated soluble full-length protein, or a soluble protein comprising less than the full-length sequence (e.g., immunization with peptides comprising the extracellular domain/loop, ECD1 and/or ECD2 of CLDN6 alone or in combination). As used herein, the term "portion" refers to the minimum number of amino acids or nucleic acids suitable for constituting an immunogenic epitope of the antigen of interest. Any genetic vector suitable for transforming the target cell can be used, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids.

It is desirable to prepare monoclonal antibodies (mAbs) from a variety of mammalian hosts (e.g., mice, rodents, primates, humans, etc.). Techniques for preparing such monoclonal antibodies are described in, for example, Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY. Typically, spleen cells from an animal immunized with the desired antigen are immortalized and typically fused with myeloma cells, see and Milstein (196), Eur. J. Immunol., 6 (7): 511-9. Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes or retroviruses, or other methods known in the art. See, for example, Doyle et al. (1994 ed. and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROEDURES, John Wiley and Sons, New York, NY. Clones produced by single immortalized cells are screened to produce antibodies with desired specificity and affinity for antigens, and the yield of monoclonal antibodies produced by such cells can be increased by various techniques (including injection into the peritoneal cavity of a vertebrate host). Alternatively, for example, according to the general operating protocol outlined by Huse et al. (1989) Science 246: 1275-1281, DNA sequences encoding monoclonal antibodies or antigen-binding fragments thereof can be isolated by screening DNA libraries from human B cells. Therefore, antibodies can be obtained by a variety of techniques familiar to those skilled in the art.

Other suitable techniques include selecting antibody libraries in phage, yeast, viruses or similar vectors. See, for example, Huse et al., supra; and Ward et al. (1989) Nature 341: 544-546. The polypeptides and antibodies disclosed herein can be used with or without modification, including chimeric or humanized antibodies. Typically, the polypeptides and antibodies will be labeled by covalently or non-covalently linking to a substance that provides a detectable signal. A variety of labeling and coupling techniques are known and widely reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,9396,345; 4,277,437; 4,275,149; and 4,366,241. In addition, recombinant immunoglobulins can be produced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10023; or made in transgenic mice, see Nils Lonberg et al. (1994), Nature 368:856-859; and Mendez et al. (1997) Nature Genetics 15:146-156; TRANSGENICANIMALS AND METHODS OF USE (WO 2012/62118), Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour and other technologies.

In some embodiments, the ability of the generated antibodies to bind to CLDN6 and/or other claudin family-related members can be assessed using standard binding assays, such as surface plasmon resonance (SPR), FoteBio (BLI), Gator (BLI), ELISA, Western blot, immunofluorescence, flow cytometric analysis (FACS), or internalization assays.

Antibody compositions prepared from hybridomas or host cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a common purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (see, for example, Lindmark et al., 1983 J. Immunol. Meth. 62: 1-13). Protein G is recommended for all mouse isotypes and human γ3 (see, for example, Guss et al., 1986 EMBO J. 5: 1567-1575). The matrix to which the affinity ligand is attached is typically agarose, but other matrices may also be used. Mechanically stable matrices, such as controlled pore glass or poly (styrenedivinyl) benzene, can achieve faster flow rates and shorter processing times than agarose. When the antibody comprises a CH3 domain, Bakerbond ABX ^™ resin (JT Baker, Phillipsburg, NJ) is used for purification. Other protein purification techniques may also be used, depending on the antibody to be recovered, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin SEPHAROSE ^™ chromatography, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

Following any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, typically at a low salt concentration (e.g., about 0-0.25 M salt).

Also included are nucleic acids that hybridize under low, medium, and high stringency conditions as defined herein to all or part of a nucleotide sequence represented by an isolated polynucleotide sequence encoding an antibody or antibody fragment of the present disclosure (e.g., a portion encoding a variable region). The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid has at least 80%, such as at least 90%, at least 95%, or at least 98% identity to a portion or all of a nucleic acid encoding an anti-CLDN6 polypeptide (e.g., a heavy chain or light chain variable region), or its complementary sequence. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers (e.g., PCR primers), or diagnostic probes.

Polynucleotides, vectors and host cells

Other embodiments include isolated polynucleotides comprising sequences encoding anti-CLDN6 antibodies or antibody fragments thereof, vectors and host cells comprising the polynucleotides, and recombinant techniques for producing antibodies. The isolated polynucleotides may encode any desired form of anti-CLDN6 antibody, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab') ₂ and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

Some embodiments include isolated polynucleotides comprising a sequence encoding a heavy chain variable region of an antibody or antibody fragment having an amino acid sequence of SEQ ID NOs: 1, 3, 23, 24, 26, 28, and 30. Some embodiments include isolated polynucleotides comprising a sequence encoding a light chain variable region of an antibody or antibody fragment having an amino acid sequence of any one of SEQ ID NOs: 2, 4, 25, 27, 29, and 31.

In one embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having light and heavy chain variable regions comprising the following amino acid sequences:

(a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2;

(b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

(c) a variable heavy chain sequence comprising SEQ ID NO: 23 and a variable light chain sequence comprising SEQ ID NO: 2;

(d) a variable heavy chain sequence comprising SEQ ID NO: 24 and a variable light chain sequence comprising SEQ ID NO: 25;

(e) a variable heavy chain sequence comprising SEQ ID NO: 26 and a variable light chain sequence comprising SEQ ID NO: 27;

(f) a variable heavy chain sequence comprising SEQ ID NO: 28 and a variable light chain sequence comprising SEQ ID NO: 29; and

(g) a variable heavy chain sequence comprising SEQ ID NO: 30 and a variable light chain sequence comprising SEQ ID NO: 31.

In another embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having light and heavy chain variable regions comprising the following amino acid sequences:

(a) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 1 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 2; (b) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 3 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 4;

(c) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 23 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 2;

(d) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 24 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 25;

(e) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 26 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 27;

(f) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 28 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO: 29; and

(g) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:30 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:31.

As known in the art, polynucleotides comprising sequences encoding anti-CLDN6 antibodies or antibody fragments thereof may be fused to one or more regulatory or control sequences and may be contained in a suitable expression vector or host cell as known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain may be independently fused to a polynucleotide sequence encoding a constant domain (e.g., a human constant domain) to enable the production of a complete antibody. Alternatively, polynucleotides or portions thereof may be fused together to provide a template for the production of a single-chain antibody.

For recombinant production, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or expression. Many suitable vectors for expressing recombinant antibodies are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Anti-CLDN6 antibodies or antibody fragments thereof can also be produced as fusion polypeptides, wherein the antibody or fragment is fused to a heterologous polypeptide (e.g., a signal sequence, or other polypeptide having a specific cleavage site at the amino terminus of a mature protein or polypeptide). The selected heterologous signal sequence is generally a sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the anti-CLDN6 antibody signal sequence, the signal sequence can be replaced by a prokaryotic signal sequence. The signal sequence can be, for example, an alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leader sequence, etc. For yeast secretion, the natural signal sequence can be replaced by, for example, a leader sequence obtained from yeast invertase α-factor (including Saccharomyces and Kluyveromyces α-factor leaders), acid phosphatase, Candida albicans glucoamylase, or the signal described in WO90/13646. In mammalian cells, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, can be used. The DNA of this precursor region is linked in reading frame to the DNA encoding the anti-CLDN6 antibody.

Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, the sequence is a sequence that enables the vector to replicate independently of the host chromosome DNA, including a replication origin or an autonomous replication sequence. Such sequences of various bacteria, yeasts and viruses are well known. The replication origin from plasmid pBR322 is applicable to most gram-negative bacteria, the 2-υ. plasmid origin is applicable to yeast, and various viral origins (SV40, polyoma virus, adenovirus, VSV and BPV) can be used to clone vectors in mammalian cells. In general, mammalian expression vectors do not require a replication origin assembly (the SV40 origin can usually be used, just because it contains an early promoter).

Expression and cloning vectors may contain genes encoding selectable markers to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), or alternatively, encode proteins that complement auxotrophic deficiencies, or alternatively, encode proteins that provide specific nutrients not present in complex media, such as the gene encoding D-alanine racemase for Bacilli.

Compositions and methods of treatment

The present disclosure also provides compositions, including, for example, pharmaceutical compositions comprising anti-CLDN6 antibodies or antibody fragments thereof, for use as therapeutic agents for treating patients with primary or metastatic cancers originating from epithelial cells. In specific embodiments, the compositions described herein are administered to cancer patients to kill tumor cells. For example, the compositions described herein can be used to treat patients with solid tumors characterized by the presence of cancer cells expressing or overexpressing CLDN6. In certain aspects, the compositions of the present disclosure can be used to treat breast cancer, lung cancer, ovarian cancer, testicular cancer, pancreatic cancer, gastric cancer, gallbladder cancer, and urothelial cancer.

In some aspects, the treatment of cancer represents an area where combined strategies are particularly needed, because the combined effects of two, three, four or even more cancer drugs/therapies often produce synergistic effects, which are much stronger than the effects of a single treatment method. The reagents and compositions (e.g., pharmaceutical compositions) provided herein can be used alone or in combination with conventional treatment regimens such as surgery, radiotherapy, chemotherapy and/or bone marrow transplantation (autologous, isogenic, allogeneic or unrelated). The reagents and compositions can also be used in combination with one or more of the following: antitumor agents, chemotherapeutic agents, growth inhibitors, cytotoxic agents, immune checkpoint inhibitors, co-stimulatory molecules, kinase inhibitors, angiogenesis inhibitors, small molecule targeted therapeutic drugs and multi-epitope strategies. Therefore, in another embodiment, cancer treatment can be effectively combined with various other drugs.

In a method of treatment, a pharmaceutical composition comprising an anti-CLDN6 antibody may further comprise a therapeutic agent or a toxic agent coupled or uncoupled to the anti-CLDN6 antibody or antibody fragment. In a specific embodiment, the anti-CLDN6 antibody is used to target an ADC with a cytotoxic payload to a tumor that expresses and/or overexpresses CLDN6. In an alternative embodiment, the anti-CLDN6 antibody is used to target an ADC with a cytotoxic payload to a tumor that expresses and/or overexpresses CLDN6 and CLDN9.

The anti-CLDN6 antibodies disclosed herein can be administered alone or in combination with other compositions for treating cancer. In one embodiment, the antibodies disclosed herein can be administered alone or in combination with other immunotherapeutics (including other antibodies for treating cancer). For example, in one embodiment, other immunotherapeutics are antibodies against immune checkpoint molecules selected from the following: human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, lymphocyte activation gene 3 (LAG3), NKG2A, B7-H3, B7-H4, CTLA-4, GITR, VISTA, CD137, TIGIT and any combination thereof. In an alternative embodiment, the second immunotherapeutic agent is an antibody against a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). Each combination represents a separate embodiment of the present disclosure.

The combination of therapeutic agents discussed herein can be administered simultaneously as components of a bispecific or multispecific binding agent or fusion protein, or as a single component in a pharmaceutically acceptable carrier. Alternatively, the combination of therapeutic agents can be administered simultaneously as separate compositions, each containing each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

The pharmaceutical composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques, for example, those disclosed in Remington: The Science and Practice of Pharmacy, 19th edition, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1995. In some aspects, the pharmaceutical composition is administered to a subject to treat cancer.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody, bispecific and multispecific molecule) can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

Typically, the composition for injection is a solution in a sterile isotonic aqueous buffer. If necessary, the drug may also include a solubilizer and a local anesthetic (such as lidocaine) to relieve pain at the injection site. Typically, the ingredients are provided separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or pouch indicating an active dose). When the drug is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline may be used for distribution. When the drug is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed before administration.

The compositions of the present disclosure can be applied by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and/or mode will vary according to the desired result. The active compound can be prepared together with a carrier (such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems) that will protect the compound from rapid release. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such preparations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, compiled by J.R.Robinson, Marcel Dekker, Inc., New York, 1978.

In an alternative embodiment, conventional viral and non-viral gene transfer methods can be used to import nucleic acids encoding antibodies or fragments thereof described herein in mammalian cells or target tissues. Such methods can be used to apply nucleic acids encoding antibodies to cells in vitro. In some embodiments, nucleic acids encoding antibodies or fragments thereof are administered for in vivo or ex vivo gene therapy purposes. In other embodiments, gene delivery technology is used to study the activity of antibodies based on cells or animal models. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids compounded with delivery vectors (such as liposomes). Viral vector delivery systems include DNA and RNA viruses, which have free or integrated genomes after delivery to cells. Such methods are well known in the art.

Non-viral delivery methods for nucleic acids encoding engineered polypeptides of the present disclosure include lipofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial viral particles, and agent-enhanced DNA uptake. Lipofection methods and lipofection agents are well known in the art (e.g., Transfectam ^TM and Lipofectin ^TM ). Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid: nucleic acid complexes containing targeted liposomes (such as immunolipid complexes) is well known to those skilled in the art.

The use of RNA or DNA virus-based systems to deliver nucleic acids encoding antibodies described herein utilizes a highly evolved process for targeting specific cells in the virus body and transporting the viral payload to the nucleus. Viral vectors can be directly applied to patients (in vivo), or they can be used for in vitro treatment of cells and for applying modified cells to patients (ex vivo). Conventional viral-based systems for delivering polypeptides disclosed herein can include retroviruses, slow viruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Viral vectors are the most effective and versatile methods for gene transfer in target cells and tissues at present. By retrovirus, slow virus, and adeno-associated virus gene transfer methods, it is possible to be integrated into the host genome, usually resulting in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues.

The dosage level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in the medical field.

The pharmaceutical compositions described herein can be administered in an effective amount. An "effective amount" refers to an amount that achieves a desired response or desired effect alone or together with other doses. In the case of treating a specific disease or a specific condition, the desired response preferably involves inhibition of the disease process. This includes slowing the progression of the disease, particularly interrupting or reversing the progression of the disease.

The broad scope of the present disclosure may be best understood with reference to the following examples, which are not intended to limit the present disclosure to the specific examples. The specific embodiments described herein are provided by way of example only, and the present disclosure will be limited to the terms of the appended claims and the full range of equivalents to which these claims are entitled.

Example

General approach

Methods for protein purification are described, including immunoprecipitation, chromatography, and electrophoresis. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are described. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) Bio Directory, Piscataway, N.J., pp. 384-391. The production, purification and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane, supra.

According to the manufacturer's method, the hybridoma or cell culture supernatant containing anti-tight junction protein-6 antibody was purified by HiTrap protein G column (GE, catalog number 17040401). In short, the supernatant was balanced with 5CV of DPBS (Gibco, catalog number 14190--136) and loaded by a syringe/infusion pump (Legato200, KDS) at ambient temperature and a residence time of 3 minutes. The column was washed with 5CV of DPBS and eluted with 4CV of pH 2.8 elution buffer (Fisher Scientific, catalog number PI21004). The eluate was fractionated, and the fractions were neutralized with 1M Tris-HCL, pH 8.5 (FisherScientific, catalog number 50-843-270) and measured with A280 (DropSense96, Trinean). The peak fractions were combined and the buffer was exchanged to DPBS. The centrifugal filter (EMD Millipore, catalog number UFC803024) was equilibrated in DPBS at 4,000×g for 2 minutes. The purified sample was loaded, DPBS was added, and the sample was spun at 4,000×g for 5 to 10 minutes until the total DPBS volume reached ≥6 DV. The final pool was analyzed by A280.

Standard methods of molecular biology are described. See, for example, Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods also appear in Ausbel et al., (2001) Current Protocols in Molecular Biology, Vols. 1 to 4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

By using electroporation-based or lipid-based transfection, the selected host cells (i.e., CHO-K1 or HEK293) are transfected with a plasmid based on pcDNA3.1 expressing Homo sapiens tight junction proteins (reference protein sequences in Table 3) to produce stable cell lines expressing human tight junction proteins-6, tight junction proteins-9, tight junction proteins-3 or tight junction proteins-4. Geneticin or puromycin is used to select integrated cells. After 7-10 days of antibiotic selection, stable clones are isolated by FACS or serial dilution using labeled antibodies. After amplification, stable clones are further confirmed to express tight junction proteins by flow cytometry. Using lipid-based transfection, mouse and cynomolgus monkey tight junction proteins-6 (reference sequences are shown in Table 3) are transiently expressed in HEK293T cells, respectively.

NEC8\CLDN6 knockout cell lines were generated using the CRISPR-Cas9 system. Briefly, NEC8 cells were transfected by electroporation using sgRNA targeting CLDN6 exon 2 as a ribonucleoprotein complex. Knockout cell pools were obtained by sorting and verified by NGS. KO cell pools were further confirmed by flow cytometry.

Table 3 : Tight junction protein sequences

As described below, the sequences of the heavy and light chain variable regions of hybridoma clones were determined. Total RNA was extracted from 1-2×10 ⁶ hybridoma cells using the RNeasy Plus micro kit from Qiagen (Germantown, MD, USA). 5'RACE reaction was performed to generate cDNA using the SMARTer RACE 5'/3' kit from Takara (Mountainview, CA, USA). Gene-specific primers for the 3' mouse constant region of the appropriate immunoglobulin were combined using Takara Universal Primer Mix, and PCR was performed using Q5 High-Fidelity DNA polymerase from NEB (Ipswitch, MA, USA) to amplify the variable regions of the heavy and light chains. The amplified heavy and light chain variable regions were run on a 2% agarose gel, the appropriate bands were cut, and the gel was then purified using the Mini Elute Gel Extraction kit from Qiagen. Purified PCR products were cloned using the Zero Blunt PCR Cloning kit from Invitrogen (Carlsbad, CA, USA) and transformed into Stellar competent E. coli cells from Takara and plated on LB agar + 50ug/ml kanamycin plates. Direct colony Sanger sequencing was performed by GeneWiz (South Plainfield, NJ, USA). The resulting nucleotide sequences were analyzed using IMGT V-QUEST to identify productive rearrangements and analyze translated protein sequences. CDRs were determined based on Kabat numbering.

The selected VH or VL chain was PCR amplified and cloned into an expression vector based on pcDNA3.4 with constant regions from human IgG1 (Uniprot P01857) or human kappa light chain (Uniprot P01834). According to the supplier's Expi293 expression system protocol, plasmids expressing paired heavy and light chains were transfected into Expi293 cells (ThermoFisher Scientific). Five days after transfection, the culture supernatant was collected by centrifugation. The recombinant antibody was purified by 1-step affinity purification using a protein A column and the buffer was exchanged to PBS pH 7.2.

Flow cytometry methods are available, including fluorescence-activated cell sorting detection systems See, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd Edition; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides and antibodies, are available, for example, for use as diagnostic reagents. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

Standard techniques for characterizing ligand/receptor interactions are available. See, for example, Coligan et al. (2001) Current Protocols in Immunology, Volume 4, John Wiley, Inc., New York. Standard methods for characterizing antibody function suitable for characterizing antibodies with a specific mechanism of action are also well known to those skilled in the art.

An internal CLDN6 antibody based on the anti-CLDN6 antibody (64A) was prepared based on publicly available information published in WO 2012/156018, referred to herein as "NR.N6.PC1" (PC1) (wherein VH, SEQ ID NO: 36; and VL, SEQ ID NO: 35). The PC1 antibody was used to confirm the expression of claudin-6 by transfected cells and tumor cell lines used in the examples, and to establish binding and functional assays for evaluating and characterizing the anti-CLDN6 specific antibodies disclosed herein. A second internal claudin 6/9 reactive antibody (hsC27.22) was prepared based on publicly available information published in WO2015/069794, referred to herein as "NR.N6.PC2" (PC2) (wherein VH, SEQ ID NO: 67; and VL, SEQ ID NO: 65).

Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites and sequence alignments.

Example 1: Production of anti-CLDN-6 antibodies

Full-length human anti-human CLDN6 antibodies were generated by immunizing human Ig transgenic mice (Trianni mice) expressing human antibody VH and VL genes (see, e.g., WO 2013/063391, mice).

Immunization - The above-mentioned TRIANI mice were immunized by injection of immunogens, which included DNA containing the human claudin-6 gene and CHO cells stably transfected with the human claudin-6 gene. TRIANI mice were immunized with DNA by tail vein injection. CHO cells transfected with human claudin-6 were immunized by intraperitoneal (IP), subcutaneous (SC) tail-based or footpad injection.

Immune responses were monitored by retro-orbital bleeding. Plasma was screened by flow cytometry (FACS) or imaging (described below). Mice with adequate anti-claudin-6 titers were used for fusions. Mice were boosted with the immunogen intraperitoneally, at the base of the tail, or intravenously, then sacrificed and the spleen and lymph nodes removed.

Selection of mice producing anti-Claudin-6 antibodies - To select mice producing antibodies that bind to Claudin-6, sera from immunized mice were screened by FACS or imaging for binding to cells expressing Claudin-6 protein (CHO cells transfected with the Claudin-6 gene) but not to control cells that do not express Claudin-6 (CHO cells).

For FACS, briefly, Claudin-6-CHO cells or parental CHO cells were incubated with dilutions of immune mouse sera at 4°C for 2 hours. Cells were fixed with 2% PFA (Alfa Aesar, catalog number: J61899) at 4°C for 15 minutes and then washed. After incubation at 4°C for one hour, specific antibody binding was detected using Alexa 647-labeled goat anti-mouse IgG antibody (ThemoFisher Scientific, catalog number: A-21235). Flow cytometric analysis was performed on a flow cytometric instrument (Intellicyte, IQue plus, Sartorius).

In addition, mouse sera were tested by imaging. Briefly, tight junction protein-6-CHO cells were incubated with serum dilutions from immunized mice. Cells were washed, fixed with paraformaldehyde, washed, and specific antibody binding was detected with Alexa488 goat anti-mouse secondary antibody and Hoechst dye (Invitrogen). The plates were scanned and analyzed on an imaging instrument (Cytation 5, Biotek).

Generation of hybridomas producing antibodies against CLDN6 - To generate hybridomas producing human antibodies of the present disclosure, spleen cells and lymph node cells are isolated from immunized mice and fused with a suitable immortalized cell line (e.g., a mouse myeloma cell line). The resulting hybridomas are screened for the production of antigen-specific antibodies. For example, a single cell suspension of spleen cells and lymph node cells from immunized mice is fused by electrofusion with an equal number of Sp2/0 non-secreting mouse IgG myeloma cells (ATCC, CRL 1581). The cells are placed in a flat-bottomed 96-well tissue culture plate and then incubated in a selection medium (HAT medium) for about one week before switching to hybridoma culture medium. After about 10-14 days of cell plating, the supernatant in each well is screened by imaging or FACS as described above. The antibody-secreting hybridomas are transferred to 24-well plates and screened again, and if the hybridoma is still positive for anti-claudin-6, the positive hybridomas are subcloned by sorting using a single cell sorter. The subclones are screened again by imaging or FACS as described below. The stable subclones are then cultured in vitro to generate small amounts of antibody for purification and characterization.

Example 2: Binding specificity of anti-CLDN6 antibodies

The binding specificity of the anti-claudin-6 antibodies (NR.N6.Ab1 and NR.N6.Ab2) of the present disclosure was evaluated by FACS using the cell line claudin-6 transfected with claudin-6-CHO-K1 (GenScript, catalog number U3288DL180_3) and parental CHO cells (CHO-K1, ATCC, CCL-61). Briefly, claudin-6-CHO-K1 cells were incubated with parental CHO-K1 cells and anti-CLDN6 antibodies at 4°C for 2 hours. The cells were fixed with 2% PFA (Alfa Aesar, catalog number: J61899) at 4°C for 15 minutes and then washed. After incubation at 4°C for one hour, specific antibody binding was detected using Alexa 647-labeled goat anti-human IgG antibody (ThemoFisher Scientific, catalog number: A21445). Flow cytometric analysis was performed on a flow cytometric instrument (Intellicyte, IQue plus, Sartorius).

Figures 2A and 2B show that the anti-claudin-6 antibodies (NR.N6.Ab1 and NR.N6.Ab2) of the present disclosure bind to claudin-6-CHO-K1 transfected cells (GenScript, catalog number U3288DL180_3) at 28-fold and 24-fold MFI, respectively, compared to the isotype control antibody staining at 5ug/ml. The control antibodies NR.N6.PC1 and NR.N6.PC2 bind to claudin-6-CHO-K1 at 25-fold MFI compared to the isotype control antibody. None of the antibodies bind to the parental CHO-K1 cells (Figures 2A and 2B).

The binding specificity of the anti-claudin-6 antibody disclosed in the present invention to claudin-9 was further evaluated by FACS. Briefly, claudin-9-HEK293 cells (GenScript, catalog number U3288DL180_4) were incubated with recombinant claudin-6 antibodies at 4°C for 2 hours. The cells were fixed with 2% PFA (Alfa Aesar, catalog number: J61899) at 4°C for 15 minutes and then washed. After incubation at 4°C for one hour, specific antibody binding was detected using a goat anti-human IgG secondary antibody coupled to Alexa Fluor 647 (ThermoFisher Scientific, catalog number: A21445). Flow cytometry analysis was performed on a flow cytometry instrument (Intellicyte, IQue plus, Sartorius).

Figures 3A and 3B show the binding activity of the disclosed claudin-6 antibodies (NR.N6.Ab1 and NR.N6.Ab2) and positive controls (NR.N6.PC1 and NR.N6.PC2) to claudin-9-HEK293 cells and HEK293 parental cells by FACS (antibody concentration is 5 μg/ml). Compared with the isotype control, the MFI of NR.N6.Ab1 binding to claudin-9-HEK293 cells is 16 times; compared with the isotype control, the MFI of NR.N6.Ab2 binding to claudin-9-HEK293 cells is 53 times. Compared with the isotype control antibody, the MFI of the control antibodies NR.N6.PC1 and NR.N6.PC2 binding to human claudin-9-HEK293 cells is 15 times and 32 times higher, respectively. The binding pattern of NR.N6.Ab1 is similar to that of NR.N6.PC1, and the binding pattern of NR.N6.Ab2 is similar to that of NR.N6.PC2. None of the antibodies tested bound to parental HEK293 cells (Figures 3A and 3B). Previous studies have shown that the amino acid sequence of tight junction protein-6 is highly homologous to tight junction protein-9, 3, and 4 in the extracellular (ECL) loop 1 (ECL-1) and loop 2 (ECL-2) (see Tables 4 and 5, summarizing the percentage identity between the amino acid sequences of the ECL1 and EC2 loops of human CLDN6, 9, 3, and 4) (Biochemical et Biophysica Acta 1778 (2008) 631-645). Therefore, it is very important to evaluate the ability of anti-tight junction protein-6 antibodies to bind to cells expressing these tight junction protein family members.

Table 4 : Percent identity of human CLDN6 to ECL1 (53 aa) of CLDN 9, 3 and 4

CLDN6 CLDN9 CLDN4 CLDN3 CLDN6 98.1% 84.9% 81.1% CLDN9 98.1% 83.0% 79.2% CLDN4 84.9% 83.0% 94.3% CLDN3 81.1% 79.2% 94.3%

Table 5 : Percent identity of human CLDN6 to ECL2 (23aa) of CLDN 9, 3 and 4

CLDN6 CLDN9 CLDN4 CLDN3 CLDN6 91.3% 78.3% 73.9% CLDN9 91.3% 78.3% 65.2% CLDN4 78.3% 78.3% 65.2% CLDN3 73.9% 65.2% 65.2%

To further evaluate the binding characteristics of the anti-CLDN6 antibodies NR.N6.Ab1 and NR.N6.Ab2 of the present disclosure (purified from hybridoma supernatants) and two internal positive controls NR.N6.PC1 and NR.N6.PC2, these antibodies were tested for binding to Claudin-3-CHO-K1 and Claudin-4-CHO-K1 cells. As described above, binding was evaluated by FACS using anti-Claudin-3 antibody (R&D, catalog number: MAB4620) and anti-Claudin-4 antibody (R&D, catalog number: MAB4219) that recognize the native epitope as positive control antibodies for Claudin-3 and 4.

Figures 4A and 4B show that NR.N6.Ab1 binds to cells transfected with tight junction protein-3-CHO-K1, and its MFI is 12 times that of the isotype control. In contrast, the anti-tight junction protein 3 control antibody MAB4620 binds to tight junction protein-3-CHO-K1 cells, and its MFI is 61 times that of the isotype control at a concentration of 5ug/ml. This observation suggests that NR.N6.Ab1 can be characterized as selective for CLDN3, however, in subsequent FACS analysis using MCF7 cells endogenously expressing human CLDN3, no binding of NR.N6.Ab1 was observed. This difference may be attributed to the conformational differences in CLDN6 expression of CHO-K1 transfected cells compared to endogenous expression of human cells. Another anti-tight junction protein-6 antibody NR.N6.Ab2 does not bind to CHO-K1 cells transfected with tight junction protein-3. The two positive control antibodies NR.N6.PC1 and NR.N6.PC2 also did not bind to Claudin-3-CHO-K1 cells.

Figures 5A and 5B show that NR.N6.Ab1 and NR.N6.Ab2 do not bind to Claudin-4-CHO-K1 cells. The positive control anti-Claudin-4 antibody MAB4219 binds to Claudin-4-CHO-K1 with an MFI 33 times that of the isotype control. NR.N6.PC1 does not bind to Claudin-4-CHO-K1 cells, while NR.N6.PC2 binds to Claudin-4-CHO-K1 cells with an MFI 4.5 times that of the isotype control.

Previous studies have determined that NEC8 (testicular germ cell tumor cell line) highly expresses endogenous human tight junction protein-6 and OV90 (ovarian cancer cell line) expresses lower levels of tight junction protein-6. In order to determine whether the anti-tight junction protein 6 antibodies NR.N6.Ab1 and NR.N6.Ab2 of the present disclosure can bind to CLDN6 expressed on NEC8 and OV90 cells, these two antibodies and two positive control antibodies NR.N6.PC1 and NR.N6.PC2 were evaluated by FACS. In brief, NEC8 and OV90 cells were incubated with tight junction protein-6 recombinant antibodies NR.N6.Ab1, NR.N6.Ab2, NR.N6.PC1 and NR.N6.PC2 at 4°C for 2 hours. The cells were fixed with 2% PFA (Alfa Aesar, catalog number: J61899) at 4°C for 15 minutes and then washed. After one hour incubation at 4°C, specific antibody binding was detected using a goat anti-human IgG secondary antibody coupled to Alexa Fluor 647 (Thermo Fisher Scientific, catalog number: A21445). Flow cytometric analysis was performed on a flow cytometric instrument (Intellicyte, IQue plus, Sartorius).

The results of Figures 6A and 6B show that the anti-claudin 6 antibodies NR.N6.Ab1 and NR.N6.Ab2 of the present disclosure can bind to NEC8 cells, and their MFIs are 27 and 25 times that of the isotype control, respectively. The binding activity of the positive control antibodies NR.N6.PC1 and NR.N6.PC2 to NEC8 cells is 19 and 20 times that of the isotype control, respectively.

Figures 7A and 7B show that the anti-claudin-6 antibodies NR.N6.Ab1 and NR.N6.Ab2 of the present disclosure bind to OV90 cells, and their MFIs are 19 and 17 times that of the isotype control antibodies, respectively. Positive control antibodies NR.N6.PC1 and NR.N6.PC2 bind to OV90 cells, and their MFIs are 20 and 15 times that of the isotype control, respectively.

The binding of the disclosed anti-claudin 6 antibodies NR.N6.Ab1 and NR.N6.Ab2 (purified from hybridomas) and two positive control antibodies NR.N6.PC1 and NR.N6.PC2 to the MCF7 cell line (a cell line known to express endogenous claudin-3 and 4, WO 2019/056023) was also evaluated by FACS using anti-claudin-3 (R&D, MAB4620) and anti-claudin-4 (R&D, MAB4219) antibodies as positive control antibodies.

Figures 8A and 8B show that the anti-claudin-6 antibodies NR.N6.Ab1 and NR.N6.Ab2 of the present disclosure do not bind to MCF7 cells, while anti-claudin-3 (MAB4620) and anti-claudin-4 (MAB4620) antibodies bind to claudin-3 and claudin-4 with MFIs of 20 and 15 times that of the isotype control, respectively. Control antibodies NR.N6.PC1 and NR.N6.PC2 also do not bind to MCF7 cells.

To verify the expression levels of Claudin-9 in the three tumor cell lines NEC8, OV90 and MCF7, these three cell lines as well as the Claudin-9-HEK293 cell line were tested by FACS, using an anti-Claudin-9 polyclonal antibody specific for the intracellular C-terminal epitope (Invitrogen, catalog number: PA5-67431) as a positive control, and the FACS operation protocol described above.

Figure 9 shows that, compared with the isotype control antibody, the anti-claudin-9 positive control antibody does not bind to the NEC8 and OV90 cell lines, has a very low binding signal on the MCF7 cell line, while the anti-claudin-9 antibody strongly binds to the claudin-9-HEK293 cells (MFI is 13 times that of the isotype control). These results indicate that the human cell lines NEC8, OV90 and MCF7 do not express claudin-9.

Overall, the results of the above FACS binding experiments show that the antibody NR.N6.Ab1 of the present disclosure binds strongly to CLDN6 and weakly to Claudin-9 compared to the isotype control. NR.N6.Ab1 binds to CLDN3 with a detectable but weak binding signal (20% of the positive control), while the CLDN3 positive control antibody has a higher binding signal (61 times) compared to the isotype control. NR.N6.Ab1 does not bind to CLDN4-CHO-K1 cells and MCF7 cells (expressing CLDN3 and CLDN4). These results indicate that NR.N6.Ab1 selectively binds to Claudin-6.

The data further demonstrated that the anti-CLDN6 antibody NR.N6.Ab2 strongly bound to claudin-6 and 9, but not to claudin-3 or claudin-4.

The binding specificity of the anti-claudin-6 antibodies NR.N6.Ab1 and NR.N6.Ab2 and the relevant positive control (PC) antibodies to the following cells is summarized in Table 6 below: Claudin-6-CHO-K1 cells, Claudin-9-HEK293 cells, Claudin-3-CHO-K1 cells, Claudin-4-CHO-K1 cells, cell lines NEC8 and OV90 endogenously expressing Claudin-6, and cell line MCF7 endogenously expressing Claudin-3 and 4. Binding selectivity was determined by comparing the MFI of the anti-CLDN antibody with the MFI of the isotype control antibody. Note: [-] indicates that no binding was observed compared to the isotype control; n/d indicates no data; entries marked with an asterisk (*) provide data not shown in the figure.

Table 6 : Summary of Anti-CLDN6 Binding Properties

The binding affinity of NR.N6.Ab1 and NR.N6.Ab2 to cell lines overexpressing Claudin-6 was evaluated by FACS. Briefly, NR.N6.Ab1 and NR.N6.Ab2 as well as NR.N6..PC1 and NR.N6.PC2 were serially diluted and tested for binding to HEK293 overexpressing Claudin-6, HEK293 overexpressing Claudin-9, and CHO overexpressing Claudin-6 by FACS as described above.

Figures 10A, 10B and 10C show that NR.N6.Abl and NR.N6.Ab2 bind to the cell lines tested in a dose-dependent manner. The _EC50 values are summarized in Table 7 below.

The results of FACS experiments showed that NR.N6.Ab1 bound to Claudin-6 with high affinity (EC ₅₀ was 0.55 nM on Claudin-6-HEK293 cells and 0.97 _nM on Claudin-6-CHO cells). Compared with the binding to Claudin-6-HEK293 cells, NR.N6.Ab1 had a lower binding affinity to Claudin-9-HEK293 cells (6.72 nM). The binding pattern of NR.N6.Ab1 on these tested cell lines was similar to that of the positive control NR.N6.PC1.

The anti-claudin-6 antibody NR.N6.Ab2 binds to claudin-6 and claudin-9 with similar affinity, with an EC ₅₀ of 1.00 nM on claudin-6-HEK293 cells and an EC ₅₀ of 1.49 nM on claudin-9-HEK293 cells, respectively. It binds to claudin-6-CHO cells with a low nM EC50 (6.88 nM). The binding pattern and affinity on these cell lines are similar to that of the positive control NR.N6.Ab2.

Table 7 : Summary of binding (EC ₅₀ values) of NR.N6.Ab1 and NR.N6.Ab2 on cell lines expressing Claudin-6 and Claudin-9

Example 3: Antibody-dependent cellular cytotoxicity (ADCC) in tumor cells endogenously expressing claudin-6

The ADCC activity of anti-CLDN6 antibodies NR.N6.Ab1 and NR.N6.Ab2 binding to various human claudin-6 positive cells was measured by bioluminescence assay. Briefly, anti-CLDN6 antibodies were serially diluted in assay buffer containing RPMI + 4% low IgG FBS and added to a mixture of a single target cell line and ADCC effector cells. ADCC effector cells are Jurkat cells expressing CD16A, which are activated after recognizing the Fc portion of the bound claudin-6 antibody. The activation of effector cells was detected using the Promega bioluminescence assay according to the manufacturer's instructions (Promega, catalog number: E6130).

ADCC activity was measured on the NEC8 cell line, which has endogenous levels of claudin-6 expression and lacks expression of other claudin family members such as claudin 3, 4, and 9.

As shown in Figure 11A and Table 8, both NR.N6.Ab1 and NR.N6.Ab2 enhanced ADCC activity against NEC8 cells. The EC ₅₀ values of NR.N6.Ab1 and NR.N6.Ab2 were 0.64 nM and 2.77 nM, respectively.

Table 8 : ADCC activity of anti-CLDN6 antibodies in NEC8 cell line

As described in the above examples, tight junction protein-6 is expressed at a low level in OV90 cells. As shown in Figure 11B and Table 9, ADCC activity of NR.N6.Ab1 and NR.N6.Ab2 on OV90 cells was also observed. The EC ₅₀ levels of NR.N6.Ab1 and NR.N6.Ab2 were 0.3 nM and 0.75 nM, respectively, which is comparable to the EC ₅₀ values of 0.28 nM and 0.52 nM of NR.N6.PC1 and NR.N6.PC2.

Table 9 : ADCC activity of anti-human claudin-6 antibodies in OV90 cell line

Example 4: Antibody-mediated endocytosis (ADC)

The endocytosis of the Claudin-6-specific antibodies of the present disclosure binding to Claudin-6-positive cells was measured by a cytotoxicity-based endocytosis assay using co-internalization of the target-bound antibodies with an anti-human IgG Fc-MMAF antibody.

NEC8, OV90 and HEK-Claudin-6 cells were cultured in growth media (RPMI1640 + 10% FBS, MCDB with medium 199 (1: 1) + 15% FBS, DMEM + 10% FBS with 0.5 μg / mL puromycin, respectively). The cells were harvested, resuspended in their respective growth media, and seeded into the assay plate. The cells were incubated overnight at 37 ° C. The anti-CLDN6 antibody was pre-incubated with MMAF-coupled Fab anti-hFc fragment (Moradec, catalog number: AH-202AF-50), then added to the cell plate and incubated for another 96 hours. CellTiter-Glo (Promega, catalog number: G7570) was added to assess cell viability in each well. The signal was quantified using a Neo2 microplate reader (BioTek).

As shown in Table 10 and Figure 12A, NR.N6.Ab1 and NR.N6.Ab2 as well as the internal positive control antibody induced endocytosis-mediated cytotoxicity in NEC8 cells endogenously expressing Claudin-6 with _EC50 values ranging from 0.1 to 0.2 nM.

Table 10 : _EC50 values for antibody-dependent endocytosis on NEC8 cells

OV90 is a cell line that endogenously expresses claudin-6, although at a lower level than NEC8 cells. As shown in Table 11 and Figure 12B, endocytosis-mediated cytotoxicity was similar in all tested antibodies. The EC ₅₀ values for NR.N6.Ab1 and NR.N6.Ab2 were 1.08 nM and 2.32 nM, respectively.

Table 11 : _EC50 values for antibody-dependent endocytosis on OV90 cells

HEK-293 cell lines recombinantly expressing claudin-6 were also used to test endocytosis-mediated cytotoxicity. As shown in Figure 12C and Table 12, NR.N6.Ab1 and NR.N6.Ab2 as well as the internal positive control antibody all induced endocytosis-mediated cytotoxicity. The EC ₅₀ values ranged from 1.73 to 2.19 nM, respectively.

Table 12 : _EC50 values for antibody-dependent endocytosis of CLDN6-HEK293 cells

Example 5: Binding specificity of anti-CLDN6 antibodies

The binding of anti-claudin-6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 to claudin-6-HEK293 (GenScript, catalog number U3288DL180_3) and claudin-9-HEK293 cells (GenScript, catalog number U3288DL180_4) and negative HEK293 (ATCC, CRL1573) cells was evaluated by FACS. Briefly, claudin-6-HEK293, claudin-9-HEK293 and HEK293 cells were incubated with claudin-6 antibodies (NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 recombinant antibodies and NR.N6.Ab6 (purified from hybridoma supernatant)) at 4°C for 2 hours. Cells were fixed with 2% PFA (Alfa Aesar, catalog number: J61899) at 4°C for 15 minutes and then washed. As used herein, the term "recombinant antibody" refers to an antibody engineered to contain a human IgG1 constant region. After incubation at 4°C for one hour, goat anti-human IgG secondary antibodies (ThermoFisher Scientific, catalog number: A21445) coupled to Alexa Fluor 647 were used to detect the specific antibody binding of recombinant antibodies (NR.N6.Ab3 to Ab5), and goat anti-mouse IgG (ThermoFisher Scientific, catalog number: A21235) coupled to Alexa Fluor 647 was used to detect the specific antibody binding of purified antibody NR.N6.Ab6. Flow cytometry analysis was performed on a flow cytometry instrument (Intellicyte, IQue plus, Sartorius).

Figures 13A, 13B, 13C and 13D show that the anti-claudin-6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 of the present disclosure bound to claudin-6-HEK293 transfected cells, and their MFIs were 31 times, 23 times, 24 times and 60 times that of the isotype control antibodies stained at 5 μg/ml (NR.N6.Ab3-5) and 10 μg/ml (NR.N6.Ab6), respectively. These antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 bound to Claudin-9-HEK293 transfected cells with MFIs 5-fold, 2-fold, 2-fold and 47-fold higher than the isotype control antibodies stained at 5 μg/ml (NR.N6.Ab3-5) and 10 μg/ml (NR.N6.Ab6), respectively.

The results showed that the anti-CLDN6 antibody preferentially bound to claudin-6 over claudin-9.

To determine whether the anti-claudin-6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5, NR.N6.Ab6, and NR.N6.Ab1 can bind to CLDN6 expressed on endogenous NEC8 and CLDN6 gene knockout NEC8 (NEC8 claudin-6KO) cells, these antibodies and isotype control antibodies were evaluated by FACS. Briefly, NEC8 and NEC8 claudin-6KO cells were incubated with claudin-6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 (recombinant), NR.N6.Ab6 (purified from hybridoma supernatant), and NR.N6.Ab1 (recombinant) at 4°C for 2 hours. The cells were fixed with 2% PFA (Alfa Aesar, catalog number: J61899) at 4°C for 15 minutes and then washed. After one hour incubation at 4°C, goat anti-human IgG secondary antibody (Thermo Fisher Scientific, catalog number: A21445) coupled to Alexa Fluor 647 was used to detect the specific antibody binding of recombinant antibodies (NR.N6.Ab1 and NR.N6.Ab3 to Ab5), and anti-mouse IgG (Thermo Fisher Scientific, catalog number: A21235) coupled to Alexa Fluor 647 was used to detect the specific antibody binding of NR.N6.Ab6 (purified from hybridoma supernatant). Flow cytometric analysis was performed on a flow cytometric instrument (Intellicyte, IQue plus, Sartorius).

Figures 14A, 14B, 14C, 14D and 14E show that the anti-CLDN6 antibodies NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5, NR.N6.Ab6 and NR.N6.Ab1 of the present disclosure bind to NEC8 cells endogenously expressing claudin-6 with MFIs of 20-fold, 19-fold, 21-fold, 23-fold and 32-fold, respectively. These antibodies do not bind to NEC8 CLDN6 knockout cells compared to isotype control antibodies stained at 5 μg/ml (for NR.N6.A3 to Ab5) or 10 μg/ml (for NR.N6.Ab6). NR.N6.Ab1 shows a similar binding pattern as previously reported in Example 2 (14E).

To further evaluate the binding characteristics of NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 (recombinant antibodies) and NR.N6.Ab6 (purified from hybridoma supernatants), their binding to Claudin-6-CHO-K1, Claudin-3-CHO-K1 and Claudin-4-CHO-K1 cells was tested by FACS as described in Example 2. Anti-claudin antibodies recognize native epitopes (mouse anti-claudin 3 IgG2a (R&D, MAB4620), mouse anti-claudin 4 IgG2a (R&D, MAB4219)). U.S. Patent 2016/0222125A1 antibody was used as a positive control for Claudin 3 and Claudin 4 to confirm the expression levels of Claudin 3 and Claudin 4 on CHO-K1 cells.

Figures 15A, 15B, 15C and 15D demonstrate that NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 bind to Claudin-6-CHO-K1 cells in a dose-dependent manner, but they do not bind to Claudin-3-CHO-K1 and Claudin-4-CHO-K1 cells. Figures 15E and 15F show that positive control antibodies MBA4620 (anti-Claudin-3) and MBA4219 (anti-Claudin-4) bind to Claudin-3-CHO-K1 and Claudin-4-CHO-K1, respectively, in a dose-dependent manner.

The data further demonstrate that NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 bind strongly to Claudin-6 but not to Claudin-3 or Claudin-4, or are characterized by limited binding activity to Claudin-4 (NR.N6.Ab3, NR.N6.Ab6).

Table 13 summarizes the binding properties of NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 and the relevant positive control antibodies to Claudin-6-HEK293 cells, Claudin-6-CHO-K1 cells, Claudin-9-HEK293 cells, Claudin-3-CHO-K1 cells, Claudin-4-CHO-K1 cells, NEC8 cell lines endogenously expressing Claudin-6, and NEC8 cells with the Claudin-6 gene knocked out. Binding selectivity was determined by comparing the MFI of the anti-CLDN6 antibody with the MFI of the isotype control antibody. Note: [-] indicates that no binding was observed compared to the isotype control; * indicates cells with the CLDN6 gene knocked out.

Table 13 : Summary of anti-CLDN6 antibody binding properties

The binding affinity of anti-CLDN6 antibodies to cell lines overexpressing Claudin-6 was also evaluated by FACS. Briefly, NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 (recombinant antibodies) and NR.N6.Ab6 (purified from hybridoma supernatant) were serially diluted and tested by FACS for binding to HEK293 overexpressing Claudin-6, HEK293 overexpressing Claudin-9, CHO overexpressing Claudin-6, and NEC8 endogenously expressing Claudin-6 as described above.

Figures 16A and 16C show that NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab6 bind to Claudin-6-HEK293 cells in a dose-dependent manner, while they only minimally bind (Figure 16B) or weakly bind (16C) to Claudin-9-HEK293 cells. Figures 16A and 16B summarize the binding activity of NR.N6.Ab1.

No significant binding was detected on parental HEK293 cells (Figure 16D). A mouse IgG isotype control was included for NR.N6.Ab6, which did not bind to any cell line (data not shown).

Figure 17A demonstrates that NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 bind to NEC8 cells endogenously expressing Claudin-6 in a dose-dependent manner, and do not bind to NEC8 cells knocked out of Claudin-6 at all (Figure 17B). Figure 17C shows that NR.N6.Ab6 binds to NEC8 cells, but does not bind to NEC8 cells knocked out of Claudin-6.

These results indicate that antibodies NR.N6.Ab3, 4, 5, and 6 of the present disclosure specifically and preferentially bind to Claudin-6.

The EC ₅₀ values (obtained from duplicate experiments) of these anti-Claudin-6 antibodies binding to Claudin-6-HEK293, Claudin-6-CHO-K1 and NEC8 are shown in the following Table 14. Note: # indicates human cell lines that endogenously express CLDN6.

Table 14 : Summary of _EC50 values of Claudin-6 obtained by FACS

The data demonstrate that NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5, and NR.N6.Ab6 bind to cell lines expressing Claudin-6 with _EC50 values ranging from 4.11 nM to 15.67 nM for Claudin-6-HEK293; 3.06 nM to 4.79 nM for Claudin-6-CHO cells; and 3.27 nM to 8.34 nM for NEC8 cells.

Sequenced VH and VL were routinely checked for obvious defects, including N-linked glycosylation sites and added/deleted cysteine residues. As an example, the VH of NR.N6.Ab1 (SEQ ID NO: 1) contains an N-linked glycosylation site in FR3 (N73 counting from the N-terminus). N-linked glycosylation was removed by mutating Asn to Asp under the guidance of the homologous germline sequence, resulting in SEQ ID NO: 23.

As described above, the binding specificity and affinity of NR.N6.Ab1 variant N73D as well as NR.N6.Ab1 and isotype control antibodies to Claudin-6-HEK293, Claudin-9-HEK293, NEC8 cells endogenously expressing Claudin-6, and NEC8 cells with Claudin-6 knocked out were evaluated by FACS.

The EC ₅₀ values of these anti-Claudin-6 antibodies NR.N6.Ab1 N73D and NR.N6.Ab1 for binding to Claudin-6-HEK293 cells and NEC8 cells endogenously expressing Claudin-6 were obtained from duplicate experiments and are summarized in Table 15.

Table 15 : Summary of the binding of NR.N6.Ab1 and NR.N6.Ab1 N73D to cell lines expressing CLDN6 by FACS

Figures 18A, 18B and 18C show that NR.N6.Ab1 and NR.N6.Ab1 variant N73D bind to tight junction protein-6-HEK293 cells (Figure 18A) and NEC8 cells (Figure 18C) in a dose-dependent manner, and NR.N6.Ab1 variant N73D shows similar binding properties to parent NR.N6.Ab1. They bind to tight junction protein-9-HEK293 cells with much lower activity (Figure 18B) and do not bind to NEC8 cells with tight junction protein-6 knocked out (Figure 18D).

Example 6: Antibody-dependent cellular cytotoxicity (ADCC) in tumor cells endogenously expressing claudin-6

ADCC activity of NR.N6.Ab3, NR.N6.Ab4, NR.N6.Ab5 and NR.N6.Ab1 was measured by bioluminescence assay. Briefly, anti-CLDN6 antibodies were serially diluted in assay buffer containing RPMI + 4% low IgG FBS and added to a mixture of a single target cell line and ADCC effector cells. ADCC effector cells are Jurkat cells expressing CD16A, which are activated after recognizing the Fc portion of the bound claudin-6 antibody. The activation of effector cells was detected using the Promega bioluminescence assay according to the manufacturer's instructions (Promega, catalog number: E6130).

ADCC activity was measured on NEC8 and NEC Claudin-6KO (NEC8 cell line with Claudin-6 knockout), which have endogenous levels of Claudin-6 expression and lack the expression of other Claudin family members such as Claudin 3, 4 and 9 (Sahin, U. et al., 2016).

As shown in Figure 19A and Table 16, NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 induced ADCC activity on NEC8 cells with EC ₅₀ values of 6.43nM, 9.83nM and 3.78nM, respectively. NR.N6.Ab1 (as a positive control) always showed ADCC activity with an EC ₅₀ value of 0.40nM, while the previous EC ₅₀ value was 0.64nM (Example 3, Table 8). All EC ₅₀ values were obtained from duplicate experiments. No ADCC activity was detected in NEC8 tight junction protein-6KO cells (Figure 19B).

Table 16: ADCC activity of anti-claudin-6 antibodies on NEC8 cells

Antibody EC50(nM) NR.N6.Ab1 0.40 NR.N6.Ab3 6.43 NR.N6.Ab4 9.83 NR.N6.Ab5 3.78

Example 7: Internalization of anti-CLDN6 antibodies

The internalization of NR.N6.Ab1, NR.N6.Ab3, NR.N6.Ab4 and NR.N6.Ab5 was measured by immunofluorescence imaging assay using NEC8 or NEC8 cells with tight junction protein-6 knocked out. The cells were plated in complete medium containing RPMI-1640 and 10% FBS and then incubated overnight at 37°C. First, Alex Fluor ^™ 488 antibody labeling kit (ThermoFisher, A20181) was used to chemically couple the antibody to a fluorescent dye. Excess uncoupled dyes were removed using Zeba ^™ spin desalting columns, 40KMWCO (ThermoFisher, 87766). The cells were then incubated at 4°C for 4 hours with 10 μg/ml fluorescently labeled antibodies. After antibody pre-binding, the corresponding plates were incubated at 37°C for 0, 4 and 24 hours, and then the cells were fixed at room temperature for 15 minutes with paraformaldehyde. The fixed cells were washed three times with PBS and then incubated with anti-Alexa Fluor 488 antibody (ThermoFisher, A11094) at room temperature for 1 hour to quench the extracellular cell surface signal. The fluorescence signal of the internalized antibody was assessed by imaging the cells and quantifying the fluorescence intensity using a Cytation Imager (Biotek, VT).

Figures 20A and 20B show that the antibodies of the present disclosure are internalized into NEC8 cells, but not into NEC8 Claudin-6 KO cells. When the human IgG1 isotype control antibody was incubated with NEC8 cells or NEC8 Claudin-6 KO cells, no internalization signal was detected. This observation indicates that the internalization of the antibodies of the present disclosure is specific by binding to the Claudin-6 protein on the cell surface.

Example 8: Antibody-mediated endocytosis by anti-CLDN6 antibodies

Internalization of NR.N6.Ab1, NR.N6.Ab3, NR.N6.Ab4, and NR.N6.Ab5 antibodies bound to Claudin-6 positive cells was measured by a cytotoxicity-based endocytosis assay based on co-internalization of target-bound antibodies with anti-human IgG Fc-MMAF antibody.

NEC8 and NEC8 tight junction protein-6 knockout cells were cultured in growth medium (RPMI1640+10% FBS). The cells were harvested, resuspended in growth medium, and seeded into the assay plate. The cells were incubated overnight at 37°C. The anti-tight junction protein-6 antibody was pre-incubated with MMAF-coupled Fab anti-hFc fragment (Moradec, catalog number: AH-202AF-50), then added to the cell plate and incubated for another 72 hours. CellTiter-Glo (Promega, catalog number: G7570) was added to assess cell viability in each well. The signal was quantified using a Neo2 microplate reader (BioTek).

As shown in Figure 21A and Table 17, the anti-Claudin 6 antibodies of the present disclosure induced antibody-mediated endocytosis in the NEC8 cell line with EC ₅₀ values ranging from 0.14 to 0.51 nM. No endocytosis-derived cytotoxicity was detected in the NEC8 Claudin 6 KO cell line (Figure 21B).

Table 17 : Summary of _EC50 values for antibody-dependent endocytosis killing of NEC8 cells

mAbs EC50[nM] NR.N6.Ab1 0.14 NR.N6.Ab3 0.51 NR.N6.Ab4 0.41 NR.N6.Ab5 0.26

Unless otherwise indicated, all numbers used in the specification and claims expressing quantities of ingredients, properties (such as molecular weight, reaction conditions, etc.) should be understood as being modified by the term "about" in any case. Therefore, unless indicated to the contrary, the numerical parameters listed in the specification and the attached claims are approximate values, which may vary depending on the desired properties sought to be obtained by the present disclosure. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0013] While the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

Unless otherwise indicated herein or clearly contradictory to the context, the terms "a", "an", "the" and similar references used in the context of describing the present disclosure (especially in the context of the following claims) should be interpreted as covering both the singular and the plural. The description of the numerical range herein is intended only to be used as a shorthand method for referring to each individual numerical value falling within the range individually. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were quoted individually herein. Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure and does not limit the scope of the present disclosure otherwise claimed. Any language in the specification should not be interpreted as indicating any unclaimed elements essential to the implementation of the present disclosure.

The grouping of the alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. The member of each group can be quoted and claimed separately, or can be used in combination with other members of the group or other elements present herein. It is expected that one or more members in the group may be included in the group or deleted from the group for convenience and/or patentability. When any such inclusion or deletion occurs, the specification is considered to include the group modified, thereby satisfying the written description of all Markush groups used in the appended claims.

Certain embodiments of the present disclosure are described herein, including the best mode known to the inventor for carrying out the present disclosure. Of course, after reading the foregoing description, the changes in the embodiments described will become apparent to those of ordinary skill in the art. The inventor expects that a skilled person will appropriately adopt such variations, and the inventor wishes to practice the present disclosure in a manner different from that specifically described herein. Therefore, the present disclosure includes all modifications and equivalents of the subject matter recorded in the appended claims as permitted by applicable law. Moreover, unless otherwise noted herein or clearly contradictory to the context, the present disclosure encompasses any combination of the above-mentioned elements in all possible variations thereof.

Specific embodiments disclosed herein may be further limited in the claims using "consisting of" or "consisting essentially of" language. When used in the claims, whether as filed or added by amendment, the transitional term "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The transitional term "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those that do not materially affect the basic and novel characteristics. The embodiments of the present disclosure so claimed are inherently or expressly described and used herein.

It should be understood that the embodiments of the present disclosure disclosed herein are illustrations of the principles of the present disclosure. Other modifications that may be adopted are within the scope of the present disclosure. Therefore, by way of example and not limitation, alternative configurations of the present disclosure may be utilized according to the teachings herein. Therefore, the present disclosure is not limited to those precisely shown and described.

Although the disclosure has been described and illustrated herein by reference to various specific materials, methods and embodiments, it should be understood that the disclosure is not limited to the specific combination of materials and methods selected for this purpose. As will be appreciated by those skilled in the art, many variations of these details may be implied. The description and embodiments are intended to be considered as exemplary only, and the true scope and spirit of the disclosure are indicated by the appended claims. All references, patents and patent applications cited in this application are incorporated herein by reference in their entirety.

Sequence Listing

<110> Neostone Biopharmaceutical Co., Ltd.

<120> Antibodies against tight junction protein-6 and their uses

<130> 122863-5006-WO

<150> 63/155,304

<151> 2021-03-02

<150> 63/240,399

<151> 2021-09-03

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Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Cys Val Ile Ala Leu Leu Leu Ala Leu Leu Gly Leu Leu

85 90 95

Val Ala Ile Thr Gly Ala Gln Cys Thr Thr Cys Val Glu Asp Glu Gly

100 105 110

Ala Lys Ala Arg Ile Val Leu Thr Ala Gly Val Ile Leu Leu Leu Ala

115 120 125

Gly Ile Leu Val Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile

130 135 140

Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu

145 150 155 160

Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ala Ala Leu Leu Met Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Pro Pro Gln Val Glu Arg

180 185 190

Pro Arg Gly Pro Arg Leu Gly Tyr Ser Ile Pro Ser Arg Ser Gly Ala

195 200 205

Ser Gly Leu Asp Lys Arg Asp Tyr Val

210 215

<210> 21

<211> 209

<212> PRT

<213> Homo sapiens

<400> 21

Met Ala Ser Met Gly Leu Gln Val Met Gly Ile Ala Leu Ala Val Leu

1 5 10 15

Gly Trp Leu Ala Val Met Leu Cys Cys Ala Leu Pro Met Trp Arg Val

20 25 30

Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser Gln Thr Ile Trp Glu

35 40 45

Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Val Ile Ile Ser Ile Ile Val Ala Ala Leu Gly Val Leu

85 90 95

Leu Ser Val Val Gly Gly Lys Cys Thr Asn Cys Leu Glu Asp Glu Ser

100 105 110

Ala Lys Ala Lys Thr Met Ile Val Ala Gly Val Val Phe Leu Leu Ala

115 120 125

Gly Leu Met Val Ile Val Pro Val Ser Trp Thr Ala His Asn Ile Ile

130 135 140

Gln Asp Phe Tyr Asn Pro Leu Val Ala Ser Gly Gln Lys Arg Glu Met

145 150 155 160

Gly Ala Ser Leu Tyr Val Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Asn Cys Pro Pro Arg Thr Asp Lys Pro

180 185 190

Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Ala Ala Ala Ser Asn Tyr

195 200 205

Val

<210> 22

<211> 220

<212> PRT

<213> Homo sapiens

<400> 22

Met Ser Met Gly Leu Glu Ile Thr Gly Thr Ala Leu Ala Val Leu Gly

1 5 10 15

Trp Leu Gly Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser

20 25 30

Ala Phe Ile Gly Ser Asn Ile Ile Thr Ser Gln Asn Ile Trp Glu Gly

35 40 45

Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys Lys

50 55 60

Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg

65 70 75 80

Ala Leu Ile Val Val Ala Ile Leu Leu Ala Ala Phe Gly Leu Leu Val

85 90 95

Ala Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Asp Thr Ala

100 105 110

Lys Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala

115 120 125

Leu Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg

130 135 140

Asp Phe Tyr Asn Pro Val Val Pro Glu Ala Gln Lys Arg Glu Met Gly

145 150 155 160

Ala Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly

165 170 175

Gly Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Lys Tyr Thr

180 185 190

Ala Thr Lys Val Val Tyr Ser Ala Pro Arg Ser Thr Gly Pro Gly Ala

195 200 205

Ser Leu Gly Thr Gly Tyr Asp Arg Lys Asp Tyr Val

210 215 220

<210> 23

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable heavy chain sequence

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Thr Phe Thr Ser Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Tyr Tyr Asn Tyr Val Trp Gly Ser Phe Arg Phe Asp Asp Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable heavy chain sequence

<400> 24

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ala Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Trp Tyr Ser Ser Gly Trp Ser Met Tyr Asp Val Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 25

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable light chain sequence

<400> 25

Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

Tyr Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr

35 40 45

Leu Ile Tyr Ser Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Val Leu Tyr Met Gly Ser

85 90 95

Gly Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 26

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable heavy chain sequence

<400> 26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Arg Tyr Phe Asp Trp Gly Gly Ser Tyr Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 27

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable light chain sequence

<400> 27

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable heavy chain sequence

<400> 28

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Trp Phe Asp Gly Arg Asn Lys Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Arg Tyr Phe Asp Trp Gly Gly Ser Tyr Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable light chain sequence

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 30

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable heavy chain sequence

<400> 30

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Ser Ile Pro Val Ala Gly Thr Ser Tyr Phe Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 31

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_variable light chain sequence

<400> 31

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 32

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR1

<400> 32

Gly Tyr Gly Met His

1 5

<210> 33

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR2

<400> 33

Ala Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 34

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR3

<400> 34

Glu Trp Tyr Ser Ser Gly Trp Ser Met Tyr Asp Val Phe Asp Ile

1 5 10 15

<210> 35

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR1

<400> 35

Gly Leu Ser Ser Gly Ser Val Ser Thr Ser Tyr Tyr Pro Ser

1 5 10

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR2

<400> 36

Ser Thr Asn Thr Arg Ser Ser

1 5

<210> 37

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR3

<400> 37

Val Leu Tyr Met Gly Ser Gly Ile Trp Val

1 5 10

<210> 38

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR1

<400> 38

Ser Tyr Gly Met His

1 5

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR2

<400> 39

Val Ile Trp Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 40

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR3

<400> 40

Asp Gly Arg Tyr Phe Asp Trp Gly Gly Ser Tyr Tyr Tyr Tyr Tyr Gly

1 5 10 15

Met Asp Val

<210> 41

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR1

<400> 41

Gly Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR2

<400> 42

Asp Thr Ser Ser Arg Ala Thr

1 5

<210> 43

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR3

<400> 43

Gln Gln Tyr Gly Ser Ser Tyr Thr

1 5

<210> 44

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR2

<400> 44

Ile Ile Trp Phe Asp Gly Arg Asn Lys Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 45

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR2

<400> 45

Asp Ala Ser Ser Arg Ala Thr

1 5

<210> 46

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR1

<400> 46

Ser Tyr Tyr Trp Ser

1 5

<210> 47

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR2

<400> 47

Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 48

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_heavy chain variable region containing CDR3

<400> 48

Gly Ser Ile Pro Val Ala Gly Thr Ser Tyr Phe Tyr Tyr Tyr Gly Met

1 5 10 15

Asp Val

<210> 49

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR1

<400> 49

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ser

1 5 10

<210> 50

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR2

<400> 50

Gly Ala Ser Thr Arg Ala Thr

1 5

<210> 51

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence_Light chain variable region containing CDR3

<400> 51

Gln Gln Asp Tyr Asn Leu Pro Trp Thr

1 5

Claims (18)

1. An anti-CLDN6 antibody, comprising: (a) (i) VH: CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 6, CDR3: SEQ ID NO: 7, VL: CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 9, CDR3 :SEQ ID NO:10; (b) VH: CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, CDR3: SEQ ID NO: 13, VL: CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, CDR3: SEQ ID NO:16; (c) VH: CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, CDR3: SEQ ID NO: 34, VL: CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, CDR3: SEQ ID NO:37; (d) VH: CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, CDR3: SEQ ID NO: 40, VL: CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 42, CDR3: SEQ ID NO:43; (e) VH: CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 44, CDR3: SEQ ID NO: 40, VL: CDR1: SEQ ID NO: 41, CDR2: SEQ ID NO: 45, CDR3: SEQ ID NO:43; and (f) VH: CDR1: SEQ ID NO: 46, CDR2: SEQ ID NO: 47, CDR3: SEQ ID NO: 48, VL: CDR1: SEQ ID NO: 49, CDR2: SEQ ID NO: 50, CDR3: SEQ ID NO:51. 2. The anti-CLDN6 antibody of claim 1, wherein the antibody comprises: (a) a heavy chain variable region having the sequence shown in SEQ ID NO: 1 and a light chain variable region having the sequence shown in SEQ ID NO: 2; (b) A heavy chain variable region having the sequence shown in SEQ ID NO:3 and a light chain variable region having the sequence shown in SEQ ID NO:4 (c) a heavy chain variable region having the sequence shown in SEQ ID NO: 23 and a light chain variable region having the sequence shown in SEQ ID NO: 2; (d) a heavy chain variable region having the sequence shown in SEQ ID NO: 24 and a light chain variable region having the sequence shown in SEQ ID NO: 25; (e) a heavy chain variable region having the sequence shown in SEQ ID NO: 26 and a light chain variable region having the sequence shown in SEQ ID NO: 27; (f) a heavy chain variable region having the sequence shown in SEQ ID NO: 28 and a light chain variable region having the sequence shown in SEQ ID NO: 29; or (g) A heavy chain variable region having the sequence shown in SEQ ID NO: 30 and a light chain variable region having the sequence shown in SEQ ID NO: 31. 3. The anti-CLDN6 antibody of claim 1, wherein the antibody is a human antibody. 4. The anti-CLDN6 antibody of claim 1, wherein the antibody is a chimeric antibody. 5. The anti-CLDN6 antibody of any one of claims 1 to 4, wherein the antibody is conjugated to a cytotoxic agent. 6. The anti-CLDN6 antibody of claim 1, wherein the antibody is a full-length antibody. 7. The anti-CLDN6 antibody of claim 1, wherein the antibody is an antibody fragment. 8. The anti-CLDN6 antibody of claim 7, wherein the antibody fragment is selected from the group consisting of: Fab, Fab, F(ab)2, Fd, Fv, scFv and scFv-Fc fragments, single chain antibodies, minibodies and double Antibody. 9. A pharmaceutical composition comprising as an active ingredient at least one antibody according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 10. A pharmaceutical composition comprising as an active ingredient an antibody according to any one of claims 5 and a pharmaceutically acceptable carrier. 11. The pharmaceutical composition according to any one of claims 9 or 10, for use in the treatment of cancer. 12. A method of treating cancer, comprising administering a pharmaceutical composition according to claim 9 or 10 to a subject in need thereof. 13. A method of diagnosing cancer in a subject, the method comprising contacting a biological sample with the antibody or antibody fragment of any one of claims 1 to 2. 14. An isolated polynucleotide comprising a sequence encoding the anti-CLDN6 antibody of claim 1. 15. The isolated polynucleotide of claim 14, encoding a sequence set forth in any one of SEQ ID NOs: 1 to 4. 16. A vector comprising the polynucleotide of claim 15. 17. A host cell comprising the polynucleotide according to claim 15 and/or the vector according to claim 16. 18. A method for producing the anti-CLDN6 antibody of claim 1, comprising culturing the host cell of claim 17.