Anti- CLDN6 antibodies and uses thereof

Abstract

To provide anti- CLDN6 antibodies and anti- CLDN6 and / or anti- CLDN9 antibodies having higher specificity and low toxicities and side effects.SOLUTION: Provided are anti- CLDN6 antibodies or antigen-binding fragments thereof; nucleic acids encoding the same; immunoconjugates, bispecific molecules, chimeric antigens receptors and pharmaceutical compositions comprising the same; and uses thereof for the prevention and / or treatment of tumors.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the fields of disease treatment and immunology. Specifically, the present invention relates to an anti-CLDN6 antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the same, an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, and a pharmaceutical composition comprising the same, as well as to their use for the prevention and / or treatment of tumors. [Background technology]

[0002] Ovarian cancer (OC) is a common gynecological malignancy. It is a highly heterogeneous epithelial tumor with distinct histological subtypes and genetic and biological characteristics, including serous carcinoma, endometrial carcinoma, clear cell sarcoma, and mucinous carcinoma. Globally, 310,000 new cases of ovarian cancer occur annually, resulting in more than 200,000 deaths. Approximately 75% of ovarian cancer cases are serous carcinoma, with a 5-year survival rate of 35%. It is a highly aggressive type of cancer and is usually diagnosed at an advanced stage. This type of tumor exhibits exceptional aggressiveness. Regarding targeted therapy, antiangiogenic therapy has been clinically proven to improve progression-free survival in patients with stage III-IV serous ovarian cancer. However, advanced serous ovarian cancer initially responds to platinum-based chemotherapy, but recurs shortly after the initial response. Primary treatment is surgery in approximately 70-80% of cases. In recent years, research into the pathogenesis and molecular characteristics of ovarian cancer has provided scientific evidence and insights into targeted ovarian cancer therapy. Approximately 90% of ovarian cancers are associated with spontaneous somatic mutations in TP53, and more importantly, BRCA1 and BRCA2 mutations, which cause genetic instability and homozygous deficiency of homologous DNA repair, which are the primary causes of cellular malignancy. These studies provide a rationale for treating ovarian cancer with drugs that target poly (ADP-ribose) polymerase (PARP), although their actual effectiveness has not yet been clinically proven.

[0003] Claudins (CLDNs) are transmembrane proteins located in the tight junctions of epithelial and endothelial cells. The distribution of CLDN family proteins is tissue- and organ-specific, and their main functions are involved in cell-cell adhesion, maintaining cell polarity, controlling extracellular permeability, and regulating cell proliferation and differentiation. Recent studies have shown that some CLDN family members are upregulated during carcinogenesis and ectopically activated in tissues where they are not normally distributed. This characteristic has led scientists to further explore the possibility of CLDN proteins as tumor targets. In 2016, the American Society of Clinical Oncology (ASCO) reported the results of a phase II clinical trial of zolbetui-mab, a monoclonal antibody drug targeting CLDN18.2, and found that the combination of zolbetui-mab with chemotherapy significantly extended overall survival and progression-free survival. The drug is currently undergoing phase III clinical trials for gastric cancer and phase II clinical trials for pancreatic cancer. The success of drugs targeting CLDN18.2 has further increased scientists' confidence in CLDN family members as tumor targets.

[0004] Data from multiple TCGA studies have shown that CLDN6 levels are significantly upregulated in ovarian cancer patients compared with normal ovarian tissue. CLDN6 levels in normal ovarian tissue are 0 TPM (transcripts per million, n=88), whereas CLDN6 levels in ovarian tumors are 31.4 TPM (n=426). CLDN6 is also highly expressed only during embryonic development and is not expressed in normal adult tissues. Among normal adult tissues, the testis shows the highest CLDN6 expression, with an expression level of only 0.83 TPM. In addition to its high expression in ovarian cancer, CLDN6 is also highly expressed in testicular cancer (159.9 TPM, n=137), uterine sarcoma (8.4 TPM, n=57), and some endometrial, gastric, and lung cancers. Studies have shown that high CLDN6 expression in endometrial cancer is associated with multiple clinicopathological factors and is an independent prognostic factor. Kaplan-Meier analysis showed a significant difference in overall survival and recurrence-free survival between the high and low CLDN6 expression groups, with the 5-year survival rate being approximately 30% in the high CLDN6 expression group and 89% in the low CLDN6 expression group. In addition to endometrial cancer, high CLDN6 expression is also negatively correlated with prognosis in gastric cancer and urothelial cancer. The lack of expression in normal tissues, high expression in tumor tissues, and negative correlation with tumor prognosis make CLDN6 an ideal tumor target.

[0005] The CLDN6 protein is a quadruple transmembrane protein with four transmembrane hydrophobic regions and two extracellular loops. This makes recombinant protein expression extremely difficult, and there is no suitable protein antigen for immunization, making immunization and screening for antibodies against CLDN6 challenging. Furthermore, CLDN family proteins share high homology. When targeting CLDN6, it is necessary to avoid binding to CLDN3 and CLDN4, which are widely expressed in normal tissues and share high homology with CLDN6, and to avoid cross-linking, which could lead to toxicity issues. These are two major challenges in developing anti-CLDN6 antibodies.

[0006] CLDN9 is the protein with the highest homology to CLDN6 in the CLDN family. TCGA data show that CLDN9 is expressed at low levels in the pancreas (4.23 TPM, n=4), kidney (1.99 TPM, n=25), and is barely expressed in other normal tissues (the highest expression level is in the bile duct, 0.82 TPM, n=9). At the same time, CLDN9 is upregulated in ovarian cancer (5.68 TPM, n=426) and cholangiocarcinoma (8.44 TPM, n=36).

[0007] Therefore, there is an urgent need and necessity to develop anti-CLDN6 antibodies and / or anti-CLDN6 and / or anti-CLDN9 antibodies with higher specificity and lower toxicity and side effects, which will provide cancer patients with more drug options. Summary of the Invention

[0008] Contents of the invention The antibodies of the present invention can specifically recognize / bind to human CLDN6 and / or CLDN9 and induce the death of cells expressing CLDN6 (e.g., tumor cells) via ADCC and / or CDC. Therefore, the antibodies of the present invention have potential for use in the prevention and / or treatment of tumors and are of great clinical value. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1A shows the binding activity of the anti-CLDN6 mouse antibody 15H2 to different cell surface CLDN proteins: Figure 1A: CHOS-hCLDN6; Figure 1B: CHOS-CLDN3; Figure 1C: CHOS-CLDN4; and Figure 1D: CHOS-CLDN9. [Figure 2] Figure 2 shows the binding activity of the anti-CLDN6 mouse antibody 9H3 to different cell surface CLDN proteins: Figure 2A: CHOS-hCLDN6; Figure 2B: CHOS-CLDN3; Figure 2C: CHOS-CLDN4; and Figure 2D: CHOS-CLDN9. [Figure 3]Figure 3 shows the binding results of humanized antibodies 7008-01 and 7008-03 to four tumor cells that naturally express human CLDN6: Figure 3A: OV90; Figure 3B: NEC8; Figure 3C: NTERA2; Figure 3D: Bewo. [Figure 4] Figure 4 shows the results of antibody-dependent cell-mediated cytotoxicity of effector cells induced by humanized antibodies 7008-01 and 7008-03 against four tumor cell lines that naturally express human CLDN6: Figure 4A: OV90; Figure 4B: NEC8; Figure 4C: NTERA2; Figure 4D: Bewo. [Figure 5] 1 shows the results of the complement-dependent cytotoxicity effect of components derived from humanized antibodies 7008-01 and 7008-03 on NTERA2 tumor cells, which naturally express human CLDN6. [Figure 6] Figure 6A shows the binding results of antibodies with hotspot mutations to different CLDN6-expressing tumor cells. Figure 6A shows the binding results of the 15H2 antibody with hotspot mutations to NEC8; Figure 6B shows the binding results of the 15H2 antibody with hotspot mutations to Bewo; Figure 6C shows the binding results of the 9H3 antibody with hotspot mutations to NEC8; and Figure 6D shows the binding results of the 9H3 antibody with hotspot mutations to Bewo. Specific Description of the Invention

[0010] Antibodies of the Invention Thus, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6, comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 4 or 33, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0011] In certain preferred embodiments, the substitutions according to any one of (i) to (vi) are conservative substitutions.

[0012] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0013] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0014] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO:3, a VH CDR2 having the sequence set forth in SEQ ID NO:4, and a VH CDR3 having the sequence set forth in SEQ ID NO:5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO:6, a VL CDR2 having the sequence set forth in SEQ ID NO:7, and a VL CDR3 having the sequence set forth in SEQ ID NO:8.

[0015] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO:3, a VH CDR2 having the sequence set forth in SEQ ID NO:33, and a VH CDR3 having the sequence set forth in SEQ ID NO:5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO:6, a VL CDR2 having the sequence set forth in SEQ ID NO:7, and a VL CDR3 having the sequence set forth in SEQ ID NO:8.

[0016] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (eg, human immunoglobulin FR sequences).

[0017] In certain preferred embodiments, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences.

[0018] In a particularly preferred embodiment, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Includes:

[0019] In certain preferred embodiments, the substitutions according to any one of (i) to (vi) are conservative substitutions.

[0020] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0021] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0022] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO:3, a VH CDR2 having the sequence set forth in SEQ ID NO:4, and a VH CDR3 having the sequence set forth in SEQ ID NO:5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO:6, a VL CDR2 having the sequence set forth in SEQ ID NO:7, and a VL CDR3 having the sequence set forth in SEQ ID NO:8.

[0023] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (eg, human immunoglobulin FR sequences).

[0024] In certain preferred embodiments, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences.

[0025] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has the following biological functions: (a) binds to human CLDN6 with an EC50 of 5 μg / mL or less (e.g., 3 μg / mL or less); (b) does not bind to human CLDN3, CLDN4, and CLDN9; (c) induce the killing of cells expressing human CLDN6 (e.g., tumor cells (e.g., tumor cells expressing CLDN6)) via antibody-dependent cell-mediated cytotoxicity (ADCC); (d) Inducing the death of cells expressing human CLDN6 (e.g., tumor cells (e.g., tumor cells expressing CLDN6)) via complement-dependent cytotoxicity (CDC). (e) preventing and / or treating a tumor (e.g., a CLDN6-expressing tumor) in a subject; It has one or more of the following.

[0026] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 1; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 1; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:1; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 2; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 2; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:2; Includes:

[0027] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0028] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:1 and a VL having the sequence set forth in SEQ ID NO:2.

[0029] In a particularly preferred embodiment, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 33, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Includes:

[0030] In certain preferred embodiments, the substitutions according to any one of (i) to (vi) are conservative substitutions.

[0031] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0032] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0033] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 3, a VH CDR2 having the sequence set forth in SEQ ID NO: 33, and a VH CDR3 having the sequence set forth in SEQ ID NO: 5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 6, a VL CDR2 having the sequence set forth in SEQ ID NO: 7, and a VL CDR3 having the sequence set forth in SEQ ID NO: 8.

[0034] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (eg, human immunoglobulin FR sequences).

[0035] In certain preferred embodiments, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences.

[0036] In some exemplary embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 19; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 19; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 19; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 20; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 20; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 20; Includes:

[0037] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0038] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:19 and a VL having the sequence set forth in SEQ ID NO:20.

[0039] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6 and / or CLDN9, wherein the antibody or antigen-binding fragment thereof is (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 14 or 23, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 16 or 24, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Includes:

[0040] In certain preferred embodiments, the substitutions according to any one of (i) to (vi) are conservative substitutions.

[0041] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0042] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0043] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 16, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18.

[0044] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 23, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 24, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18.

[0045] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (eg, human immunoglobulin FR sequences).

[0046] In certain preferred embodiments, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences.

[0047] In some preferred embodiments, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6 and / or CLDN9, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 14, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 16, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Includes:

[0048] In certain preferred embodiments, the substitutions according to any one of (i) to (vi) are conservative substitutions.

[0049] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0050] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0051] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 16, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18.

[0052] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (eg, human immunoglobulin FR sequences).

[0053] In certain preferred embodiments, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences.

[0054] In some preferred embodiments, the antibody or antigen-binding fragment thereof has the following biological functions: (a) binds to human CLDN6 with an EC50 of 5 μg / mL or less (e.g., 3 μg / mL or less); (b) does not bind to human CLDN3 and CLDN4; (c) induce the killing of cells expressing human CLDN6 (e.g., tumor cells (e.g., tumor cells expressing CLDN6)) via antibody-dependent cell-mediated cytotoxicity (ADCC); (d) Inducing the death of cells expressing human CLDN6 (e.g., tumor cells (e.g., tumor cells expressing CLDN6)) via complement-dependent cytotoxicity (CDC). (e) preventing and / or treating a tumor (e.g., a CLDN6-expressing tumor) in a subject; It has one or more of the following.

[0055] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 11; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 11; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 11; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 12; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 12; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 12; Includes:

[0056] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0057] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:11 and a VL having the sequence set forth in SEQ ID NO:12.

[0058] In some preferred embodiments, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6 and / or CLDN9, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 23, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 24, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Includes:

[0059] In certain preferred embodiments, the substitutions according to any one of (i) to (vi) are conservative substitutions.

[0060] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0061] In certain preferred embodiments, the CDRs set forth in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0062] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 23, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 24, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18.

[0063] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (eg, human immunoglobulin FR sequences).

[0064] In certain preferred embodiments, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences.

[0065] In some preferred embodiments, the antibody or antigen-binding fragment thereof exhibits one of the following biological functions: (a) binds to human CLDN6 with an EC50 of 5 μg / mL or less (e.g., 3 μg / mL or less); (b) does not bind to human CLDN3 and CLDN4; (c) induce the killing of cells expressing human CLDN6 (e.g., tumor cells (e.g., tumor cells expressing CLDN6)) via antibody-dependent cell-mediated cytotoxicity (ADCC); (d) Inducing the death of cells expressing human CLDN6 (e.g., tumor cells (e.g., tumor cells expressing CLDN6)) via complement-dependent cytotoxicity (CDC). (e) preventing and / or treating a tumor (e.g., a CLDN6-expressing tumor) in a subject; It has one or more of the following.

[0066] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 9; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 9; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:9; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 10; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 10; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 10; Includes:

[0067] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0068] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:10.

[0069] In certain preferred embodiments, the antibody or antigen-binding fragment thereof is humanized.

[0070] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) framework region (FR) derived from a human immunoglobulin, and / or the VH of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) framework region (FR) derived from a human immunoglobulin. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or antigen-binding fragment thereof may comprise one or more non-human (e.g., murine) amino acid residues. For example, the heavy chain framework region FR and / or the light chain framework region FR may comprise one or more amino acid backmutations corresponding to murine amino acid residues.

[0071] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; and (b) a human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to the wild-type sequence from which it is derived; Further includes:

[0072] In certain preferred embodiments, the heavy chain constant region is an IgG heavy chain constant region (eg, an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region).

[0073] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:21.

[0074] In certain preferred embodiments, the light chain constant region is a kappa or lambda light chain constant region.

[0075] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO:22.

[0076] In certain preferred embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, an Fv, a disulfide-linked Fv, a scFv, a diabody, and a single domain antibody (sdAb); and / or the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0077] In the present invention, the antibodies or antigen-binding fragments thereof of the present invention may include variants that differ from the antibody or antigen-binding fragment thereof from which they are derived only in one or more conservative amino acid substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 conservative amino acid substitutions), have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the antibody or antigen-binding fragment thereof from which they are derived, and substantially retain the above-mentioned biological functions of the antibody or antigen-binding fragment thereof from which they are derived.

[0078] Antibody production The antibodies of the present invention can be produced by various methods known in the art (e.g., genetic engineering and recombinant technology). For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, which is then transfected into host cells. The transfected host cells are then cultured under suitable conditions to express the antibodies of the present invention.

[0079] Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Furthermore, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture medium. Other techniques for the production of these antigen-binding fragments are known to those skilled in the art.

[0080] Thus, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof of the present invention. In a particularly preferred embodiment, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof of the present invention.

[0081] In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain preferred embodiments, the vector of the invention is, for example, a plasmid, cosmid, phage, etc. In certain preferred embodiments, the vector is capable of expressing an antibody or antigen-binding fragment of the invention in a subject (e.g., a mammal (e.g., a human)).

[0082] In another aspect, the present invention provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention. Such host cells include, but are not limited to, prokaryotic cells (e.g., E. coli cells), eukaryotic cells (e.g., yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells)). In a particularly preferred embodiment, the host cell of the present invention is a mammalian cell such as a CHO cell (e.g., CHO-K1, CHO-S, CHO DG44).

[0083] In another aspect, a method for producing an antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing a host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cells.

[0084] derivatized antibodies Antibodies or antigen-binding fragments thereof of the present invention can be derivatized (e.g., linked to another molecule (e.g., another polypeptide or protein)). Typically, derivatization (e.g., labeling) of antibodies or antigen-binding fragments thereof does not adversely affect binding to CLDN6 and / or CLDN9 (particularly human CLDN6 and / or human CLDN9). Thus, the antibodies or antigen-binding fragments thereof of the present invention are intended to encompass such derivatized forms. For example, antibodies or antigen-binding fragments thereof of the present invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent bonding, etc.) to one or more other molecular moieties (e.g., other antibodies (e.g., to form bispecific antibodies), detection reagents, pharmaceutical reagents, and / or proteins or polypeptides capable of mediating binding between antibodies or antigen-binding fragments thereof and other molecules (e.g., avidin or polyhistidine tags)). Furthermore, antibodies or antigen-binding fragments thereof of the present invention can be derivatized with chemical groups (e.g., polyethylene glycol (PEG), methyl or ethyl, glycosyl). These groups may be used to improve the biological characteristics of the antibody, eg, increasing serum half-life.

[0085] Thus, in certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are labeled. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin. A detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds), colorimetric markers such as magnetic beads (e.g., Dynabeads®), gold colloids, or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin that binds to the above-mentioned labeled-modified avidin (e.g., streptavidin). Patents that teach the use of such labels include, but are not limited to, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference in their entirety). The detectable labels can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate and detecting the product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. In certain embodiments, such labels may be adapted for use in immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.) In certain embodiments, the detectable labels may be attached to the antibodies or antigen-binding fragments thereof of the invention via linkers of various lengths to reduce potential steric hindrance.

[0086] Dual- or multi-property molecules Antibodies or antigen-binding fragments thereof of the present invention can be used to form bispecific or multispecific molecules. Antibodies or antigen-binding fragments thereof may be part of bispecific or multispecific molecules that contain a second functional module (e.g., a second antibody) that differs in binding specificity from the antibody or antigen-binding fragment thereof of the present invention, thereby enabling binding to at least two different binding sites and / or target molecules. For example, antibodies or antigen-binding fragments thereof of the present invention may be linked to a second antibody or antigen-binding fragment thereof capable of specifically binding to any protein that may be used as a potential target for combination therapy. To establish bispecific or multispecific molecules, antibodies or antigen-binding fragments thereof of the present invention may be linked (e.g., by chemical conjugation, genetic fusion, noncovalent bonding, or other methods) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0087] Thus, in another aspect, there is provided a bispecific or multispecific molecule comprising an antibody or antigen-binding fragment thereof of the invention.

[0088] In certain preferred embodiments, the bispecific or multispecific molecule specifically binds to CLDN6 and also specifically binds to one or more other targets.

[0089] In certain preferred embodiments, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0090] Immunoconjugates The antibodies or antigen-binding fragments thereof of the present invention can be linked to therapeutic agents to form immunoconjugates. The ability of the immunoconjugates to selectively deliver one or more therapeutic agents to target tissues (e.g., tumor-associated antigens, such as tumors expressing CLDN6 and / or CLDN9) can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention in treating diseases (e.g., cancer).

[0091] Thus, in another aspect, the invention provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

[0092] In certain preferred embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0093] In certain preferred embodiments, the therapeutic agent is a cytotoxic agent. In the present invention, a cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells.

[0094] In certain preferred embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof.

[0095] Examples of alkylating agents useful in the immunoconjugates of the invention include, but are not limited to, nitrogen mustards (e.g., mechlorethamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethylenimines (e.g., thiotepa, etc.), sulfate esters and polyols (e.g., busulfan, dibromomannitol, etc.), nitrosoureas (e.g., carmustine, l-bromomannitol, etc.), platinum-based antitumor agents (e.g., cisplatin, oxaliplatin, carboplatin, etc.), and the like.

[0096] Examples of antimitotic agents useful in the immunoconjugates of the invention include, but are not limited to, maytansinoids (e.g., maytansine, maytansinol, C-3 esters of maytansinol, etc.), taxanes (e.g., docetaxel, paclitaxel, or nanoparticulate paclitaxel, etc.), vinca alkaloids (e.g., vindesine sulfate, vincristine, vinblastine, or vinorelbine, etc.).

[0097] Examples of anti-tumor antibiotics useful in the immunoconjugates of the invention include, but are not limited to, actinomycin, anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), calicheamicin, duocarmycin, etc.

[0098] Examples of antimetabolites useful in the immunoconjugates of the invention include, but are not limited to, folate antagonists (e.g., methotrexate, etc.), pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine, cytarabine, capecitabine, gemcitabine, etc.), purine antagonists (6-mercaptopurine, 6-thioguanine, etc.), adenosine deaminase inhibitors (cladribine, fludarabine, nelarabine, pentostatin, etc.).

[0099] Examples of topoisomerase inhibitors useful in the immunoconjugates of the invention include, but are not limited to, camptothecin and its derivatives (e.g., irinotecan, topotecan, etc.), amsacrine, daunomycin, adriamycin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane, teniposide, etc.

[0100] Examples of tyrosine kinase inhibitors useful in the immunoconjugates of the invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, ritotinib, nilotinib, semaxanib, sunitinib, vandetanib, and the like.

[0101] Examples of radionuclide agents useful in the immunoconjugates of the present invention include, but are not limited to: 131 I, 111 In, 90 Y, 177 Lu and others.

[0102] In certain exemplary embodiments, the therapeutic agent is selected from the group consisting of platinum-based anti-cancer agents, anthracycline antibiotics, taxane compounds, nucleoside analogs, camptothecin compounds, and analogs or homologs thereof, and any combination thereof.

[0103] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention are conjugated to a therapeutic agent, optionally via a linker.

[0104] In the present invention, cytotoxic agents can be attached to the antibodies or antigen-binding fragments thereof using linker technology available in the art. Examples of linker types that have been used to attach cytotoxic agents to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. The selected linker is susceptible to cleavage, for example, by the low pH in the lysosomal compartment or by proteases (e.g., proteases preferentially expressed in tumor tissues, such as cathepsins (cathepsins B, C, D, etc.)).

[0105] Further discussion of types of cytotoxic agents, types of linkers, and methods of attaching therapeutic agents to antibodies can also be found in Saito, G. et al (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, PA et al., (2003) Cancer Immunol. Immunother. 52: 328-337; Payne, G. (2003) Cancer Cell 3: 207-212; Allen, TM (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug Deliv. Rev. 53: 247-264.

[0106] Chimeric Antigen Receptor The antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptors (CARs) comprising an extracellular antigen-binding domain (e.g., scFv) capable of specifically binding to CLDN6 and / or CLDN9, linkable to a transmembrane domain, and linkable to one or more intracellular T cell signaling domains. The intracellular T cell signaling domain can comprise, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or a combination thereof. The T cell receptor signaling domain refers to the portion of the CAR comprising the intracellular domain of a T cell receptor (e.g., the intracellular portion of the CD3ζ protein). The costimulatory signaling domain refers to the portion of the CAR comprising the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen.

[0107] A feature of the CARs of the present invention is their ability to induce T cell specificity and responsiveness to cells (e.g., tumor cells) expressing CLDN6 and / or CLDN9 in a non-MHC-restricted manner. The non-MHC-restricted ability to recognize CLDN6 and / or CLDN9 confers antigen recognition ability independent of antigen processing to T cells expressing the CARs of the present invention.

[0108] Thus, in another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of an antibody or antigen-binding fragment thereof of the present invention.

[0109] In certain preferred embodiments, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof of the invention.

[0110] In certain preferred embodiments, the antigen binding domain is an scFv.

[0111] In certain preferred embodiments, the chimeric antigen receptor comprises an antigen-binding fragment (e.g., scFv) of an antibody of the invention.

[0112] In certain preferred embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

[0113] In certain preferred embodiments, a spacer domain comprising a polypeptide sequence may be present between the antigen-binding domain and the transmembrane domain of the CAR. The spacer domain may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, the spacer domain may comprise an immunoglobulin domain (e.g., a human immunoglobulin sequence). In certain exemplary embodiments, the immunoglobulin domain comprises immunoglobulin CH2 and CH3 domain sequences. In such embodiments, without being bound by theory, it is believed that the CH2 and CH3 domains extend the antigen-binding domain of the CAR from the membrane of the CAR-expressing cell, more accurately mimicking the size and domain structure of a native TCR.

[0114] In certain preferred embodiments, the transmembrane domain may be derived from natural or synthetic sources. In such embodiments, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains useful in the CARs of the invention may comprise at least the transmembrane region of the α, β, or ζ chain of a T cell receptor, The receptor may be selected from the group consisting of CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain may be a synthetic domain, in which case it is composed primarily of hydrophobic residues such as leucine and valine.

[0115] In certain exemplary embodiments, the transmembrane domain comprises a transmembrane domain of a T cell receptor (eg, the CD8 transmembrane domain).

[0116] In certain exemplary embodiments, the transmembrane domain comprises the transmembrane domain of a T cell costimulatory molecule (eg, CD137 or CD28).

[0117] In certain preferred embodiments, examples of intracellular T cell domains useful in CARs include the cytoplasmic sequence of the T cell receptor (TCR) and a costimulatory molecule, which cooperate to initiate signaling following engagement with an antigen receptor, and any derivative or variant of these sequences, as well as any synthetic sequence having the same functional capability.

[0118] In certain preferred embodiments, the intracellular region of the CAR may comprise a primary cytoplasmic signal sequence that acts in stimulation, which may include what are known as immunoreceptor tyrosine-based activation motifs or ITAM signal motifs. Examples of ITAMs containing primary cytoplasmic signal sequences that can be included in a CAR include those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, and CD66d proteins.

[0119] In certain preferred embodiments, the intracellular region of the CAR may comprise an ITAM comprising a primary cytoplasmic signaling domain (e.g., CD3ζ) by itself or in combination with any other desired cytoplasmic domain that can be used in the context of a CAR. For example, the cytoplasmic domain of a CAR comprises a CD3ζ chain portion and an intracellular costimulatory signaling domain. A costimulatory signaling domain refers to the portion of a CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, and ICO. These include S, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0120] In certain preferred embodiments, the CAR may comprise a CD3ζ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or any combination thereof. The order of one or more T cell signaling domains on the CAR can be varied by one skilled in the art as needed.

[0121] Methods for establishing chimeric antigen receptors, T cells containing such receptors, and their uses (e.g., for the treatment of cancer) are known in the art, see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4,666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pp 1-9; Till et al., 2008, Blood, 112:2261-2271; Park et al., Trends Biotechnol, 29:550-557, 2011; Grupp et al., NEnglJMed, 368:1509-1518, 2013; Han et al., J.Hematol Oncol., 6:47, 2013; WO 2012 / 079000, WO 2013 / 126726; and U.S. Patent Application Publication No. 2012 / 0213783, all of which are incorporated herein by reference in their entireties. For example, a nucleic acid molecule encoding a chimeric antigen-binding receptor of the present invention can be included in an expression vector (e.g., a lentiviral vector) for expression in a host cell, such as a T cell, to produce a CAR. In certain exemplary embodiments, a method of using a chimeric antigen receptor includes isolating T cells from a subject, transforming the T cells with an expression vector (e.g., a lentiviral vector) encoding the chimeric antigen receptor, and administering the engineered T cells expressing the chimeric antigen receptor to the subject for therapeutic purposes (e.g., to treat a tumor in the subject).

[0122] Thus, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor of the present invention. In a particularly preferred embodiment, the isolated nucleic acid molecule encodes a chimeric antigen receptor of the present invention.

[0123] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule described above. In certain preferred embodiments, the vector of the present invention is, for example, a plasmid.

[0124] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule or vector. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0125] Therapeutic methods and pharmaceutical compositions The antibodies or antigen-binding fragments of the present invention can induce ADCC and / or CDC by binding to CLDN6 and / or CLDN9, thereby killing cells, and can therefore be used in the prevention and / or treatment of tumors.

[0126] Accordingly, in another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and a pharmaceutically acceptable carrier and / or excipient.

[0127] In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0128] In certain preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity (e.g., an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer (e.g., gemcitabine, 5-fluorouracil, taxane, cisplatin, etc.), an anti-angiogenic agent, a cytokine (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), a molecularly targeted drug (e.g., a CD20 antibody such as rituximab, a Her2 antibody such as trastuzumab, a VEGF antibody such as bevacizumab, an EGFR antibody such as cetuximab, etc.), an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, etc.), or an oncolytic virus, etc.).

[0129] In certain preferred embodiments, the pharmaceutical composition provides the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and the additional pharmaceutically active agent as separate components or as components in the same composition. Thus, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately, or sequentially.

[0130] In certain exemplary embodiments, the pharmaceutical composition comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof.

[0131] In another aspect, the present invention provides a method for reducing expression of CLDN6 and / or CLDN9 on the surface of a cell, comprising contacting the cell with an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention, so as to reduce expression of CLDN6 and / or CLDN9 on the surface of the cell; wherein the cell expresses CLDN6 and / or CLDN9 on the cell surface.

[0132] In certain preferred embodiments, the cells are tumor cells that express CLDN6 and / or CLDN9.

[0133] In certain preferred embodiments, the methods are used to reduce the expression of CLDN6 and / or CLDN9 on the surface of cells in vitro for non-diagnostic purposes.

[0134] In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof, or bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition of the invention in the manufacture of a medicament for reducing expression of CLDN6 and / or CLDN9 on the surface of a cell.

[0135] In another aspect, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition of the invention is provided for reducing the expression of CLDN6 and / or CLDN9 on the surface of a cell.

[0136] In another aspect, the present invention provides a method for inhibiting the growth of and / or killing tumor cells that express CLDN6 and / or CLDN9, comprising contacting the tumor cells with an effective amount of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition, chimeric antigen receptor, or a host cell expressing the chimeric antigen receptor (e.g., a chimeric antigen receptor T cell (CAR-T)) of the present invention.

[0137] The methods can be used for therapeutic or non-therapeutic purposes. In some preferred embodiments, the methods can be used for non-therapeutic purposes, in which the methods are used to inhibit the growth of and / or kill tumor cells that express CLDN6 and / or CLDN9 in vitro.

[0138] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or protein, host cell expressing the chimeric antigen receptor (e.g., chimeric antigen receptor T cell (CAR-T)) of the present invention in the manufacture of a medicament for inhibiting the growth of and / or killing tumor cells expressing CLDN6 and / or CLDN9.

[0139] In another aspect, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or proteins expressing said chimeric antigen receptors, host cells (e.g., chimeric antigen receptor T cells (CAR-T)) of the present invention are provided for inhibiting the growth of and / or killing tumor cells expressing CLDN6 and / or CLDN9.

[0140] In another aspect, the present invention provides a method for preventing and / or treating a tumor in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor (e.g., chimeric antigen receptor T cell (CAR-T)) of the present invention.

[0141] In certain preferred embodiments, the tumor refers to tumor cells that express CLDN6 and / or CLDN9. In certain preferred embodiments, CLDN6 and / or CLDN9 are expressed on the surface of tumor cells.

[0142] In certain preferred embodiments, the tumor expresses CLDN6 and / or CLDN9.

[0143] In certain preferred embodiments, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis, or lymph node metastasis).

[0144] In certain preferred embodiments, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor of the present invention (e.g., chimeric antigen receptor T cell (CAR-T)) is used in combination with an additional drug having anti-tumor activity. The additional drug having anti-tumor activity may be administered before, simultaneously with, or after administration of the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor of the present invention (e.g., CAR-T).

[0145] In certain preferred embodiments, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor (e.g., CAR-T) of the present invention may be administered in combination with an additional therapy. The additional therapy may be any known therapy used for tumors (e.g., surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy, or palliative therapy). The additional therapy may be administered before, simultaneously with, or after administration of the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, or chimeric antigen receptor, or host cell expressing the chimeric antigen receptor (e.g., CAR-T) of the present invention.

[0146] The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing the chimeric antigen receptors of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gelling agents, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, and sprays. The preferred dosage form depends on the intended mode of administration and therapeutic application. The pharmaceutical compositions of the present invention are sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injection may be a sterile injectable solution. For example, sterile injectable solutions can be prepared by incorporating the required amount of an antibody of the present invention in an appropriate solvent, optionally along with other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity adjusters, preservatives, diluents, or any combination thereof), followed by filter sterilization. Furthermore, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle (e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer (e.g., phosphate-buffered saline), Ringer's solution, and any combination thereof) prior to use.

[0147] Furthermore, the above-described antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing the chimeric antigen receptors may be presented in pharmaceutical compositions in unit dosage form for ease of administration.

[0148] The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing a chimeric antigen receptor of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., as a powder, ointment, or drops), or nasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). One of skill in the art will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells expressing chimeric antigen receptors (e.g., T cells) of the invention are administered by intravenous injection or bolus injection.

[0149] Pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell (e.g., T cell) expressing a chimeric antigen receptor of the invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, hinder, or delay the onset of disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient (e.g., age, weight, sex), mode of administration, other therapies administered concomitantly, etc.

[0150] In the present invention, dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single dose may be administered, multiple doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0151] In the present invention, the subject may be a mammal such as a human.

[0152] Detection Methods and Kits The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to CLDN6 and / or CLDN9, and can therefore be used to detect the presence or level of CLDN6 and / or CLDN9 in a sample.

[0153] Thus, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label. In certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0154] In the present invention, the detectable label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. It is particularly preferred that such labels are suitable for use in immunological assays (e.g., ELISA, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds), calorimetric markers such as magnetic beads (e.g., Dynabeads®), gold colloids, and colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the above-mentioned labeled-modified avidin (e.g., streptavidin). Patents that teach the use of such labels include, but are not limited to, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference in their entirety). Such detectable labels can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate and detecting the product produced by the action of the enzyme on the substrate, and thermal labels are detected by simply visualizing the colored label. In certain embodiments, the detectable labels may be attached to the antibodies or antigen-binding fragments of the invention via linkers of various lengths to reduce potential steric hindrance.

[0155] In another aspect, the present invention provides a method for detecting the presence or amount of CLDN6 and / or CLDN9 in a sample, the method comprising the steps of: (1) contacting the antibody or antigen-binding fragment thereof of the present invention with the sample; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and CLDN6 and / or CLDN9, or detecting the amount of the complex; The present invention provides a method comprising:

[0156] The formation of the above complex refers to the presence of CLDN6 and / or CLDN9, or cells expressing CLDN6 and / or CLDN9.

[0157] In certain preferred embodiments, the sample is a cell sample (i.e., a sample containing cells (e.g., tumor cells)). In such embodiments, a complex is preferably formed between the antigen, antigen-binding fragment, or conjugate and CLDN6 and / or CLDN9 expressed by cells in the sample.

[0158] In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a detectable label. In another preferred embodiment, in step (2), the antibody or antigen-binding fragment thereof of the present invention is detected using an agent comprising a detectable label.

[0159] The methods can be used for diagnostic or non-diagnostic purposes (e.g., the sample is a cell sample rather than a patient-derived sample). In certain preferred embodiments, the CLDN6 and / or CLDN9 are human CLDN6 and / or CLDN9.

[0160] In another aspect, there is provided use of an antibody or antigen-binding fragment thereof of the present invention in the manufacture of a kit for detecting the presence or amount of CLDN6 and / or CLDN9 (e.g., human CLDN6 and / or human CLDN9) in a sample. In a particularly preferred embodiment, the CLDN6 and / or CLDN9 is human CLDN6 and / or CLDN9.

[0161] In another aspect, the present invention provides a method for determining whether a tumor is treatable by an anti-tumor therapy targeting CLDN6 and / or CLDN9, comprising the steps of: (1) contacting a sample containing tumor cells with the antibody or antigen-binding fragment thereof of the present invention; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and CLDN6 and / or CLDN9; The present invention provides a method comprising:

[0162] In certain preferred embodiments, a complex is formed between the antigen or antigen-binding fragment and CLDN6 and / or CLDN9 expressed by tumor cells in the sample.

[0163] In certain preferred embodiments, the sample is derived from a subject who has, is suspected of having, or is at risk of having a tumor. In certain preferred embodiments, the sample is derived from a tissue or organ whose cells do not substantially express CLDN6 and / or CLDN9 if the tissue or organ is not cancerous. In certain preferred embodiments, the tissue is selected from the group consisting of stomach tissue, lung tissue, esophageal tissue, pancreatic tissue, or breast tissue, and the tissue has optionally been diagnosed as being affected by cancer (e.g., diagnosed by visual inspection or culture of tissue or organ cells). In certain preferred embodiments, the tissue is a tissue other than stomach tissue. In certain preferred embodiments, the tissue is lung tissue, esophageal tissue, pancreatic tissue, or breast tissue. In such an embodiment, the presence of CLDN6 and / or CLDN9, or cells expressing CLDN6 and / or CLDN9, and / or an increase in the amount of CLDN6 and / or CLDN9, or cells expressing CLDN6 and / or CLDN9, compared to a reference level (e.g., compared to a patient without a neoplastic disease) indicates that the subject is suitable for anti-tumor therapy targeting CLDN6 and / or CLDN9.

[0164] In a preferred embodiment, the antibody or antigen-binding fragment thereof further comprises a detectable label. In another preferred embodiment, in step (2), the antibody or antigen-binding fragment thereof of the present invention is detected using an agent comprising a detectable label.

[0165] In certain preferred embodiments, the CLDN6 and / or CLDN9 is human CLDN6 and / or CLDN9.

[0166] In certain preferred embodiments, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastases such as Krukenberg tumors, peritoneal metastases, or lymph node metastases).

[0167] In another embodiment, there is provided use of the antibody or antigen-binding fragment thereof of the present invention in the manufacture of a kit for determining whether a tumor is treatable by an anti-tumor therapy that targets CLDN6 and / or CLDN9.

[0168] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0169] In certain preferred embodiments, the CLDN6 and / or CLDN9 is human CLDN6 and / or CLDN9.

[0170] In certain preferred embodiments, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastases such as Krukenberg tumors, peritoneal metastases, or lymph node metastases).

[0171] Definition of Terms In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operating procedures used herein in cell culture, biochemistry, nucleic acid chemistry, and immunology research are all common procedures widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0172] As used herein, the term "CLDN6 (claudin 6)" has the meaning commonly understood by those skilled in the art. It is a transmembrane protein belonging to the claudin family, present in epithelial and endothelial tight junctions, and may be involved in autophagy. The sequence of CLDN6 is well known in the art and can be found under NCBI database accession number P56747. As used herein, the term "CLDN9 (claudin 9)" has the meaning commonly understood by those skilled in the art. It is a transmembrane protein belonging to the claudin family, present in epithelial and endothelial tight junctions. The sequence of CLDN9 is well known in the art and can be found under NCBI database accession number O95484.

[0173] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (κ) light chains and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected by a "J" region consisting of about 12 or more amino acids, and heavy chains also contain a "D" region consisting of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions are not directly involved in antibody-antigen binding but exhibit various effector functions, such as mediating the interaction of immunoglobulins with host tissues and factors (e.g., binding of various cells of the immune system (e.g., effector cells) to the first component of the classical complement system (C1q)). The VH and VL regions can also be subdivided into highly variable regions (also called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the respective antigen-binding sites. The assignment of amino acids to regions or domains may follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342:878-883.

[0174] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The heavy and light chain variable regions each contain three CDRs, designated CDR1, CDR2, and CDR3, respectively. The exact boundaries of these CDRs can be defined by various numbering systems known in the art (e.g., the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003)). For a given antibody, one of ordinary skill in the art would be able to readily determine the CDRs defined by each numbering system. Additionally, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0175] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention preferably comprises CDRs determined by the Kabat, Chothia, or IMGT numbering system. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention preferably comprises CDRs determined by the IMGT numbering system.

[0176] As used herein, the term "framework region" or "FR" residues refers to amino acid residues of antibody variable regions other than the CDR residues defined above.

[0177] The term "antibody" is not limited to a particular method of antibody production. It includes, for example, recombinant antibodies, monoclonal antibodies, polyclonal antibodies, etc. Antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0178] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody (e.g., a polypeptide that is a fragment of a full-length antibody and that retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen), also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989) (incorporated by reference in its entirety for all purposes). Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (Domantis technology), domain antibodies (Ablynx technology), and such polypeptides that comprise at least a sufficient portion of an antibody to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136. do.

[0179] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains." Here, a "full-length heavy chain" refers to a polypeptide chain comprising, from N- to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, if the full-length antibody is an IgE isotype, optionally a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting, from N- to C-terminus, of a VH, CH1, HR, CH2, and CH3. A "full-length light chain" is a polypeptide chain consisting, from N- to C-terminus, of a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are linked by a disulfide bond between the CL and CH1 of the two full-length heavy chains and a disulfide bond between the HRs. The full-length antibodies of the present invention may be derived from a single species such as human, or may be chimeric or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites, each formed by a pair of VH and VL, which specifically recognize and bind to the same antigen.

[0180] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains, and the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al, Nature 341: 544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond linking the two heavy chain fragments of the F(ab')2 fragment and consisting of an intact light chain and heavy chain Fd fragment (consisting of the VH and CH1 domains).

[0181] As used herein, the term "Fv" refers to an antibody fragment consisting of the separate VL and VH domains of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment capable of forming an intact antigen-binding site. The six CDRs are generally believed to confer antigen-binding specificity to an antibody. However, even variable regions (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind antigen, although possibly with lower affinity than the intact binding site.

[0182] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bond linkage between the second and third constant regions of a first heavy chain and the second and second constant regions of a second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0183] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 and its variants (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448) can be used. Other linkers useful in the present invention are described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may exist between the VH and VL of the scFv.

[0184] As used herein, the term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but which uses a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on the other chain and forming two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448(1993), Poljak RJ et al., Structure 2:1121-1123(1994)).

[0185] As used herein, the term "single domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable antibody domain) that retains the ability to specifically bind to the same antigen as a full-length antibody. Single domain antibodies are also called nanobodies.

[0186] As used herein, the term "probody" has the meaning commonly understood by those skilled in the art, and refers to a masked antibody that remains inactive in healthy tissue but is specifically activated in a disease environment (e.g., by proteolytic cleavage by a protease concentrated in or specific to the disease environment). A detailed description thereof can be found, for example, in Desnoyers et al., Sci. Transl. Med., 5: 207ral44, 2013. Similar masking techniques can be used with any of the antibodies or antigen-binding portions thereof described herein.

[0187] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen.

[0188] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies are screened for specificity in the same manner as intact antibodies.

[0189] Herein, unless the context clearly indicates otherwise, when the term "antibody" is referred to, it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0190] As used herein, the terms "monoclonal antibody," "McAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or antibody fragment obtained from a population of highly homologous antibody molecules, i.e., antibody molecules that are identical except for natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies are generally the counterpart of monoclonal antibodies and generally include at least two or more different antibodies that recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies and should not be construed as requiring a particular method for producing the antibody.

[0191] The monoclonal antibodies of the present invention can be produced by a variety of techniques, including hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Patent Application Publication No. 4,816,567), or bacteriophage antibody library technology (see, e.g., Clackson et al., Nature 352: 624-628, 1991 or Marks et al., J. Mol. Biol. 222: 581-597, 1991).

[0192] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently, or alternatively, a specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and immunospecific antibodies can be purified by immunoaffinity chromatography. For immunoglobulin purification, see, for example, D. Wilkinson (The Scientist, Inc., Philadelphia, Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0193] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light and / or heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and other portions of the light and / or heavy chain are derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which still retains binding activity for a target antigen (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a mouse antibody) and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody) (e.g., a human-mouse chimeric antibody).

[0194] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been altered to increase homology with that of a human antibody. Typically, all or a portion of the CDRs of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable FRs and / or constant regions) are derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the expected properties of the donor antibody (including, but not limited to, antigen specificity, affinity, reactivity, etc.). The donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the expected properties (antigen specificity, affinity, reactivity, etc.).

[0195] In the present application, expected properties of the antibodies of the present invention include: (1) specific recognition / binding to CLDN6 and / or CLDN9 (particularly human CLDN6 and / or human CLDN9); (2) mediation of CLDN6 internalization; (3) induction of death of cells expressing human CLDN6 via antibody-dependent cell-mediated cytotoxicity (ADCC); (4) induction of death of cells expressing CLDN6 via complement-dependent cytotoxicity (CDC); and (5) ability to prevent and / or treat tumors. The antibodies of the present invention have one or more of the above-mentioned desired properties.

[0196] Chimeric or humanized antibodies of the invention can be produced using the sequences of the mouse monoclonal antibodies prepared above. DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and modified to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0197] To produce chimeric antibodies, mouse immunoglobulin variable regions can be linked to human immunoglobulin constant regions by methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly, et al.). For example, VH-encoding DNA is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (e.g., Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, the DNA encoding the VL is operably linked to another DNA molecule encoding the light chain constant region, CL, to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be either a kappa constant region or a lambda constant region, with a kappa constant region generally being preferred.

[0198] To produce humanized antibodies, murine CDRs can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, eds., Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins but are capable of producing a fully human antibody repertoire after immunization can be used. For example, it has been reported that endogenous antibody production can be completely suppressed by homozygously deleting the antibody heavy-chain joining region (JH) gene in chimeric and germline mutant mice, and that subsequent introduction of a human germline immunoglobulin gene array into the germline mutant mice results in the mice producing human antibodies upon antigen stimulation (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of transgenic animals include the HuMAb mouse (Medarex, Inc.), which contains human immunoglobulin gene microlocuses encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, and targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994), Nature 368(6474):856-859); or the "KM mouse™," which has a human heavy chain transgene and a human light chain transchromosome (see WO 02 / 43478).Other methods for humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., Man, 1996, Nature Biotech 14:309).

[0199] As used herein, "germline antibody genes" or "germline antibody gene fragments" refer to immunoglobulin-encoding sequences present in the genome of an organism that have not undergone the mutational maturation process and gene rearrangement that leads to the expression of a specific immunoglobulin. In the present invention, the term "heavy chain germline genes" refers to germline antibody genes or gene fragments encoding immunoglobulin heavy chains, including V (variable), D (diverse), J (joining), and C (constant) genes; similarly, the term "light chain germline genes" refers to germline antibody genes or gene fragments encoding immunoglobulin light chains, consisting of V (variable), J (joining), and C (constant) genes. In the present invention, the amino acid sequences encoded by germline antibody genes or germline antibody gene fragments are also referred to as "germline sequences." Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those of skill in the art and can be obtained or queried from specialized databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0200] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of that interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the stronger the binding between the antibody and the antigen and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one in which the antibody binds to a specific antigen at a dissociation equilibrium constant of about 10 -9 Less than M (e.g., about 10 -9 Under M, 10 -10 M, 10 -11 M, 10 -12 M or less). Specific binding properties between two molecules can be determined using methods well known in the art (e.g., surface plasmon resonance (SPR) in a BIACORE instrument).

[0201] As used herein, the term "cytotoxic agent" includes any agent that is detrimental to (eg, kills) cells, such as chemotherapeutic drugs, bacterial toxins, plant toxins, radioactive isotopes, etc.

[0202] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material element carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-derived artificial chromosomes (PACs); phages such as lambda phage and M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication site.

[0203] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell (such as E. coli or Bacillus subtilis), a fungal cell (such as a yeast cell or Aspergillus), an insect cell (such as an S2 Drosophila cell or Sf9), or an animal cell (such as a fibroblast, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, a human cell, etc.).

[0204] As used herein, the term "identity" refers to the sequence identity between two polypeptides or two nucleic acids. If a position in both compared sequences is occupied by the same base or amino acid monomer subunit (e.g., if each position in two DNA molecules is occupied by adenine, or if each position in two polypeptides is occupied by lysine), the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of identical positions shared by the two sequences divided by the number of compared positions x 100. For example, if two sequences match at 6 out of 10 positions, the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT are 50% identical (3 out of 6 total positions are identical). Typically, comparison is performed when the two sequences are aligned to maximize their identity. Such alignment can be achieved, for example, using the method of Needleman et al., (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). To determine percent identity between two amino acid sequences, the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0) can be used, in which case the PAM120 residue weighting table with a gap length penalty of 12 and a gap penalty of 4 can be used. Additionally, to determine percent identity between two amino acid sequences, the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used, in which case a Blossum62 matrix or a PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 can be used.

[0205] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the intended properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions with amino acid residues having similar side chains (e.g., substitutions with residues that are physically or functionally similar to the corresponding amino acid residue (e.g., have similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.)). Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace corresponding amino acid residues with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al., Protein Eng. 12(10):879-884(1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417(1997), which are incorporated herein by reference).

[0206] The 20 conventional amino acids referred to herein are written according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0207] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered T cell receptor that has an extracellular antibody-derived targeting domain (e.g., scFv) linked to one or more of the intracellular signaling domains of the T cell receptor. In the present invention, the term "chimeric antigen receptor T cell" refers to a T cell that expresses a CAR and has antigen specificity determined by the targeting domain of the CAR. Methods of producing CARs (e.g., for the treatment of cancer) are known in the art, see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., NEnglJMed., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; WO 2012 / 079000, WO 2013 / 059593; and U.S. Patent Application Publication No. 2012 / 0213783, all of which are incorporated herein by reference in their entireties.

[0208] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmaceutically and / or physiologically compatible with the subject and active ingredient. These carriers and / or excipients are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption retarders, preservatives, etc. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants (e.g., Tween-80). Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents (e.g., p-hydroxybenzoic acid esters, chloretone, phenol, sorbic acid, etc.). Osmotic agents include, but are not limited to, sugars, NaCl, etc. Absorption delaying agents include, but are not limited to, monostearate salts, gelatin, etc. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents (e.g., thimerosal, 2-phenoxyethanol, parabens, chloretone, phenol, sorbic acid, etc.).The stabilizer has the meaning commonly understood by those skilled in the art and is capable of stabilizing the desired activity of the active ingredient in the drug, such as sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, glucan, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable fluids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof.

[0209] As used herein, the term "prevention" refers to a method performed to prevent or delay the onset of a disease or disorder or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method performed to obtain beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), slowing or delaying the progression of disease, improvement or remission of the disease state, and alleviation of symptoms (whether partial or total, detectable or undetectable). Furthermore, "treatment" can also mean prolonging survival compared to the expected survival if not receiving treatment.

[0210] As used herein, the term "subject" refers to a mammal, such as a primate mammal (such as a human). In certain embodiments, the subject (e.g., a human) has a tumor (e.g., a tumor that expresses CLDN6 and / or CLDN9) or is at risk of suffering from the above-mentioned disease.

[0211] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients with the disease. Determining such an effective amount is within the capabilities of those skilled in the art. For example, the effective amount in therapeutic applications depends on the severity of the disease to be treated, the general condition of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration of the drug, and other therapies administered at the same time.

[0212] As used herein, the term "immune effector cell" includes cells of hematopoietic origin that function in an immune response (e.g., lymphocytes such as B cells and T cells; natural killer cells; myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes). In certain preferred embodiments, the immune effector cell is a T cell.

[0213] As used herein, the term "metastasis" refers to the spread of cancer cells from their original site to other parts of the body. The formation of metastases is a highly complex process, dependent on the detachment of malignant cells from the primary tumor, their invasion into the extracellular matrix, their penetration through the endothelial basement membrane into body cavities and blood vessels, and their subsequent infiltration into target organs. Finally, the growth of new tumors (i.e., secondary or metastatic tumors) at the target site depends on angiogenesis. Tumor metastasis often occurs even after resection of the primary tumor, as tumor cells or components may persist and develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention refers to "distant metastasis," which refers to metastasis that has spread beyond the primary tumor and the regional lymph node system. The cells of secondary or metastatic tumors are similar to those of the original tumor. For example, if ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells (rather than abnormal liver cells). Liver tumors are referred to as metastatic ovarian cancer (not liver cancer).

[0214] Beneficial Effects of the Invention Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The antibodies of the present invention can specifically recognize / bind to CLDN6 and / or CLDN9 and induce the death of cells expressing CLDN6 (e.g., tumor cells) via ADCC and / or CDC. Therefore, the antibodies of the present invention can potentially be used for the prevention and / or treatment of tumors (particularly tumors expressing CLDN6). The humanized antibodies of the present invention not only retain the functions and properties of the parent antibodies, but are also highly humanized and can be safely administered to human subjects without eliciting an immunogenic response. In particular, the antibodies of the present invention hardly bind to other proteins in the CLDN family (e.g., CLDN3 and CLDN4). Therefore, the antibodies of the present invention (particularly humanized antibodies) have high clinical value.

[0215]

[0023] The present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and do not limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

[0216] Sequence information Information regarding some of the sequences involved in the present invention is provided in the table below.

[0217] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0218] Specific Model for Implementing the Invention The present invention will now be described with reference to the following examples, which are intended to illustrate but not limit the invention.

[0219] Unless otherwise specified, the molecular biological experimental methods and immunoassays used in the present invention were essentially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used according to the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples are provided to illustrate the present invention and are not intended to limit the invention described in the claims. [Example]

[0220] Example 1: Production of anti-CLDN6 mouse antibodies Human CLDN6 (hCLDN6) or mouse CLDN6 (mCLDN6), human CLDN3 (CLDN3), human CLDN4 (CLDN4), or human CLDN9 (CLDN9) were overexpressed in HEK293 cells (ATCC) and CHOS cells (Invitrogen) by lentiviral infection (MOI = 3-10, 5 μg / ml polybrene). Lentivirus was obtained from Shanghai Genechem Co. Ltd. 72 hours after infection, the cells were cultured with the corresponding antibiotic for 2-4 weeks, amplified, and frozen to obtain nine cell lines: HEK293-hCLDN6, HKE293-mCLDN6, HEK293-CLDN3, HEK293-CLDN4, HEK293-CLDN9, CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, and CHOS-CLDN9. These were used in subsequent experiments.

[0221] To generate anti-human CLDN6 antibodies, Balb / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., strain code: 216) were immunized with the constructed CHOS-hCLDN6 cells overexpressing human CLDN6. The adjuvant used for the primary immunization was complete Freund's adjuvant CFA (InvivoGen Company, Art. No. vac-cfa-60); the adjuvant used for all subsequent immunizations was IFA (InvivoGen Company, Art. No. vac-ifa-60); immunizations were administered subcutaneously at multiple sites. After multiple immunizations, spleen cells from the immunized mice were fused with mouse myeloma cells SP2 / 0 using the polyethylene glycol method. B cell fusions expressing antibodies and capable of in vitro proliferation were obtained, and these fusions were cultured in HAT selection medium. The fused hybridoma cells were seeded into 96-well cell culture plates, and the ability of the antibodies in the supernatant to bind to CLDN3 / 4 / 6 / 9 was detected at the cellular level. Positive clones of interest (antibodies capable of binding to CLDN6 but not to CLDN3 / 4 / 9; antibodies capable of simultaneously binding to CLDN6 / 9 but not to CLDN3 / 4) were screened by two to three rounds of subcloning.

[0222] High-throughput screening of mouse antibody binding to cells: For screening, human CLDN3 / 4 / 6 / 9-expressing cells (CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, and CHOS-CLDN9) were individually seeded. 10,000 cells were diluted with 100 μL of complete medium and placed in a flat-bottom 96-well plate. The cells were allowed to adhere to the well walls or sink to the bottom overnight, and the supernatant was discarded the following day. 100 μL of the hybridoma supernatant to be screened was added to the cell plate and incubated at room temperature for 1 hour. After removing the supernatant, 100 μL of secondary antibody (DyLight488 goat anti-mouse IgG (Abcam, Cat. No.: ab97015)) was added to each well at a concentration of 5 μg / mL and incubated at room temperature for 0.5 hours. After staining, the supernatant was discarded and 100 μL of DPBS was added to each well. The plate was then placed in a reader. Experimental plates were measured and read using a full-field cell scanning analyzer (Nexcelom, Model Celigo® Image Cytometer). During measurement, the fluorescent channel corresponding to the secondary antibody and the bright-field channel were simultaneously selected, and high-speed scanning imaging of cells in the wells was performed. In the fluorescent channel, antibody-bound cells were counted according to the set parameters of fluorescently labeled cell morphology and fluorescence intensity. In the bright-field channel, adherent cells were counted according to the set parameters of cell morphology. The two sets of data were then divided to determine the percentage of antibody-bound, fluorescent cells relative to the total cell count. From this percentage, the binding effect of the antibodies in the fusion tumor supernatant on CLDN3 / 4 / 6 / 9-expressing cells was calculated.

[0223] Flow cytometry evaluation of mouse antibody binding to CLDN3 / 4 / 6 / 9: 0.5 million CLDN6-expressing cells (CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, CHOS-CLDN9) were placed in FACS buffer (PBS + 2% FBS) per well, and the mouse antibody to be tested was added and incubated for 1 hour at 4°C. The supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer. The secondary antibody (DyLight488 goat anti-mouse IgG, Abcam, Cat. No.: ab97015) was added and incubated for 0.5 hours at 4°C. After staining, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer. The cells were resuspended in FACS buffer and loaded onto the reader. Experimental cells were measured and read using a flow cytometer (BD, Model Canto II). During the measurement, the cells were first located according to FCS and SSC, and then the fluorescent channels corresponding to the secondary antibody and SSC were selected to analyze the cells.

[0224] The binding results of mouse antibody 15H2 to CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, and CHOS-CLDN9 are shown in Figure 1. As shown in the figure, the isotype control antibody ISO (Beyotime, CatA7028) did not bind to CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, or CHOS-CLDN9 cells, whereas antibody 15H2 could bind to CHOS-hCLDN6 cells but hardly to CHOS-CLDN3, CHOS-CLDN4, or CHOS-CLDN9 cells; the binding results of mouse antibody 9H3 to CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, and CHOS-CLDN9 are shown in Figure 2. As shown in the figure, the isotype control antibody ISO did not bind to CHOS-hCLDN6, CHOS-CLDN3, CHOS-CLDN4, or CHOS-CLDN9 cells, whereas antibody 9H3 was able to bind to CHOS-hCLDN6 and CHOS-CLDN9 cells but showed little binding to CHOS-CLDN3 and CHOS-CLDN4 cells.

[0225] Example 2: Determination of variable region sequences of anti-CLDN6 mouse antibodies Hybridoma cells were collected by centrifugation and diluted to 5–10 × 10 6 For each cell, 1 ml of TRIzol and 0.2 ml of chloroform were added, shaken vigorously for 15 seconds, and incubated at room temperature for 3 minutes. The aqueous phase was then centrifuged to remove the residue. 0.5 ml of isopropanol was added, incubated at room temperature for 10 minutes, and the precipitate was collected, washed with ethanol, and dried to obtain RNA. The template RNA and primers were added to a centrifuge tube cooled in an ice bath. After proper primer-template pairing, reverse transcription was performed and PCR amplification was performed. 2.5 μl of dNTP / ddNTP mixture was added to four microtubes, and the mixture was incubated at 37°C for 5 minutes for further use. 1 pmol of PCR-amplified double-stranded DNA, 10 pmol of sequencing primer, and 2 μl of 5x sequencing buffer were added to an empty microcentrifuge tube, and double-distilled water was added to bring the total volume to 10 μl. The mixture was heated at 96°C for 8 minutes, cooled in an ice bath for 1 minute, and centrifuged at 10,000 x g for 10 seconds at 4°C. 2 μl of pre-chilled labeling mixture (dCTP, dGTP, dTTP, each 0.75 μmol / L) was added, 1 μl of 0.1 mol / L DDT, and 2 U of sequencing enzyme were added, and water was added to a total volume of 15 μl. The mixture was mixed well and placed on ice for 2 minutes to label the newly synthesized DNA strands. 3.5 μl of the labeling reaction mixture was added to four microcentrifuge tubes and incubated at 37°C for 5 minutes. 4 μl of stop solution was then added to each tube. The samples were heat-denatured in an 80°C water bath for 5 minutes, after which 2 μl of the mixture was added to each lane of a sequencing gel. The fragments were separated by electrophoresis and sequence information was recovered.

[0226] The VH and VL sequences of the two mouse antibodies are shown in Table 1. In addition, the CDR sequences of the two mouse monoclonal antibodies were determined according to the IMGT numbering system.

[0227] [Table 2]

[0228] Example 3: Recombinant expression of chimeric anti-CLDN6 antibodies After confirming the antibody gene sequence, the variable region of the mouse antibody was linked to the constant region of a human antibody (human IgG1), and the expression plasmid containing the antibody gene was transfected into mammalian cells. The supernatant (containing the antibody clone) of mammalian cells grown in a culture flask was collected and purified on a Protein A column. The antibody protein was then eluted with 100 mM acetic acid at pH 3.0. The purified antibody protein was loaded onto a size-exclusion chromatography column for further separation and purification. The antibody protein corresponding to the monomer was formulated in PBS buffer supplemented with 20% glycerol.

[0229] Example 4: Humanization of mouse anti-human CLDN6 antibody and hotspot mutations for post-transcriptional modification To increase sequence homology between candidate antibodies and human antibodies and reduce the immunogenicity of the antibodies to humans, the murine antibodies provided in the above examples can be designed and prepared for humanization, and the murine CDR regions can be inserted into human framework sequences using methods known in the art (Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., eds., Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0230] Specifically, the heavy and light chain CDR regions of mouse antibodies 15H2 and 9H3 were constructed on the FR frameworks of the corresponding humanized templates, and a series of back mutations of amino acid residues in the FR regions of the humanized templates were performed so that the humanized antibodies retained as much of the antigen-binding ability of the mouse antibodies as possible.

[0231] At the same time, considering the possible presence of post-translational modification hotspots (PTM hotspots) in the CDR regions of the mouse antibody (e.g., asparagine isomerization hotspot DG in 15H2 HCDR2, asparagine isomerization hotspot DG in 9H3 HCDR2, deamination hotspot NG in 9H3 LCDR1, etc.), these hotspots were mutated by methods known in the art. For example, the D in the asparagine isomerization hotspot DG was mutated to E / S / G or the G in DG was mutated to A / S / D or the N in the deamidation hotspot NG was mutated to Q / S / A or the G in NG was mutated to A / S / D or the like. Experiments have confirmed that most of the mutations described above do not alter the antibody's binding ability to CLDN6-expressing cells (e.g., 15H2 HCDR2 DG mutation to EG / SG / GG; 9H3 LCDR1 NG mutation to QG / NA / SG; 9H3 LCDR1 NG mutation to QG with simultaneous HCDR2 DG mutation to DA), or alter the binding ability by less than threefold (9H3 LCDR1 NG mutation to NA with simultaneous HCDR2 DG mutation to DA; 9H3 LCDR1 NG mutation to SG with simultaneous HCDR2 DG mutation to DA). Figures 6A and 6B show the binding of several 15H2 hotspot mutant antibodies to the CLDN6-expressing tumor cell line NEC8 / Bewo. Figures 6C and 6D show the binding of several 9H3 hotspot mutant antibodies to the CLDN6-expressing tumor cell line NEC8 / Bewo.

[0232] According to the above method, the present inventors produced a series of humanized hot spot mutant antibodies of the mouse antibody 15H2 and the mouse antibody 9H3 and screened them for their ability to bind to CLDN6-expressing cells. As a result, it is preferred that one humanized antibody and one hot spot mutant antibody exist for each mouse antibody, which are designated as 7008-01 (the heavy chain variable region and light chain variable region are set forth in SEQ ID NOs: 19 and 20, respectively) and 7008-03 (the heavy chain variable region and light chain variable region are set forth in SEQ ID NOs: 9 and 10, respectively). For each antibody, the heavy chain constant region is set forth in SEQ ID NO: 21, and the light chain constant region is set forth in SEQ ID NO: 22.

[0233] Example 5: Evaluation of antigen-binding activity of humanized anti-CLDN6 antibodies 500,000 tumor cells naturally expressing CLDN6 (OV90, ATCC; NEC8, JCRB; NTERA2, Nanjing Cobioer Biosciences Co., Ltd.; Bewo, Nanjing Cobioer Biosciences Co., Ltd.) were placed in FACS buffer for subsequent use (round-bottom, low-adsorption 96-well plates were used). Antibody samples were serially diluted in FACS buffer. The diluted antibody was added to the cell plate, and FACS buffer was added to the corresponding negative control wells and incubated at 4°C for 1 hour. After centrifugation, the supernatant was removed and the wells were washed twice with FACS buffer. Secondary antibody (DyLight488 goat anti-human IgG, Abcam, Cat. No.: ab97003) was added to each well and incubated at 4°C for 0.5 hours. After staining, the supernatant was removed by centrifugation, washed twice with FACS buffer, and the cells were resuspended in FACS buffer and loaded into the reader. Cells in the experimental plate were measured and read using a flow cytometer (BD, Model Canto II). During measurement, cells were first positioned according to FCS and SSC, and then analyzed by selecting the fluorescence channel corresponding to the secondary antibody and SSC. Data were analyzed using GraphPad, with the horizontal axis representing the logarithm of antibody concentration and the vertical axis representing the mean fluorescence intensity. The EC50 of the anti-CLDN6 antibody was fitted according to the curve.

[0234] The binding of humanized antibodies 7008-01 and 7008-03 to four tumor cell lines (OV90 / NEC8 / NTERA2 / Bewo) that naturally express human CLDN6 is shown in Figure 3. Table 2 shows the EC50 and maximum binding values ​​(maximum MFI) of the two antibodies that bound to the tumor cells. The results showed that humanized antibodies 7008-01 and 7008-03 exhibited good binding activity to membrane surface CLDN6.

[0235] [Table 3]

[0236] Example 6: Effect of antibody-induced ADCC (antibody-dependent cell-mediated cytotoxicity) Tumor cells naturally expressing CLDN6 (OV90, ATCC; NEC8, JCRB; NTERA2, Nanjing Kebai Bio; Bewo, Nanjing Kebai Bio) were used as target cells, and a Jurkat-NFAT-Luc-CD16 cell line constructed in-house and stably transfected with the CD16 receptor and nuclear factor of activated T-cells (NFAT) response element was used as effector cells. Experiments were performed in 96-well flat-bottom cell plates (Corning 3903). Serially diluted antibodies were added to the target cells and incubated at 37°C for 30 minutes. 60,000 effector cells were added per 10,000 target cells and incubated at 37°C for 4–6 hours. After incubation, One-Glo™ reagent (Promega, E6110) was added for fluorescence development, and the luminescence intensity of the cell plate was measured using a Tecan Spark10 microplate reader. GraphPad was used for data analysis, and the horizontal axis was the logarithm of the antibody concentration, and the vertical axis was the luminescence value of the corresponding well, and the EC50 of the antibody-dependent cell-mediated cytotoxicity of the anti-CLDN6 antibody was calculated by curve fitting.

[0237] Figure 4 shows that humanized antibodies 7008-01 and 7008-03 induced antibody-dependent cell-mediated cytotoxicity of effector cells against four tumor cell lines (OV90 / NEC8 / NTERA2 / Bewo) that naturally express human CLDN6. Table 3 shows the EC50 values ​​and maximum values ​​(max Lum) of the two antibodies in ADCC of different tumor cells. These results demonstrate that both humanized antibodies 7008-01 and 7008-03 can effectively induce effector cells that kill CLDN6-expressing tumor cells.

[0238] [Table 4]

[0239] Example 7: Antibody-induced CDC (complement-dependent cytotoxicity) effect NTERA2 tumor cells, which naturally express CLDN6, were used as target cells. Target cells were incubated with the BATDA fluorescence-enhanced ligand reagent (PerkinElmer, AD0116) from the EuTDA cytotoxicity detection kit at 1 × 10 6 The treated target cells (5 × 10 cells / 1 mL) were mixed in advance and incubated at 37°C for 20 minutes. 3 ) was mixed with various concentrations of antibodies and a 1:8 mixture of human serum (TPCS, A515) as a complement source in a 96-well round-bottom plate (Coring, GLS3799) and incubated at 37°C for 3 hours. After incubation, the supernatant from the 96-well round-bottom plate was transferred to a 96-well flat-bottom plate (Coring, Cat. No. 3903) containing europium solution (PerkinElmer, AD0116) and incubated for an additional 15 minutes at room temperature. The fluorescent donor released into the supernatant after cell death in each well and subsequently transferred to the receptor was excited with energy, and the emitted light from the receptor was measured using a multifunctional microplate reader (excitation: 320 / 340 nm, emission: 615 nm).

[0240] Cytotoxicity is expressed by the following formula:

number

[0241] The complement-dependent cytotoxicity effect of complement induced by humanized antibodies 7008-01 and 7008-03 against NTERA2 tumor cells, which naturally express human CLDN6, is shown in Figure 5. The results showed that both humanized antibodies 7008-01 and 7008-03 could effectively induce complement to kill CLDN6-expressing tumor cells.

[0242] Example 8: CLDN6 internalization mediated by anti-CLDN6 antibodies In this example, anti-CLDN6 antibody-mediated CLDN6 internalization was examined by flow cytometry. To measure the relationship between antibody-induced internalization and time, tumor cells (OV90 / Bewo) that naturally express CLDN6 were incubated with 10 μg / mL of antibody at 37°C / 4°C for different periods of time. After washing several times with PBS containing 2% FBS, the cells were stained with 10 μg / mL of secondary antibody at 4°C for 30 minutes, and intracellular CLDN6 expression was analyzed by flow cytometry.

[0243] MFI37 is the MFI of the sample incubated at 37°C; MFI4 is the MFI of the sample incubated at 4°C, under which conditions only binding occurs, not endocytosis. MFI background was the MFI of the secondary antibody alone. The percentage of antibody-mediated cell surface CLDN6 endocytosis was calculated using the following formula:

number

[0244] The results are shown in Table 4. Humanized antibodies 7008-01 and 7008-03 mediated the internalization of CLDN6 at the tumor cell surface, although to different extents.

[0245] [Table 5]

[0246] Although the specific model for carrying out the present invention has been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the details in accordance with all the teachings disclosed, and all of these modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and their equivalents. <Additional Notes> The present invention includes the following aspects. <Section 1> An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6, (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 4 or 33, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Including, Preferably, the substitution according to any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO:3, a VH CDR2 having the sequence set forth in SEQ ID NO:4, and a VH CDR3 having the sequence set forth in SEQ ID NO:5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO:6, a VL CDR2 having the sequence set forth in SEQ ID NO:7, and a VL CDR3 having the sequence set forth in SEQ ID NO:8; or the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO:3, a VH CDR2 having the sequence set forth in SEQ ID NO:33, and a VH CDR3 having the sequence set forth in SEQ ID NO:5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO:6, a VL CDR2 having the sequence set forth in SEQ ID NO:7, and a VL CDR3 having the sequence set forth in SEQ ID NO:8; Preferably, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (e.g., human immunoglobulin FR sequences); Preferably, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences. An antibody or antigen-binding fragment thereof. <Section 2> (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Including, Preferably, the substitution according to any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO:3, a VH CDR2 having the sequence set forth in SEQ ID NO:4, and a VH CDR3 having the sequence set forth in SEQ ID NO:5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO:6, a VL CDR2 having the sequence set forth in SEQ ID NO:7, and a VL CDR3 having the sequence set forth in SEQ ID NO:8; Preferably, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (e.g., human immunoglobulin FR sequences); Preferably, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences. The antibody or antigen-binding fragment thereof according to item 1. <Section 3> (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 1; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 1; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:1; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 2; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 2; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:2; Including, Preferably, the substitutions according to (ii) or (v) are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 1 and a VL having the sequence set forth in SEQ ID NO: 2. The antibody or antigen-binding fragment thereof according to item 2. <Section 4> (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 33, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Including, Preferably, the substitution according to any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 3, a VH CDR2 having the sequence set forth in SEQ ID NO: 33, and a VH CDR3 having the sequence set forth in SEQ ID NO: 5; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 6, a VL CDR2 having the sequence set forth in SEQ ID NO: 7, and a VL CDR3 having the sequence set forth in SEQ ID NO: 8; Preferably, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (e.g., human immunoglobulin FR sequences); Preferably, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences. The antibody or antigen-binding fragment thereof according to item 1. <Section 5> (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 19; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 19; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 19; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 20; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 20; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 20; Including, Preferably, the substitutions according to (ii) or (v) are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 19 and a VL having the sequence set forth in SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to item 4. <Section 6> An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6 and / or CLDN9, (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 14 or 23, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 16 or 24, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Including, Preferably, the substitution according to any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 16, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18; or the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 23, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 24, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18; Preferably, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (e.g., human immunoglobulin FR sequences); Preferably, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences. An antibody or antigen-binding fragment thereof. <Section 7> (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 14, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 16, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Including, Preferably, the substitution according to any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 16, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18; Preferably, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (e.g., human immunoglobulin FR sequences); Preferably, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences. The antibody or antigen-binding fragment thereof according to item 6. <Section 8> (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 11; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 11; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 11; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 12; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 12; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 12; Including, Preferably, the substitutions according to (ii) or (v) are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 11 and a VL having the sequence set forth in SEQ ID NO: 12. The antibody or antigen-binding fragment thereof according to item 7. <Section 9> (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 23, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 24, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) VL CDR2 consisting of the following sequence: SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; Including, Preferably, the substitution according to any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDRs according to any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 23, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and / or the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 24, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18; Preferably, the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin (e.g., human immunoglobulin FR sequences); Preferably, the human immunoglobulin is selected from the group consisting of human rearranged antibody sequences or human germline antibody sequences. The antibody or antigen-binding fragment thereof according to item 6. <Section 10> (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 9; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 9; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:9; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 10; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 10; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 10; Including, Preferably, the substitutions according to (ii) or (v) are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 9 and a VL having the sequence set forth in SEQ ID NO: 10. The antibody or antigen-binding fragment thereof according to item 9. <Section 11> (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; and (b) a human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to the wild-type sequence from which it is derived; further comprising Preferably, the heavy chain constant region is an IgG heavy chain constant region (e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 21; Preferably, the light chain constant region is a kappa or lambda light chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 22. 11. The antibody or antigen-binding fragment thereof according to any one of items 1 to 10. <Section 12> Item 12. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 11, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb); and / or the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody, or multispecific antibody. <Section 13> Item 13. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 12, wherein the antibody or antigen-binding fragment thereof comprises a label (preferably, the antibody or antigen-binding fragment thereof comprises a detectable label such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin). <Section 14> 14. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13, or the heavy chain variable region and / or light chain variable region thereof. <Section 15> A vector (preferably a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of paragraph 14. <Section 16> A host cell comprising the isolated nucleic acid molecule of paragraph 14 or the vector of paragraph 15. <Section 17> A method for producing the antibody or antigen-binding fragment thereof according to any one of Aspects 1 to 13, comprising culturing the host cell according to Aspect 16 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cells. <Section 18> A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 13, Preferably, the bispecific or multispecific molecule specifically binds to CLDN6 and also specifically binds to one or more other targets; Preferably, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (e.g., a second antibody). Bispecific or multispecific molecules. <Section 19> An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 13 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof, Preferably, said therapeutic agent is from the group consisting of cytotoxic agents; Preferably, the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; Preferably, said immunoconjugate is an antibody drug conjugate (ADC). <Section 20> A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13, the bispecific or multispecific molecule according to Item 18, or the immunoconjugate according to Item 19, and a pharmaceutically acceptable carrier and / or excipient, Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity (e.g., an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus); Preferably, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components in the same composition. Pharmaceutical compositions. <Section 21> A kit comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 13, Preferably, the antibody or antigen-binding fragment thereof comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin; Preferably, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13; Preferably, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. kit. <Section 22> A chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof according to any one of items 1 to 13, Preferably, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13; Preferably, the antigen-binding domain is an scFv; Preferably, the chimeric antigen receptor comprises an antigen-binding fragment of the antibody according to any one of Items 1 to 13; Preferably, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell). Chimeric antigen receptor. <Section 23> 23. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 22. <Section 24> A vector comprising the isolated nucleic acid molecule of item 23, preferably used for producing chimeric antigen receptor T cells. <Section 25> A host cell comprising the isolated nucleic acid molecule of Paragraph 23 or the vector of Paragraph 24, Preferably, the host cell is an immune effector cell (e.g., a T cell or an NK cell); Preferably, the host cell is a chimeric antigen receptor T cell (CAR-T). <Section 26> A method for reducing the expression of CLDN6 and / or CLDN9 on the surface of a cell, comprising contacting the cell with the antibody or antigen-binding fragment thereof according to any one of paragraphs 1 to 13, or the bispecific or multispecific molecule according to paragraph 18, or the immunoconjugate according to paragraph 19, or the pharmaceutical composition according to paragraph 20, or the chimeric antigen receptor according to paragraph 22, or the host cell according to paragraph 25, so as to reduce the expression of CLDN6 and / or CLDN9 on the surface of the cell; wherein the cells express CLDN6 and / or CLDN9 on the cell surface; Preferably, the cells are tumor cells that express CLDN6 and / or CLDN9. method. <Section 27> A method for inhibiting the growth of and / or killing tumor cells that express CLDN6 and / or CLDN9, comprising contacting tumor cells with an effective amount of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13, or the bispecific or multispecific molecule according to Item 18, or the immunoconjugate according to Item 19, or the pharmaceutical composition according to Item 20, or the chimeric antigen receptor according to Item 22, or the host cell according to Item 25. <Section 28> A method for preventing and / or treating a tumor in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof described in any one of paragraphs 1 to 13, or the bispecific or multispecific molecule described in paragraph 18, or the immunoconjugate described in paragraph 19, or the pharmaceutical composition described in paragraph 20, or the chimeric antigen receptor described in paragraph 22, or the host cell described in paragraph 25; Preferably, the tumor expresses CLDN6 and / or CLDN9; Preferably, the tumor is associated with tumor cells that express CLDN6 and / or CLDN9; preferably, CLDN6 and / or CLDN9 are expressed on the surface of the tumor cells; Preferably, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis, or lymph node metastasis); Preferably, the subject is a mammal (e.g., a human); Preferably, the method further comprises administering an additional drug having antitumor activity (e.g., an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus); Preferably, the method further comprises administering an additional anti-tumor therapy (e.g., surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy or palliative therapy), method. <Section 29> Use of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13, or the bispecific or multispecific molecule according to Item 18, or the immunoconjugate according to Item 19, or the pharmaceutical composition according to Item 20, or the chimeric antigen receptor according to Item 22, or the host cell according to Item 25 in the manufacture of a medicament for the prevention and / or treatment of a tumor in a subject (e.g., a human), Preferably, the medicament further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity (e.g., an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus); Preferably, the tumor expresses CLDN6 and / or CLDN9; Preferably, the tumor is associated with tumor cells that express CLDN6 and / or CLDN9; preferably, CLDN6 and / or CLDN9 are expressed on the surface of the tumor cells; Preferably, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis, or lymph node metastasis); Preferably, the subject is a mammal (e.g., a human). use. <Section 30> A method for detecting the presence or amount of CLDN6 and / or CLDN9 (e.g., human CLDN6 and / or human CLDN9) in a sample, comprising the steps of: (1) contacting the antibody or antigen-binding fragment thereof according to any one of items 1 to 13 with the sample; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and CLDN6 and / or CLDN9, or detecting the amount of the complex; Including, Preferably, the antibody or antigen-binding fragment thereof comprises a detectable label; Preferably, the CLDN6 is human CLDN6; Preferably, the CLDN9 is human CLDN9. method. <Section 31> 1. A method for determining whether a tumor is treatable by an anti-tumor therapy targeting CLDN6 and / or CLDN9, comprising the steps of: (1) contacting a sample containing tumor cells with the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and CLDN6 and / or CLDN9; Including, Preferably, the antibody or antigen-binding fragment thereof comprises a detectable label; Preferably, the CLDN6 is human CLDN6; Preferably, the CLDN9 is human CLDN9; Preferably, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis or lymph node metastasis). method. <Section 32> Use of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 13 in the manufacture of a kit for determining whether a tumor is treatable by an antitumor therapy targeting CLDN6 and / or CLDN9, Preferably, the antibody or antigen-binding fragment thereof comprises a detectable label; Preferably, the CLDN6 is human CLDN6; Preferably, the CLDN9 is human CLDN9; Preferably, the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof (e.g., gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis or lymph node metastasis). use.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN6 and / or CLDN9, (1) The following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 16, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18; or (2) the following three heavy chain CDRs: a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 23, and a VH CDR3 having the sequence set forth in SEQ ID NO: 15; and the following three light chain CDRs: a VL CDR1 having the sequence set forth in SEQ ID NO: 24, a VL CDR2 having the sequence set forth in SEQ ID NO: 17, and a VL CDR3 having the sequence set forth in SEQ ID NO: 18; An antibody or antigen-binding fragment thereof comprising:

2. Features include: (1) the antibody or antigen-binding fragment thereof further comprises framework region sequences of human origin; (2) the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin FR sequence; and (3) the antibody or antigen-binding fragment thereof further comprises FR sequences of a human rearranged antibody sequence or a human germline antibody sequence; The antibody or antigen-binding fragment thereof of claim 1, having one or more of the following:

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 11 and a VL having the sequence set forth in SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 9 and a VL having the sequence set forth in SEQ ID NO:

10.

5. A human immunoglobulin heavy chain constant region (CH) or a variant thereof; and a human immunoglobulin light chain constant region (CL) or a variant thereof; further comprising: An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. Features include: (1) the heavy chain constant region is an IgG heavy chain constant region; (2) the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 21; (3) the light chain constant region is a kappa or lambda light chain constant region; (4) The antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 22; (5) The antigen-binding fragment is Fab, Fab', (Fab') ２ , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb); and / or said antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody; and (6) the antibody or antigen-binding fragment thereof comprises a label; The antibody or antigen-binding fragment thereof of claim 5, having one or more of the following:

7. A bispecific or multispecific molecule comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. Features include: (1) The bispecific or multispecific molecule specifically binds to CLDN6 and also specifically binds to one or more other targets; (2) the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity for a second target; and (3) the bispecific or multispecific molecule further comprises a second antibody; 8. The bispecific or multispecific molecule of claim 7, comprising one or more of:

9. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

10. Features include: (1) the therapeutic agent is a cytotoxic agent; (2) the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; and (3) the immunoconjugate is an antibody drug conjugate (ADC); The immunoconjugate of claim 9, having one or more of:

11. A chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

12. Features include: (1) the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof; and (2) the antigen-binding domain is an scFv; The chimeric antigen receptor of claim 11, comprising one or more of:

13. (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or a heavy chain variable region and / or a light chain variable region thereof; or (ii) an isolated nucleic acid molecule encoding a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof.

14. A vector comprising the isolated nucleic acid molecule of claim 13.

15. 14. A host cell comprising the isolated nucleic acid molecule of claim 13 or a vector comprising said isolated nucleic acid molecule.

16. Features include: (1) the host cell is an immune effector cell; (2) the host cell is a T cell or an NK cell; (3) The host cell is a chimeric antigen receptor T cell (CAR-T); The host cell of claim 15, having one or more of the following:

17. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising culturing host cells under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cells.

18. A method for reducing the expression of CLDN6 and / or CLDN9 on the surface of a cell in vitro, comprising: combining the cell with one or more of the following: (1) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. (2) a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof; or (3) an immunoconjugate comprising the antibody or its antigen-binding fragment; (4) A chimeric antigen receptor comprising the antibody or its antigen-binding fragment. (5) A host cell containing an isolated nucleic acid molecule encoding the chimeric antigen receptor, or (6) Any combination thereof contacting the method.

19. The method of claim 18, wherein the cell is a tumor cell that expresses CLDN6 and / or CLDN9.

20. A pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a bispecific or multispecific molecule comprising said antibody or antigen-binding fragment thereof, an immunoconjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule encoding said antibody or antigen-binding fragment thereof, or said chimeric antigen receptor, a vector comprising said antibody or antigen-binding fragment thereof, or said chimeric antigen receptor, or a host cell comprising said antibody or antigen-binding fragment thereof, or said chimeric antigen receptor; and (ii) a pharmaceutically acceptable carrier and / or excipient, the pharmaceutical composition may or may not contain an additional pharmaceutically active agent; Pharmaceutical compositions.

21. Features include: (1) the additional pharmaceutically active agent is a drug with antitumor activity; (2) the additional pharmaceutically active agent is an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus; and (3) the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components in the same composition; 21. The pharmaceutical composition of claim 20, comprising one or more of:

22. 22. A pharmaceutical composition according to claim 20 or 21 for inhibiting the growth of and / or killing tumor cells expressing CLDN6 and / or CLDN9.

23. 22. The pharmaceutical composition according to claim 20 or 21 for the prevention and / or treatment of a tumor in a subject.

24. Features include: (1) the pharmaceutical composition further comprises an additional pharmaceutically active agent; (2) The pharmaceutical composition further comprises a drug having antitumor activity; (3) The pharmaceutical composition further comprises an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus; (4) the tumor is associated with tumor cells that express CLDN6 and / or CLDN9; (5) the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof; (6) The tumor is selected from the group consisting of gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis, and lymph node metastasis; (7) The subject is a mammal; and (8) The subject is a human; 24. The pharmaceutical composition of claim 23, comprising one or more of:

25. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, with or without a second antibody, the antibody or antigen-binding fragment thereof and / or the second antibody may or may not comprise a detectable label; kit.

26. 26. A kit according to claim 25 for detecting the presence or amount of CLDN6 and / or CLDN9 in a sample.

27. 26. The kit of claim 25 for determining whether a tumor is treatable by an antitumor therapy targeting CLDN6 and / or CLDN9.

28. Features include: (1) CLDN6 is human CLDN6; (2) CLDN9 is human CLDN9; (3) the tumor is selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, pancreatic cancer, bronchial cancer, breast cancer, ear, nose and throat (ENT) cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof; and (4) The tumor is selected from the group consisting of gastric cancer metastasis, Krukenberg tumor, peritoneal metastasis, and lymph node metastasis; 28. The kit of claim 27, comprising one or more of:

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