Cancer treatment methods

Abstract

This specification describes a method for inhibiting solid tumors expressing claudin-6 in human subjects, comprising administering to a human subject an effective amount of a composition comprising a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous portion having the following structural formula (I): [Formula 1] JPEG2026518780000042.jpg54164In formula, The first group of conjugates is bonded to four heterogeneous parts, including structural formula (I); At least about 95% of the first multiple conjugates are structurally homogeneous; and This method involves determining an effective dose within the range of approximately 1.0 mg of conjugate per kg of target weight to approximately 10 mg of conjugate per kg of target weight.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 468,817, filed on 25 May 2023, the entire contents of which are incorporated herein by this reference.

[0002] Reference to electronic sequence listings This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The above XML copy, created on 23 May 2024, is named "UCL-00525.xml" and has a size of 839,728 bytes. The sequence listing contained in this XML file is part of this specification, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Claudine-6

[0004] Claudin-6 (CLDN6) is a member of the claudin family, consisting of 27 four-transmembrane domain proteins located in tight junctions between epithelial cells, which play a crucial role in barrier function. While CLDN6 expression in non-cancerous tissues is limited to tissues involved in the early stages of development, it has been reported to be abnormally expressed in various cancer types, including ovarian cancer, gastric cancer, embryonic cancer, pediatric cancer, and endometrial cancer.

[0005] CLDN6 is expressed in the majority of ovarian, endometrial, and testicular cancers, as well as in a subset of non-small cell lung cancers using The Cancer Genome Atlas (TCGA) dataset. Analysis of cancer cell lines revealed CLDN6-positive cell lines in ovarian, lung, endometrial, and bladder cancers. Significant expression of CLDN6 was not detected in normal tissues.

[0006] Ovarian cancer

[0007] Ovarian cancer accounts for a higher mortality rate than any other cancer of the female reproductive system. The overall 5-year relative survival rate is 49.1%.

[0008] Generally, ovarian cancer is also referred to as fallopian tube cancer and abdominal cancer, as these are closely related and treated similarly. The most common type of ovarian cancer is epithelial carcinoma, which accounts for 85% to 90% of all ovarian cancers. Rare forms of ovarian cancer include germ cell malignancies and sex cord-stromal tumors.

[0009] The majority of women are diagnosed with advanced ovarian cancer, and about 80% of these patients experience tumor progression or recurrence after initial treatment. Ovarian cancer that recurs within 6 months of platinum-based chemotherapy is classified as platinum-resistant, while ovarian cancer that recurs more than 6 months after platinum-based chemotherapy is platinum-sensitive. Patients with platinum-resistant ovarian cancer typically receive additional monotherapy such as pegylated liposomal doxorubicin (PLD), topotecan, gemcitabine, and weekly paclitaxel, but the objective response rate (ORR) to these drugs is low (10-15%) and the duration of response (DOR) is short (4 months).

[0010] In addition to monotherapy, bevacizumab in combination with chemotherapy and poly(adenosine diphosphate [ADP]-ribose polymerase (PARP) inhibitors) is being studied for the treatment of advanced ovarian cancer. However, these treatments also have problems. While clinical trials have shown an increase in progression-free survival (PFS) with bevacizumab, it is unclear whether this PFS is clinically significant and whether it leads to an improvement in quality of life or overall survival (OS). Bevacizumab has also raised concerns regarding gastrointestinal (GI) toxicity. PARP inhibitors such as olaparib and niraparib are administered as maintenance therapy in patients with BRCA mutations, which represents a small proportion of patients (about 17% of patients in the hyperserous subset of ovarian cancer).

[0011] Patients with ovarian cancer ultimately experience disease progression despite all available treatments, necessitating additional therapies to extend survival. Platinum-resistant advanced ovarian cancer remains an area of ​​unmet need.

[0012] non-small cell lung cancer

[0013] Lung cancer is the second most common cancer, accounting for nearly 25% of all cancer deaths, making it by far the leading cause of cancer death. The 5-year relative survival rate is 21.7%. Smoking is the most important risk factor, accounting for 85%–90% of lung cancer cases. However, the risk of developing lung cancer is associated with the degree of smoking and the level of exposure to other carcinogens such as asbestos.

[0014] Lung cancer consists of two distinct types: non-small cell lung cancer (NSCLC) and small cell lung cancer. NSCLC accounts for the majority of cases, approximately 85% of patients. Different classifications of NSCLC include adenocarcinoma (approximately 40% of lung cancers), squamous cell carcinoma (25-30% of lung cancers), large cell carcinoma (5-10% of lung cancers), and non-small cell lung cancer that cannot be histologically identified (NSCLC - not otherwise specified) (NOS).

[0015] Systemic therapy for advanced NSCLC is selected according to the presence of specific biomarkers. Molecular changes that predict response to treatment (e.g., epidermal growth factor receptor [EGFR] mutations, anaplastic lymphoma kinase [ALK] rearrangements, ROS1 rearrangements, and BRAF V600E mutations) are present in approximately 30% of NSCLC patients. Targeted therapies for these changes improve progression-free survival compared to cytotoxic chemotherapy. Recently, a KRAS G12C inhibitor has shown antitumor activity, and sotrasib has been approved. In patients without biomarkers indicating sensitivity to specific targeted therapies, regimens containing immune checkpoint inhibitors, either as monotherapy or in combination with chemotherapy, are superior to chemotherapy alone.

[0016] Despite numerous advances in NSCLC over the past decade through targeted and immunotherapies, resistance remains common, and the development of new treatments is needed.

[0017] CLDN6-targeted therapies are needed to treat ovarian cancer and other cancers such as NSCLC in humans. [Overview of the project]

[0018] In certain embodiments, the present disclosure provides a method for inhibiting a solid tumor expressing claudin-6 in a human subject, comprising administering to a human subject an effective amount of a composition comprising a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous moiety having the following structural formula (I): [ka] During the ceremony, The first group of conjugates is bonded to approximately four heterogeneous parts, including structural formula (I); At least about 95% of the first multiple conjugates are structurally homogeneous; and The effective amount is approximately 1.7 mg / kg to 6 mg / kg; 2.0 mg / kg to 6 mg / kg; or This concerns the method, which falls within the range of 2.4 mg / kg to 6 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, and the CLDN6-specific antigen-binding protein comprises an antibody or antigen-binding fragment thereof that binds to CLDN6, and this antibody comprises: (i) HC CDR1 having sequence GFTFSNYW (sequence number 23); (ii) HC CDR2 having sequence IRLKSDNYAT (sequence number 24), (iii) HC CDR3 having sequence XDGPPSGX (sequence number 457) (wherein the formula, the X at position 1 is N and the X at position 8 is S, T, A, C, or Y), (iv) LC CDR1 having sequence ENIYSY (sequence number 20), (v) LC CDR2 having sequence NAK (sequence number 21), and (vi) LC CDR3 having sequence QHHYTVPWT (sequence number 22). [Brief explanation of the drawing]

[0019] [Figure 1A] This bar graph shows the tumor response (maximum change in tumor size according to RECIST 1.1) at dose levels of TORL-1-23 ranging from 0.2 to 2.4 mg / kg in humans. [Figure 1B] This shows TORL-1-23 at human dose levels of 0.2–3.0 mg / kg. [Figure 2A] This graph shows the change in tumor size over time in humans at dose levels of 0.2–2.4 mg / kg of TORL-1-23. [Figure 2B] This shows the tumor size over time in humans at dose levels of TORL-1-23 ranging from 0.2 to 3.0 mg / kg. [Figure 3] These are a series of line graphs evaluating the antitumor activity of TORL-1-23 administered intravenously once a week at a dose of 2.5 or 5.0 mg / kg in mouse xenograft models of human cancers, specifically CLDN6-positive ovarian cancer (OV90), bladder cancer (UMUC4), or endometrial cancer (ARK2), or CLDN6-negative melanoma (M202). [Figure 4A] Figures 4A and 4A (continuation) are a series of line graphs showing the mean plasma concentrations of total TORL-1-23 antibody (conjugated and unconjugated to the four MMAEs), TORL-1-23 (containing the four MMAEs), and unconjugated (or free) MMAEs after TORL-1-23 administration every three weeks at doses of 0.2, 0.4, 0.8, 1.0, 1.3, 1.7, 2.0, and 2.4 mg / kg in Cycle 1. [Figure 4B]Figures 4B and its continuation are a series of line graphs showing the mean plasma concentrations of total TORL-1-23 antibody (conjugated and unconjugated to the four MMAEs), TORL-1-23 (containing the four MMAEs), and unconjugated (or free) MMAEs after TORL-1-23 administration every three weeks at doses of 0.2, 0.4, 0.8, 1.0, 1.3, 1.7, 2.0, 2.4, and 3.0 mg / kg in cycles 1 and 3. [Figure 5] This line graph shows dose-normalized unconjugated MMAE concentrations after a single dose of TORL-1-23 (shaded circles) at 0.2, 0.4, 0.8, 1.0, 1.3, 1.7, 2.0, and 2.4 mg / kg, or after a single dose (shaded symbols) of 2.4 mg / kg for eight other major ADCs, ADC1-ADC8, which have undergone Phase 1 clinical trials. [Modes for carrying out the invention]

[0020] overview

[0021] This specification describes a method for inhibiting solid tumors expressing claudin-6 in human subjects, comprising administering to a human subject an effective amount of a composition comprising a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous portion having the following structural formula (I): [ka] During the ceremony, The first group of conjugates is bonded to approximately four heterogeneous parts, including structural formula (I); At least 95% of the first multiple conjugates are structurally homogeneous; and This method involves determining an effective dose within the range of approximately 1.0 mg of conjugate per kg of target weight to approximately 10 mg of conjugate per kg of target weight.

[0022] As used herein, the term “approximately” when used before a numerical expression that includes a range, such as temperature, time, quantity, or concentration, indicates an approximation that may vary by plus or minus (+ / -) 5%, 4%, 3%, 2%, or 1%.

[0023] In certain embodiments, the solid tumor is ovarian cancer, primary abdominal cancer, fallopian tube cancer, non-small cell lung cancer, or testicular cancer.

[0024] In certain embodiments, after administration, human subjects do not experience peripheral neuropathy of grade 3 or higher severity, alopecia of grade 3 or higher severity, fatigue of grade 3 or higher severity, nausea, vomiting, or loss of appetite of grade 3 or higher severity, or constipation of grade 3 or higher severity, or any combination of two or more of these adverse events. In certain embodiments, after administration, human subjects do not experience peripheral neuropathy of grade 3 or higher severity. In certain embodiments, after administration, human subjects do not experience alopecia of grade 3 or higher severity. In certain embodiments, after administration, human subjects do not experience fatigue of grade 3 or higher severity. In certain embodiments, after administration, human subjects do not experience nausea, vomiting, or loss of appetite of grade 3 or higher severity. In certain embodiments, after administration, human subjects do not experience constipation of grade 3 or higher severity. For the purposes of this disclosure, widely accepted criteria for documenting and classifying adverse events (i.e., the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0) are used. For example, a "Grade 3" adverse event may be a serious or medically significant adverse event that is not immediately life-threatening but requires hospitalization. A Grade 3 adverse event may limit a patient's ability to bathe, dress, eat, defecate, or take medication independently.

[0025] In certain embodiments, after administration, less than 30% of treated patients experience peripheral neuropathy of treatment-related grade 3 or higher severity, less than 30% experience alopecia of treatment-related grade 3 or higher severity, less than 30% experience nausea, vomiting, or loss of appetite of treatment-related grade 3 or higher severity, or any combination of two or more of these treatment-related adverse events, or less than 30% experience these adverse events. Preferably, after administration, less than 20% of treated patients experience peripheral neuropathy of treatment-related grade 3 or higher severity, less than 20% experience alopecia of treatment-related grade 3 or higher severity, less than 20% experience nausea, vomiting, or loss of appetite of treatment-related grade 3 or higher severity, or less than 20% experience any combination of two or more of these treatment-related adverse events, or less than 20% experience these adverse events. More preferably, after administration, less than 10% of treated patients experience peripheral neuropathy of treatment-related grade 3 or higher severity, less than 10% of treated patients experience alopecia of treatment-related grade 3 or higher severity, less than 10% of treated patients experience nausea, vomiting, or loss of appetite of treatment-related grade 3 or higher severity, or any combination of two or more of these treatment-related adverse events, or less than 10% of treated patients experience adverse events of grade 3 or higher severity. In certain embodiments, no human subjects experienced adverse events of grade 3 or higher severity after administration.

[0026] In certain embodiments, human subjects do not experience any adverse events of grade 3 or higher severity after administration.

[0027] In certain embodiments, after administration, human subjects do not experience peripheral neuropathy of grade 2 or higher severity, alopecia of grade 2 or higher severity, fatigue of grade 2 or higher severity, nausea, vomiting, or loss of appetite of grade 2 or higher severity, or constipation of grade 2 or higher severity, or any combination of two or more of these adverse events. In certain embodiments, after administration, human subjects do not experience peripheral neuropathy of grade 2 or higher severity. In certain embodiments, after administration, human subjects do not experience alopecia of grade 2 or higher severity. In certain embodiments, after administration, human subjects do not experience fatigue of grade 2 or higher severity. In certain embodiments, after administration, human subjects do not experience nausea, vomiting, or loss of appetite of grade 2 or higher severity. In certain embodiments, after administration, human subjects do not experience constipation of grade 2 or higher severity. For the purposes of this disclosure, widely accepted criteria for documenting and classifying adverse events (i.e., the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0) are used. For example, a "Grade 2" adverse event is a moderately severe adverse event that limits an age-appropriate instrumental activity of daily life, such as preparing meals, grocery shopping or clothing shopping, using a telephone, or managing money. Patients experiencing a Grade 2 adverse event may be directed to minimal, local, or non-invasive interventions.

[0028] In certain embodiments, after administration, less than 40% of treated patients experience peripheral neuropathy of treatment-related grade 2 or higher severity, less than 40% of treated patients experience alopecia of treatment-related grade 2 or higher severity, less than 40% of treated patients experience nausea, vomiting, or anorexia of treatment-related grade 2 or higher severity, or a combination of two or more of these treatment-related adverse events, or less than 40% of treated patients experience these adverse events. In some embodiments, after administration, less than 30% of treated patients experience peripheral neuropathy of treatment-related grade 2 or higher severity, less than 30% of treated patients experience alopecia of treatment-related grade 2 or higher severity, less than 30% of treated patients experience nausea, vomiting, or anorexia of treatment-related grade 2 or higher severity, or a combination of two or more of these treatment-related adverse events, or less than 30% of treated patients experience these adverse events. Preferably, after administration, less than 20% of treated patients experience peripheral neuropathy of treatment-related grade 2 or higher severity, less than 20% of treated patients experience alopecia of treatment-related grade 2 or higher severity, less than 20% of treated patients experience nausea, vomiting, or loss of appetite of treatment-related grade 2 or higher severity, or less than 20% of treated patients experience any combination of two or more of these treatment-related adverse events. More preferably, after administration, less than 10% of treated patients experience peripheral neuropathy of treatment-related grade 2 or higher severity, less than 10% of treated patients experience alopecia of treatment-related grade 2 or higher severity, less than 10% of treated patients experience nausea, vomiting, or loss of appetite of treatment-related grade 2 or higher severity, or less than 10% of treated patients experience any combination of two or more of these treatment-related adverse events.

[0029] In other embodiments, human subjects did not experience any adverse events of grade 2 or higher severity after administration.

[0030] use

[0031] The conjugates of this disclosure are useful for inhibiting tumor growth. While not bound by any particular theory, the inhibitory effect of the conjugates provided herein makes such entities useful in methods of treating cancer.

[0032] In some embodiments, the method involves administering a hematopoietic protein, such as a colony-stimulating factor (CSF), such as granulocyte-colony-stimulating factor (G-CSF) or granulocyte-macrophage-CSF (GM-CSF), to a human subject before administering the CLDN6 conjugate of the Disclosure. In some embodiments, G-CSF pretreatment is preferred.

[0033] Accordingly, methods for inhibiting tumor growth in a subject and methods for reducing tumor size in a subject are provided herein. In various embodiments, the method comprises administering to a subject an amount of the pharmaceutical composition of the Disclosure effective in inhibiting tumor growth or reducing tumor size in the subject. In various embodiments, the growth of ovarian tumors, malignant melanomas, bladder tumors, or endometrial tumors is inhibited. In various embodiments, the size of ovarian tumors, malignant melanomas, bladder tumors, or endometrial tumors is reduced.

[0034] As used herein, the terms “inhibit” or “reduce” and words derived therefrom may not mean 100% or complete inhibition or reduction. Rather, the degree of inhibition or reduction that a person skilled in the art would recognize as having a potential benefit or therapeutic effect varies. In this regard, the antigen-binding proteins of this disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of this disclosure is at least or about 10% inhibition (e.g., at least 20% or about 20% inhibition, at least 30% or about 30% inhibition, at least 40% or about 40% inhibition, at least 50% or about 50% inhibition, at least 60% or about 60% inhibition, at least 70% or about 70% inhibition, at least 80% or about 80% inhibition, at least 90% or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is at least 10% or about 10% reduction (e.g., at least 20% or about 20% reduction, at least 30% or about 30% reduction, at least 40% or about 40% reduction, at least 50% or about 50% reduction, at least 60% or about 60% reduction, at least 70% or about 70% reduction, at least 80% or about 80% reduction, at least 90% or about 90% reduction, at least 95% or about 95% reduction, at least 98% or about 98% reduction).

[0035] This specification further provides a method for treating a subject having cancer, for example, CLDN6-expressing cancer. In various embodiments, the method involves administering to the subject an amount of the pharmaceutical composition of this disclosure that is effective in treating the cancer in the subject.

[0036] In certain embodiments, this cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer, pancreatic cancer, gastrointestinal cancer, stomach cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, this cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, or endometrial cancer. In various embodiments, this cancer is any cancer characterized by moderate to high expression of CLDN6.

[0037] As used herein, the term “to treat” and related words do not necessarily mean 100% or complete treatment. Rather, the degree of treatment that a person skilled in the art would recognize as having a potential benefit or therapeutic effect varies. In this regard, the methods for treating cancer of the present disclosure may provide any amount or any level of treatment. Furthermore, the treatment provided by the methods of the present disclosure may include treatment of one or more conditions, symptoms, or signs of the cancer being treated. Also, the treatment provided by the methods of the present disclosure may include slowing the progression of cancer. For example, the methods may treat cancer by enhancing T-cell activity or the immune response against cancer, reducing tumor or cancer growth, reducing metastasis of tumor cells, or increasing cell death of tumor or cancer cells. In various embodiments, the methods treat to delay the onset or recurrence of cancer by at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, or 4 years or more. In various aspects, this method addresses the issue by extending the lifespan of the subject.

[0038] Dosage

[0039] The activators of this disclosure are considered useful in other methods further described herein, including methods for inhibiting tumor growth and methods for treating cancer. For the purposes of this disclosure, the amount or dose of the activator administered should be sufficient, for example, to produce a therapeutic response in a human subject over a reasonable time frame.

[0040] For example, the dose of the activator of this disclosure may be about 1.0 to about 10 mg / kg, about 1.0 to about 6.0 mg / kg, about 1.0 to about 5.0 mg / kg, about 1.0 to about 4.0 mg / kg, or about 1.0 to about 3.0 mg / kg per body weight of the subject being treated.

[0041] Furthermore, in other embodiments, this dose may be approximately 1.7 to 6.0 mg / kg, 1.7 to 5.0 mg / kg, 1.7 to 4.0 mg / kg, 1.7 to 3.0 mg / kg, or 1.7 to 2.0 mg / kg per body weight of the subject.

[0042] In certain embodiments, this dose may be approximately 1.7 to 5.0 mg / kg, approximately 1.7 to 4.0 mg / kg, or approximately 1.7 to 3.0 mg / kg per body weight of the subject.

[0043] In yet another embodiment, this dose may be approximately 2.0 to 10.0 mg / kg, 2.0 to 6.0 mg / kg, 2.0 to 5.0 mg / kg, 2.0 to 4.0 mg / kg, or 2.0 to 3.0 mg / kg per body weight of the subject.

[0044] In some embodiments, this dose may be approximately 2.0 to 6.0 mg / kg, 2.0 to 5.0 mg / kg, 2.0 to 4.0 mg / kg, or 2.0 to 3.0 mg / kg per body weight of the subject.

[0045] In certain embodiments, this dose may be approximately 2.0 to 5.0 mg / kg, 2.0 to 4.0 mg / kg, or 2.0 to 3.0 mg / kg per body weight of the subject.

[0046] In further embodiments, this dose may be approximately 2.4 to 10.0 mg / kg, 2.4 to 6.0 mg / kg, 2.4 to 5.0 mg / kg, 2.4 to 4.0 mg / kg, or 2.4 to 3.0 mg / kg per body weight of the subject.

[0047] In some embodiments, this dose may be approximately 2.4 to 6.0 mg / kg, 2.4 to 5.0 mg / kg, 2.4 to 4.0 mg / kg, or 2.4 to 3.0 mg / kg per body weight of the subject.

[0048] In certain embodiments, this dose may be approximately 2.4 to 5.0 mg / kg, approximately 2.4 to 4.0 mg / kg, or approximately 2.4 to 3.0 mg / kg per body weight of the subject.

[0049] In further embodiments, this dose may be approximately 3.0 to 5.0 mg / kg, approximately 3.0 to 4.0 mg / kg, or approximately 3.0 to 3.6 mg / kg per body weight of the subject.

[0050] Conjugates and conjugation (conjugation reaction)

[0051] This disclosure relates to an antigen-binding protein having a second portion (e.g., a heterologous portion) bound, ligated, or conjugated to the following structural formula (I): [ka]

[0052] Therefore, this disclosure provides conjugates and compositions comprising an antigen-binding protein and a heterologous moiety.

[0053] The heterogeneous part, including structural formula (I), is derived from monomethyl auristatin E (MMAE), which has the following structural formula: [ka] It has. IUPAC name: (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide MMAE, or desmethyl-aulistatin E, is a synthetic antitumor agent. It is a potent mitotic inhibitor that inhibits cell division by blocking the polymerization of tubulin.

[0054] In certain embodiments, a heterogeneous moiety (e.g., MMAE) is conjugated to an antigen-binding protein via a linker. In some such embodiments, the linker comprises a peptide having valine-citrulline (Val-Cit). In some embodiments, this linker comprises valine-citrulline conjugated to a self-immolative ρ-aminobenzyl (PAB) group, which is collectively referred to as Val-Cit-PAB. Thus, in some embodiments, the conjugate of the present disclosure comprises an antigen-binding protein, a Val-Cit-PAB, and an MMAE. In preferred embodiments, the antigen-binding protein is conjugated to at least one MMAE, each MMAE being conjugated to this antigen-binding protein via a Val-Cit-PAB.

[0055] In a particular embodiment, the heterogeneous part is structural formula (II): [ka] It has, During the ceremony, [ka] This is a covalent thiol bond to an antigen-binding protein. The heterogeneous part having structural formula (II) is called vedotin using the International Common Name (INN) nomenclature.

[0056] The heterologous moiety-to-antigen-binding protein ratio (HAR) represents the number of heterologous moieties linked to each antigen-binding molecule. In some embodiments, the HAR is in the range of about 1 to about 15, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, the HAR is in the range of about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR is in the range of about 2 to about 4. The HAR can be characterized by conventional means such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC. In some embodiments, HAR is approximately 3.0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, or approximately 5.0.

[0057] In some embodiments, the present invention provides compositions comprising structurally "homogeneous" conjugates in which a substantial proportion of antigen-binding proteins are conjugated at the same specific site of the antigen-binding protein to a predetermined number of heterologous moieties. In some embodiments, this structurally homogeneous conjugate contains about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 HARs. In some embodiments, this structurally homogeneous conjugate contains about 2, about 4, about 6, or about 8 HARs. In preferred embodiments, this structurally homogeneous conjugate contains about 4 HARs. In other preferred embodiments, this structurally homogeneous conjugate contains about 2 HARs. In some embodiments, this structurally homogeneous conjugate includes HARs of about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. In some embodiments, this structurally homogeneous conjugate includes conjugates of about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent or more having a specified HAR. In some embodiments, this structurally homogeneous conjugate includes about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent conjugates having a specified HAR. In some embodiments, this structurally homogeneous conjugate includes at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent conjugates having a specified HAR. In some embodiments, the homogeneity of this structurally homogeneous conjugate is determined by a chromatogram, e.g., HPLC or any preferred chromatography. In some embodiments, this chromatogram is an HIC chromatogram. In some embodiments, the homogeneity of the homogeneous conjugate is determined by liquid chromatography and tandem mass spectrometry (e.g., nanoLC-MS / MS) analysis (Farras et al. (2020) MAbs:12(1):1702262) following protease or trypsin digestion, or further determined.This structurally homogeneous conjugate can be produced by site-specific conjugation reactions.

[0058] In some embodiments, this heterogeneous portion is site-specifically conjugated to an antigen-binding protein (e.g., an antibody). Various site-specific conjugation methods, e.g., conjugation with thiomab, TDC, or unpaired cysteine ​​residues, or conjugation after reduction of interchain sulfide bonds (WO2020 / 164561; Junutula et al. (2008) Nat. Biotechnol. 26:925-932, Dimasi et al. (2017) Mol. Pharm. 14:1501-1516, Shen et al. (2012) Nat. Biotechnol. 30:184-9); thiol crosslinking linkers (Behrens et al. (2015) Mol. Pharm. 12:3986-98); glutamine conjugation using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15; Dennler et al. (2014) Bioconjug Chem. 25:569-78); Conjugation with manipulated non-native amino acid residues (Axup et al. (2012) Proc Natl Acad Sci USA 104-16101-6; Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71; VanBrunt et al. (2015) Bioconjug Chem 26:2249-60; Zimmerman et al. (2014) Bioconjug Chem 25:351-61); Selenocysteine ​​conjugation (Li et al. (2017) Cell Chem Biol 24:433-442); Glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5); Conjugation to galactose or GalNAc analogs (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9; van Geel et al. (2015) Bioconjug Chem 26:2233-42); via glycan manipulation (Zhou et al. (2014) Bioconjug Chem 25:510-20; Tang et al.(2017) Nat Protoc 12:1702-1721); methods mediated by short peptide tags, such as manipulation of glutamine tags or saltase A-mediated peptide transfer (Strop et al. (2013) Chem Biol 20:161-7; Beerli et al. (2015) PLoS One 10:e0131177); and methods mediated by aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5) are known in the art.

[0059] Antigen-binding protein

[0060] A method is provided herein for use in cancer treatment that utilizes an antigen-binding protein, such as an antibody, that binds to claudin-6 (CLDN6) in an ADC.

[0061] As used herein, the term “antibody” refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, a variable region, and a constant region. For example, an antibody may be IgG, which has a “Y-shaped” structure of two pairs of identical polypeptide chains, each pair having one “light” chain (typically with a molecular weight of about 25 kDa) and one “heavy” chain (typically with a molecular weight of about 50–70 kDa). Antibodies have a variable region and a constant region. In the IgG format, the variable region is generally about 100–110 amino acids or more, contains three complementarity-determining regions (CDRs), and is primarily responsible for antigen recognition, substantially different from other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region consists of the N-terminal regions of each light and heavy chain, while the constant region consists of the C-terminal regions of the heavy and light chains (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4). thed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0062] The general structure and properties of antibody CDRs have been described in the art. Briefly, in the antibody backbone, CDRs are embedded within a framework in the heavy and light chain variable regions, where they constitute regions primarily responsible for antigen binding and recognition. The variable region typically contains at least three CDRs belonging to the heavy or light chain (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md., and Chothia and Lesk, 1987, J.Mol.Biol.196:901-917, and Chothia et al., 1989, Nature 342:877-883) within a framework region (FR1, FR2, FR3, and FR4 of the designated framework regions 1-4 by Kabat et al., 1991; see also Chothia and Lesk, 1987).

[0063] Antibodies may include any constant region known in the art. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region may be, for example, a kappa or lambda-type light chain constant region, e.g., a human kappa or lambda-type light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu-type heavy chain constant region, e.g., a human alpha, delta, epsilon, gamma, or mu-type heavy chain constant region. Therefore, in various embodiments, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody containing one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, the antibody includes a constant region containing one or more amino acid modifications from the naturally occurring counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various cases, the antibody includes a constant region in which the C-terminal Lys residue present in the naturally occurring counterpart has been removed or excised.

[0064] Antibodies can be monoclonal antibodies. In some embodiments, antibodies contain sequences substantially similar to naturally occurring antibodies produced by mammals, such as mice, rabbits, goats, horses, chickens, hamsters, and humans. In this respect, antibodies can be considered mammalian antibodies, such as mouse antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, and human antibodies. In certain embodiments, antibodies are chimeric antibodies or humanized antibodies. The term "chimeric antibody" refers to an antibody that contains domains derived from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, a stretch of amino acid sequences from at least two species. A chimeric antibody may also contain domains from two or more different antibodies in the same species. When used in reference to antibodies, the term "humanized" refers to an antibody that has at least a CDR region of a non-human source that has been manipulated to have a structure and immunological function more similar to a true human antibody than to the antibody of the original source. For example, humanization may involve transplanting CDRs derived from non-human antibodies, such as mouse antibodies, into human antibodies. Humanization may also involve selective amino acid substitutions to make the non-human sequence more similar to the human sequence. Information, including sequence information for the heavy and light chain constant regions of human antibodies, is publicly available through the Uniprot database and other databases well known to those skilled in the art of antibody manipulation and production. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859 (incorporated herein by reference).

[0065] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody, producing two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody, producing an F(ab')2 fragment and a pFc' fragment.

[0066] In various embodiments, the anti-CLDN6 antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies.

[0067] CLDN6 and epitopes

[0068] In various embodiments, CLDN6 is human CLDN6 having the following amino acid sequence: MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAXIRDFYNPLVAEAQKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV (where X is Ile or Val) (Sequence ID 202).

[0069] In various embodiments, human CLDN6 contains one of the amino acid sequences of sequence numbers 1, 178, and 200-202.

[0070] In various embodiments, the antigen-binding proteins of this disclosure bind to epitopes within the amino acid sequence of CLDN6. In various embodiments, CLDN6 is human CLDN6, and the antigen-binding proteins of this disclosure bind to epitopes within the amino acid sequence of human CLDN6, for example, SEQ ID NOs: 1, 178, and 200-202. "Epitope" means a region of CLDN6 or within CLDN6 to which the antigen-binding protein binds. In some embodiments, the epitope is a linear epitope. A "linear epitope" refers to a region of CLDN6 or within CLDN6 to which the antigen-binding protein binds, the region being composed of consecutive amino acids in the amino acid sequence of CLDN6. The amino acids of the linear epitope are adjacent to each other in the primary structure of CLDN6. Thus, the linear epitope is an antigen, i.e., a fragment or portion of the amino acid sequence of CLDN6. In various other embodiments, the epitope is a structural epitope or a structural epitope. A "structural epitope" or "structural epitope" refers to an epitope composed of amino acids that are located in close proximity to each other only when CLDN6 is properly folded. Unlike linear epitopes, the amino acids of a structural epitope or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. A structural epitope or structural epitope is not made up of consecutive amino acids in the amino acid sequence of the antigen (CLDN6).

[0071] In various embodiments, the epitope is located within the extracellular domain (ECD) of CLDN6, for example, human CLDN6. In various embodiments, the antigen-binding protein binds to extracellular loop 2 (EL2) of the ECD of CLDN6, which has the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2). In various embodiments, the epitope to which the antigen-binding protein binds is located within SEQ ID NO: 2. In various embodiments, the antigen-binding protein of this disclosure binds to the N-terminal portion of SEQ ID NO: 2, for example, THAIIRDFYNPL (SEQ ID NO: 3). In various embodiments, the antigen-binding protein of this disclosure binds to the C-terminal portion of SEQ ID NO: 2, for example, LVAEAQKREL (SEQ ID NO: 4). In various cases, the antigen-binding protein of this disclosure binds to EL2 but not to extracellular loop 1 (EL1) of CLDN6. In various embodiments, the epitope(s) to which the antigen-binding protein of this disclosure binds is different from the epitope to which the anti-CLDN6 antibody, which includes a light chain variable region containing the sequence of SEQ ID NO: 185 and a heavy chain variable region containing the sequence of SEQ ID NO: 186, binds. In various embodiments, the epitope(s) to which the antigen-binding protein of this disclosure binds is different from the epitope to which the anti-CLDN6 antibody, which includes a light chain variable region containing the sequence of SEQ ID NO: 181 and a heavy chain variable region containing the sequence of SEQ ID NO: 182, binds.

[0072] In various embodiments, the antigen-binding protein binds to human CLDN6 and non-human CLDN6. In various embodiments, non-human CLDN6 is CLDN6 from chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, zebrafish, or frogs. In various examples, this antigen-binding protein binds to human CLDN6 and mouse CLDN6.

[0073] Affinity and binding strength (affinity)

[0074] The antigen-binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding strength of the antigen-binding protein to CLDN6 can be described in terms of its affinity, i.e., a measure of the strength of the interaction between the binding site of the antigen-binding protein and the epitope. In various aspects, the antigen-binding proteins provided herein have a high affinity for CLDN6 and thus bind to a greater amount of CLDN6 in a shorter period of time than low-affinity antigen-binding proteins. In various aspects, the antigen-binding protein is at least 10 5 M -1 , at least 10 6 M -1 , at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , or at least 10 10 M -1 with an equilibrium association constant K A . As will be appreciated by those skilled in the art, K A may be affected by factors including pH, temperature, and buffer composition.

[0075] In various embodiments, the binding strength of the antigen-binding protein to CLDN6 can be represented by its sensitivity. K D is the equilibrium dissociation constant between the antigen-binding protein and CLDN6, i.e., the ratio of k off / k on . K D is inversely proportional to K A . The K D value relates to the concentration of the antigen-binding protein (the amount of antigen-binding protein required for a particular experiment), and thus, the lower the K D value (the lower the concentration), the higher the affinity of the antigen-binding protein. In various aspects, the binding strength of the antigen-binding protein to CLDN6 can be represented by K D . In various aspects, the K D of the antigen-binding proteins provided herein is about 10 -1 M, about 10 -2M, about 10 -3 M, about 10 -4 M, about 10 -5 M, about 10 -6 M or less. In various embodiments, the K of the antigen-binding protein provided herein. D The K of the antigen-binding protein provided herein is micromolar, nanomolar, picomolar, or femtomole. D It is about 10 -4 M~10 -6 M or 10 -7 M~10 -9 M or 10 -10 M~10 -12 M or 10 -13 M~10 -15 It is within the range of M. In various embodiments, the K of the antigen-binding protein provided herein D It is approximately 1.0 × 10 -12 M ~ approx. 1.0×10 -8 It is within the range of M. In various embodiments, the K of antigen-binding proteins D It is approximately 1.0 × 10 -11 M ~ approx. 1.0×10 -9 It is within the range of M.

[0076] In various embodiments, the affinity of antigen-binding proteins is measured or ranked using flow cytometry or fluorescence-activated cell sorting (FACS) based assays. Flow cytometry-based binding assays are well known in the art. See, for example, Cedeno-Arias et al., Sci Pharm 79(3):569-581 (2011); Rathanaswami et al., Analytical Biochem 373:52-60 (2008); and Geuijen et al., J Immunol Methods 302(1-2):68-77 (2005). In various embodiments, the affinity of antigen-binding proteins may be measured or ranked using Trikha et al., Int J Cancer 110:326-335 (2004) and Tam et al., Circulation 98(11):1085-1091 (1998), as well as the competitive assays described below. Please refer to the section titled "Competitive Assays" below. Trikh et al. used antigen-expressing cells in a radioassay. 125 The binding of I-labeled antigen-binding proteins (e.g., antibodies) to cell surface antigens is measured using cells in suspension. In various embodiments, the relative affinity of CLDN6 antibodies is determined by a FACS-based assay, in which various concentrations of CLDN6 antibodies conjugated to fluorophores are incubated with CLDN6-expressing cells, and the emitted fluorescence (a direct measure of antibody-antigen binding) is determined. A curve is created plotting the fluorescence for each dose or concentration. The maximum value is the lowest concentration at which fluorescence plateaus or reaches its highest point, where binding saturation occurs. Half of the maximum value is considered to be the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity compared to other antibodies similarly tested.

[0077] In various aspects, IC is determined in competitive binding inhibition assays. 50 The value is the K of the antigen-binding protein. DThis approximates the following. In various cases, as discussed below, the competitive assay is a FACS-based assay performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Before performing the FACS-based assay, in some embodiments, the endogenously expressing CLDN6 cells are predetermined as either CLDN6-low or CLDN6-high expression cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are derived from cell lines, such as ovarian cell lines, endometrial cell lines, bladder cell lines, lung cell lines, gastrointestinal (GI) cell lines, hepatocyte cell lines, etc. In various embodiments, cells endogenously expressing CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper gastrointestinal cells, OV-90 ovarian cells, HUH-7 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 expressed by the cells and a reference antibody, where the reference antibody is known to bind to CLDN6 but is not the antigen-binding protein of this disclosure. In various cases, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CLDN6 and a reference antibody, and this inhibition is IC 50 Characterized by the following: In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and the reference antibody, resulting in an IC of less than approximately 2500 nM. 50This indicates that, in various embodiments, antigen-binding proteins have ICs of less than approximately 2000 nM, less than approximately 1500 nM, less than approximately 1000 nM, less than approximately 900 nM, less than approximately 800 nM, less than approximately 700 nM, less than approximately 600 nM, less than approximately 500 nM, less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, or less than approximately 100 nM. 50 This indicates that, in various embodiments, the antigen-binding protein has an IC of less than approximately 90 nM, less than approximately 80 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, or less than approximately 10 nM. 50 This demonstrates that, in various cases, the antigen-binding proteins of this disclosure compete for binding to CLDN6 with reference antibodies known to bind to CLDN6 (the reference antibodies being different from any of the antigen-binding proteins of this disclosure). For further details, see "Competitive Assays" below.

[0078] The overall strength of an antibody-antigen complex can be measured from its binding affinity (affinity). This varies depending on three main parameters: the affinity of the antigen-binding protein to the epitope, the binding titer of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting regions. The higher the binding titer (number of antigen-binding sites) of the antigen-binding protein, the greater the amount of antigen (CLDN6) it can bind to. In various embodiments, the antigen-binding protein has strong avidity to CLDN6. In various embodiments, the antigen-binding protein is polyvalent. In various embodiments, the antigen-binding protein is bivalent. In various cases, the antigen-binding protein is monovalent.

[0079] Cross-reactivity

[0080] In various embodiments, the antibody of this disclosure binds to CLDN6 and does not bind to any other member of the CLDN family, for example, it does not cross-react with any other member of the CLDN family. In various cases, the antibody of this disclosure is CLDN6 specific. In various embodiments, the antibody of this disclosure has selectivity for CLDN6, and this selectivity is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of this antibody for CLDN3, CLDN4, CLDN9, or any combination thereof. In various embodiments, the antibody of this disclosure has selectivity for CLDN6, and this selectivity is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of this antibody for CLDN3, CLDN4, and CLDN9, respectively. Selectivity is the K that this antibody exhibits towards CLDN6 or a member of the CLDN family. D It may also be based on K D This can be determined by techniques known in the art, such as surface plasmon resonance and FACS-based affinity assays.

[0081] In various embodiments, the antibody of this disclosure binds to CLDN6 but not to claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various embodiments, this antibody does not bind to CLDN3, CLDN4, and CLDN9 and produces IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) in FACS-based assays using OVCA429 cells endogenously expressing CLDN6. 50In various embodiments, this antibody does not bind to any of CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved in OVCA429 cells endogenously expressing CLDN6 is less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM). In various embodiments, this antibody exhibits at least 5 times greater selectivity for CLDN6 than its selectivity for CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved in OVCA429 cells endogenously expressing CLDN6 is less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM). In various embodiments, this antibody exhibits an IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) for artificial and endogenous models of CLDN6, and a ratio of more than approximately 5 times that distinguishes the IC50 of CLDN6 from that of CLDN3, CLDN4, and / or CLDN9. In various cases, this antibody exhibits an IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) for CLDN6, and an IC50 at least 5 times greater than that for any one of CLDN3, CLDN4, and CLDN9.

[0082] Competitive assay

[0083] In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 and a reference antibody, the reference antibody being known to bind to CLDN6 but not the antigen-binding protein of this disclosure. In various cases, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope in EL2 or EL1, optionally, within the amino acid sequence of the extracellular domain of human CLDN6. In various embodiments, the reference antibody includes a light chain variable sequence encoded by SEQ ID NO: 179 and a heavy chain variable sequence encoded by SEQ ID NO: 180. In various embodiments, the reference antibody includes a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, and this inhibition is reflected in IC 50 Characterized by the following: In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and the reference antibody, resulting in an IC of less than approximately 2500 nM. 50 This indicates that, in various embodiments, antigen-binding proteins have ICs of less than approximately 2000 nM, less than approximately 1500 nM, less than approximately 1000 nM, less than approximately 900 nM, less than approximately 800 nM, less than approximately 700 nM, less than approximately 600 nM, less than approximately 500 nM, less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, or less than approximately 100 nM. 50 This indicates that, in various embodiments, the antigen-binding protein has an IC of less than approximately 90 nM, less than approximately 80 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, or less than approximately 10 nM. 50 This indicates.

[0084] In various cases, the antigen-binding protein of this disclosure competes with a reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the in vitro competitive binding assay is a FACS-based assay that measures the fluorescence of a fluorophore-conjugated secondary antibody bound to the Fc of the reference antibody in the absence of or in the presence of a specific amount of the antigen-binding protein of this disclosure. In various embodiments, the FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Before performing the FACS-based assay, in some embodiments, the endogenously expressing cells are pre-determined as either CLDN6-low-expressing or CLDN6-high-expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells derived from cell lines, such as ovarian cell lines, endometrial cell lines, bladder cell lines, lung cell lines, gastrointestinal (GI) cell lines, hepatocyte cell lines, and lung cell lines. In various embodiments, cells endogenously expressing CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper gastrointestinal cells, OV-90 ovarian cells, HUH-7 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various cases, the antigen-binding protein of this disclosure binds with high affinity to CLDN6 endogenously expressed by one or more ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various aspects, antigen-binding proteins have an IC50 of less than approximately 3000 nM, as determined in a FACS-based competitive binding inhibition assay using one or more ARK2, OVCA429, LS513, or MCF7 cells. 50In various embodiments, antigen-binding proteins exhibit IC values ​​of less than approximately 2500 nM, less than approximately 2000 nM, less than approximately 1750 nM, less than approximately 1500 nM, less than approximately 1250 nM, less than approximately 1000 nM, less than approximately 750 nM, or less than approximately 500 nM, as determined in a FACS-based competitive binding inhibition assay using one or more ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 In various embodiments, antigen-binding proteins exhibit ICs of less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, less than approximately 100 nM, less than approximately 75 nM, less than approximately 50 nM, less than approximately 25 nM, or less than approximately 10 nM, as determined in a FACS-based competitive binding inhibition assay using one or more ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 This indicates.

[0085] Other binding assays that test the ability of an antibody to compete with a second antibody for binding to an antigen or its epitope, such as competitive binding assays or competitive assays, are known in the art. See, for example, Trikha et al., Int J Cancer 110:326-335 (2004), Tam et al., Circulation 98(11):1085-1091 (1998), U.S. Patent Application Publication No. US20140178905, Chand et al., Biologicals 46:168-171 (2017); Liu et al., Anal Biochem 525:89-91 (2017); and Goolia et al., J Vet Diagn Invest 29(2):250-253 (2017). Other methods for comparing two antibodies are also known in the art, including, for example, surface plasmon resonance (SPR). SPR can be used to determine the binding constants of one antibody and a second antibody, and the two binding constants can be compared.

[0086] Antibody production method and related methods

[0087] Suitable methods for producing antibodies are known in the art. For example, standard hybridoma methods for antibody production are described in, for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5. th This is described in Ed., Garland Publishing, New York, NY (2001). In certain embodiments, methods for producing antibodies and conjugates for use in the methods disclosed herein are described in U.S. Patents 2022372134, 2022040321, and 2023049752, each of which is incorporated herein by reference in whole.

[0088] Depending on the host species, various adjuvants can be used to enhance the immunological response and induce greater antibody production by the host. Such adjuvants include, but are not limited to, Freund's adjuvants, inorganic gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, and dinitrophenols. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

[0089] Other methods for antibody production are summarized in Table 1. [Table 1]

[0090] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to the CLDN6 epitope are known in the art, and these include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blotting, immunoprecipitation, SPR, and competitive inhibition assays (see, for example, Janeway et al., below, and U.S. Patent Application Publication No. 2002 / 0197266, and the above section relating to competitive assays).

[0091] Array / Structure

[0092] CLDN6-binding proteins are known in the art. For example, U.S. Patents 2022372134 and 2023049752 (each incorporated herein by reference in its entirety) describe CLDN6-binding proteins. CLDN6 antigen-binding proteins are also known from U.S. Patents 20220168438, 20200261594, 20220168440, 20220125943 (e.g., SEQ ID NOs. 19, 21, 23, and 25 from this publication), 2020339677 (e.g., SEQ ID NOs. 34-41 and Figures 25-26 from this publication), 2021179730 (e.g., SEQ ID NOs. 34-41 and Figures 25-26 from this publication), and 20 These sequences are also listed in Nos. 21079113, Nos. 2020385460 (e.g., sequence numbers 3-12 from this publication), Nos. 2020399370, Nos. 2022306711, Nos. 2022162302 (e.g., sequence numbers 40-45 from this publication), Nos. 2019309067, and WO2022187275 (e.g., sequence numbers 1-4 and 23-31 from this publication), each of which is incorporated herein by reference in its entirety. The sequences are listed together with their sequence numbers in the sequence listing. In this specification, (a) the amino acid sequences of heavy chain (HC) complementarity-determining regions (CDR) 1 listed in Table A, or sequences selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 107, 125, and 131, or variant sequences thereof, which differ by only one or two amino acids, or have sequence identity of at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%); (b) the HC sequences listed in Table A The amino acid sequence of CDR2, or a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 108, 126, and 132, or a variant thereof, which differs by only one or two amino acids, or which has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity;(c) The amino acid sequences of HC CDR3 listed in Table A, or sequences selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 109, 127, and 133, or variant sequences thereof, which differ by only one or two amino acids, or have at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (d) The sequences listed in Table A (e) The amino acid sequence of light chain (LC) CDR1, or a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 104, 122, and 128, or a variant thereof, which differs by only one or two amino acids, or has sequence identity of at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%); (e) LC as listed in Table A (f) The amino acid sequence of CDR2, or a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 105, 123, and 129, or a variant thereof, which differs by only one or two amino acids, or has sequence identity of at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%); (f) LCs listed in Table A An antigen-binding protein is provided, comprising the amino acid sequence of CDR3, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 106, 124, and 130, or a variant thereof, which differs by only one or two amino acids, or which has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or any combination of two or more of (g)(a) to (f). [Table 2]

[0093] Scalar

[0094] D×1 (SEQ ID NO: 514): Gly Tyr Thr Phe Thr Glu Tyr Thr Met His

[0095] D×2 (SEQ ID NO: 515): Gly Val Asn Pro Asn Ser Gly Asp Thr Ser

[0096] D×3 (SEQ ID NO: 516): Pro Gly Gly Tyr Asp Val Gly Tyr Tyr Ala Met Asp Tyr

[0097] D×4 (Sequence ID 517): Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

[0098] D×5 (Sequence ID 518): Phe Thr Ser Arg Leu His Ser

[0099] D×6 (Sequence ID 519): Gln Gln Gly Tyr Pro Leu Pro Trp Thr

[0100] D×7 (SEQ ID NO: 520): Gly Tyr Thr Phe Thr Glu Tyr Thr Met His

[0101] D×8 (Sequence ID 521): Gly Val Asn Pro Asn Ser Gly Asp Thr Ser

[0102] D×9 (SEQ ID NO: 522): Pro Gly Gly Tyr Asp Val Gly Tyr Tyr Ala Met Asp Tyr

[0103] D×10 (Sequence ID 523): Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

[0104] D×11 (Sequence ID 524): Ser Thr Ser Arg Leu His Ser

[0105] D×12 (Sequence ID 525): Gln Gln Gly Tyr Pro Leu Pro Trp Thr

[0106] D×13 (SEQ ID NO: 526): Gly Tyr Thr Phe Thr Glu Tyr Thr Met His

[0107] D×14 (SEQ ID NO: 527): Gly Val Asn Pro Asn Ser Gly Asp Thr Ser

[0108] D×15 (SEQ ID NO: 528): Pro Gly Gly Tyr Asp Val Gly Tyr Tyr Ala Met Asp Tyr

[0109] D×16 (Sequence ID 529): Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

[0110] D×17 (Sequence ID 530): Phe Thr Ser Arg Leu His Ser

[0111] D×18 (Sequence ID 531): Gln Gln Gly Tyr Pro Leu Pro Trp Thr

[0112] D×19 (SEQ ID NO: 532): Gly Tyr Thr Phe Thr Glu Tyr Thr Met His

[0113] D×20 (Sequence ID 533): Gly Val Asn Pro Asn Ser Gly Asp Thr Ser

[0114] D×21 (SEQ ID NO: 534): Pro Gly Gly Tyr Asp Val Gly Tyr Tyr Ala Met Asp Tyr

[0115] D×22 (Sequence ID 535): Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

[0116] D×23 (Sequence ID 536): Ser Thr Ser Arg Leu His Ser

[0117] Dx24 (Sequence ID 537): Gln Gln Gly Tyr Pro Leu Pro Trp Thr

[0118] In various embodiments, the antigen-binding protein comprises at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence shown in Table A, as well as the HC CDR amino acid sequence shown in Table A.

[0119] In various embodiments, the antigen-binding protein includes at least three, four, or five amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein includes each of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A, and at least one or two of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein includes each of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A, and at least one or two of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein includes all six CDR amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein contains six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 50-55; SEQ ID NOs: 122-127; SEQ ID NOs: 26-31; SEQ ID NOs: 128-133; SEQ ID NOs: 38-43; SEQ ID NOs: 62-67; SEQ ID NOs: 44-49; SEQ ID NOs: 104-109; SEQ ID NOs: 56-61; SEQ ID NOs: 32-37; SEQ ID NOs: 8-13; SEQ ID NOs: 68-73; SEQ ID NOs: 14-19; and SEQ ID NOs: 20-25.

[0120] In various cases, the amino acid sequences in Table A are separated by at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acids. In various cases, there are approximately 10 to 20 amino acids between the LC CDR1 sequence and the LC CDR2 sequence, and approximately 25 to 40 amino acids between the LC CDR2 sequence and the LC CDR3 sequence. In various cases, there are approximately 14 to 16 amino acids between the LC CDR1 sequence and the LC CDR2 sequence, and approximately 30 to 35 amino acids between the LC CDR2 sequence and the LC CDR3 sequence. In various cases, there are approximately 10 to 20 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 25 to 40 amino acids between the HC CDR2 sequence and the HC CDR3 sequence. In various cases, there are approximately 14 to 16 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 30 to 35 amino acids between the HC CDR2 sequence and the HC CDR3 sequence.

[0121] In various embodiments, the antigen-binding protein is (a) a sequence selected from the group consisting of the heavy chain variable region amino acid sequences listed in Table B, or SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 167, 173, and 175, or a variant thereof, which differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (b) light chain variable region amino acid sequences listed in Table B, or sequences selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 166, 172, 174, and 176, or variant sequences thereof that differ by only one or two amino acids, or have at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) including both (a) and (b). [Table 3]

[0122] In various embodiments, the antigen-binding protein contains a pair of amino acid sequences selected from the group consisting of SEQ ID NOs: 148 and 149; SEQ ID NOs: 172 and 173; SEQ ID NOs: 140 and 141; SEQ ID NOs: 174 and 175; SEQ ID NOs: 144 and 145; SEQ ID NOs: 152 and 153; SEQ ID NOs: 146 and 147; SEQ ID NOs: 166 and 167; SEQ ID NOs: 150 and 151; SEQ ID NOs: 142 and 143; SEQ ID NOs: 164 and 165; SEQ ID NOs: 162 and 163; SEQ ID NOs: 134 and 135; SEQ ID NOs: 154 and 155; SEQ ID NOs: 136 and 137; and SEQ ID NOs: 138 and 139.

[0123] In various embodiments, antigen-binding proteins contain amino acid sequences similar to those described above, but the antigen-binding proteins still substantially retain their biological functions, such as their ability to bind to human CLDN6, reduce tumor growth, inhibit tumor growth, and / or treat cancer.

[0124] In various embodiments, the antigen-binding protein contains an amino acid sequence that differs from the above-described amino acid sequence by only one, two, three, four, five, six, or more amino acids. In various embodiments, the antigen-binding protein contains a variant sequence of the above-described sequence, the variant sequence differing from the above-described sequence by only one or two amino acids. In various embodiments, the antigen-binding protein contains one or more amino acid substitutions occurring outside the CDR, for example, one or more amino acid substitutions occurring within the heavy chain or light chain framework region(s). In various embodiments, the antigen-binding protein contains one or more amino acid substitutions, but the antigen-binding protein still retains the amino acid sequence of the six CDRs. In various embodiments, the antigen-binding protein contains an amino acid sequence that has only one, two, three, four, five, six, or more conserved amino acid substitutions compared to the above-described amino acid sequence(s). As used herein, the term “conservative amino acid substitution” means that one amino acid is substituted with another amino acid having similar properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and this includes substitutions within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Negatively charged polar residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid, and homocysteic acid; III. Polarly polar, positively charged residues: His, Arg, Lys; Ornithine (Orn) IV. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys, Norleucine (Nle), Homocysteine V. Large aromatic residues: Phe, Tyr, Trp, Acetylphenylalanine

[0125] In various forms, a conservative amino acid substitution is an exchange within one of the following groups of amino acids: I. Fatty amino acids: Gly, Ala, Val, Leu, Ile II. Non-aromatic amino acids containing side-chain hydroxyls: Ser, Thr III. Amino acids containing sulfur side chains: Cys, Met IV: Amino acids containing aromatic rings in the side chain: Phe, Tyr, Trp V: Acidic amino acids: Glu; Asp VI: Basic amino acids: Arg; Lys VII: Amino acids containing side-chain amides: Gln, Asn VIII: Amino acids containing side-chain imidazole: His, alpha-dimethylimidiazole acetate (DMIA) IX: Imino acid: Pro, 4-hydroxy-Pro, 4-amino-Pro.

[0126] In various embodiments, the antigen-binding protein includes an amino acid sequence having more than 30% or about 30%, more than 50% or about 50%, or more than 70% or about 70% sequence identity with the above-mentioned amino acid sequence. In various embodiments, the antigen-binding protein includes an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity with the above-mentioned amino acid sequence. In various embodiments, the antigen-binding protein includes an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity with the full-length amino acid sequence described above. In various embodiments, the antigen-binding protein includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the full-length amino acid sequence described above.

[0127] In various embodiments, the antigen-binding protein includes a variant sequence of the mentioned sequence, and this variant sequence has at least 70% or about 70% sequence identity with respect to the sequence described above. In various embodiments, the antigen-binding protein includes a variant sequence of the mentioned sequence, and this variant sequence has at least 80% or about 80% sequence identity with respect to the sequence described above. In various embodiments, the antigen-binding protein includes a variant sequence of the mentioned sequence, and this variant sequence has at least 90% or about 90% sequence identity with respect to the sequence described above. In various embodiments, the antigen-binding protein includes a variant sequence of the mentioned sequence, and this variant sequence has at least 95% or about 95% sequence identity with respect to the sequence described above.

[0128] In various embodiments, the antigen-binding protein includes at least one variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to one, two, three, four, or five sequences from the row of sequence numbers in Table A, and any of sequence numbers 8-133. In various embodiments, the antigen-binding protein comprises one, two, three, four, or five sequences from a set of sequences selected from SEQ ID NOs. 50-55; SEQ ID NOs. 122-127; SEQ ID NOs. 26-31; SEQ ID NOs. 128-133; SEQ ID NOs. 38-43; SEQ ID NOs. 62-67; SEQ ID NOs. 44-49; SEQ ID NOs. 104-109; SEQ ID NOs. 56-61; SEQ ID NOs. 32-37; SEQ ID NOs. 8-13; SEQ ID NOs. 68-73; SEQ ID NOs. 14-19; and SEQ ID NOs. 20-25, wherein the antigen-binding protein further comprises at least one variant sequence having at least or approximately 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences in that set.

[0129] In various embodiments, the antigen-binding protein comprises a pair of variant sequences having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs. 134-155, 166-167, and 172-175. In various cases, the antigen-binding protein contains a pair of variant sequences having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to (u)sequences 148 and 149;sequences 172 and 173;sequences 140 and 141;sequences 174 and 175;sequences 144 and 145;sequences 152 and 153;sequences 146 and 147;sequences 166 and 167;sequences 150 and 151;sequences 142 and 143;sequences 134 and 135;sequences 154 and 155;sequences 136 and 137; and (u)sequences 138 and 139. In various embodiments, the antigen-binding protein comprises a pair of sequences, one of which is the sequence shown in Table B, and the other sequence is a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs. 134-155, 166-167, and 172-175. In various embodiments, the antigen-binding protein comprises a pair of sequences: one sequence is selected from SEQ ID NOs: 148 and 149; SEQ ID NOs: 172 and 173; SEQ ID NOs: 140 and 141; SEQ ID NOs: 174 and 175; SEQ ID NOs: 144 and 145; SEQ ID NOs: 152 and 153; SEQ ID NOs: 146 and 147; SEQ ID NOs: 166 and 167; SEQ ID NOs: 150 and 151; SEQ ID NOs: 142 and 143; SEQ ID NOs: 134 and 135; SEQ ID NOs: 154 and 155; SEQ ID NOs: 136 and 137; and the other sequence is a variant sequence having at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the above sequence.For example, in various embodiments, the antigen-binding protein comprises the sequence of SEQ ID NO: 134, and this antigen-binding protein further comprises a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO: 135.

[0130] In various cases, the antigen-binding protein includes the amino acid sequence of the aforementioned amino acid sequence and has one or more amino acid substitutions to reduce or remove reactive amino acids in order to reduce or prevent undesirable side-chain reactions. For example, the antigen-binding protein includes the amino acid sequence of the aforementioned amino acid sequence having one or more of the following: (i) a Trp residue substituted with His, Tyr, or Phe; (ii) an Asn residue substituted with Gln, Ser, Ala, or Asp; (iii) an Asp residue immediately preceding a Pro residue substituted with Ala, Ser, or Glu; (iv) an Asn residue substituted with Gln, Ser, or Ala; and / or (v) a Cys residue substituted with Tyr, Ser, or Ala. In various embodiments, the antigen-binding protein includes the amino acid sequence of the aforementioned amino acid sequence having amino acid substitutions that are predicted to have greater binding affinity, greater stability, or other beneficial properties based on SHM events or statistical analysis of a large number of other similar antibody sequences. In some embodiments, the antigen-binding protein is (a) an HC CDR1 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs. 452, 455, 461, 465, and 71, or a variant thereof, which differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) an HC CDR2 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs. 475, 456, 462, 466, and 468, or a variant thereof, which differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) an HC CDR2 amino acid sequence listed in Table A1 CDR3 amino acid sequence, or a sequence selected from the group consisting of SEQ ID NOs: 453, 457, 463, 467, and 469;or a variant sequence thereof that differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (d) A sequence selected from the group consisting of the LC CDR1 amino acid sequences listed in Table A1, or SEQ ID NOs. 449, 476, 458, 464, and 68; or a variant sequence thereof that differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) LC CDR1 amino acid sequences listed in Table A1 (g) A CDR2 amino acid sequence, or a sequence selected from the group consisting of SEQ ID NOs. 450, 477, 459, 57, and 69; or a variant sequence thereof that differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (f) A LC CDR3 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs. 451, 454, 460, 58, and 70, or a variant sequence thereof that differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) A combination of any two or more of (a) to (f). [Table 4]

[0131] In some embodiments, HC CDR1 contains Gly immediately adjacent to the N-terminus of SEQ ID NO: 452, and optionally, in some embodiments, HC CDR1 contains MX immediately adjacent to the C-terminus of SEQ ID NO: 452, where X is H, N, or S. In various embodiments, HC CDR3 contains Ala immediately adjacent to the N-terminus of SEQ ID NO: 453. In various embodiments, LC CDR1 further contains TAS immediately adjacent to the N-terminus of SEQ ID NO: 449, and optionally, XH immediately adjacent to the C-terminus of SEQ ID NO: 449, where X is H, S, Y, or Q. In some embodiments, the first amino acid of SEQ ID NO: 449 is S or Q, as described below. In some embodiments, the first amino acid of SEQ ID NO: 451 is S or Q, as described below.

[0132] In various embodiments, HC CDR1 includes Gly immediately adjacent to the N-terminus of SEQ ID NO: 455, and optionally includes MX immediately adjacent to the C-terminus of SEQ ID NO: 455, where X is N, S, or H. In some embodiments, HC CDR2 includes Gln immediately adjacent to the N-terminus of SEQ ID NO: 456, and optionally includes H immediately adjacent to the C-terminus of SEQ ID NO: 456. In various embodiments, LC CDR1 includes RIS immediately adjacent to the N-terminus of SEQ ID NO: 476, and optionally includes LA immediately adjacent to the C-terminus of SEQ ID NO: 476. In various embodiments, LC CDR2 includes XLVE immediately adjacent to the C-terminus of SEQ ID NO: 477, where X is I or S.

[0133] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 461. In various embodiments, HC CDR2 contains Tyr immediately adjacent to the N-terminus of SEQ ID NO: 462, and optionally contains TH immediately adjacent to the C-terminus of SEQ ID NO: 462. In exemplary embodiments, HC CDR3 does not contain the first two amino acids of SEQ ID NO: 463. In various embodiments, LC CDR1 contains RSS immediately adjacent to the N-terminus of SEQ ID NO: 458, and optionally contains LN immediately adjacent to the C-terminus of SEQ ID NO: 458. In various embodiments, LC CDR2 contains XRFS immediately adjacent to the C-terminus of SEQ ID NO: 459, where X is Q, S, A, or D.

[0134] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 465. In various embodiments, HC CDR2 contains YI immediately adjacent to the N-terminus of SEQ ID NO: 466, and optionally contains Xaa immediately adjacent to the C-terminus of SEQ ID NO: 466, where Xaa is N, S, Q, or A. In various embodiments, LC CDR1 contains LAS immediately adjacent to the N-terminus of SEQ ID NO: 464, and optionally contains LA immediately adjacent to the C-terminus of SEQ ID NO: 464. In various embodiments, LC CDR2 contains SLAD immediately adjacent to the C-terminus of SEQ ID NO: 57.

[0135] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 71. In various embodiments, HC CDR2 contains Tyr immediately adjacent to the N-terminus of SEQ ID NO: 468, and optionally IY immediately adjacent to the C-terminus of SEQ ID NO: 468. In various embodiments, LC CDR1 contains RAS immediately adjacent to the N-terminus of SEQ ID NO: 68, and optionally SYIH immediately adjacent to the C-terminus of SEQ ID NO: 68. In various embodiments, LC CDR2 contains XLES immediately adjacent to the C-terminus of SEQ ID NO: 69, where X is N, Q, S, A, or D.

[0136] In various embodiments, the antigen-binding protein comprises the LC CDR1 amino acid sequence, the LC CDR2 amino acid sequence, and the LC CDR3 amino acid sequence described in Table A1, and at least one or two of the HC CDR amino acid sequences described in Table A1.

[0137] In various embodiments, the antigen-binding protein includes at least three, four, or five amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein includes each of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A1, and at least one or two of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein includes each of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A1, and at least one or two of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein includes all six CDR amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein contains six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 449-453 and 475; (b) SEQ ID NOs: 476-477 and 454-457; (c) SEQ ID NOs: 458-463; (d) SEQ ID NOs: 57, 58 and 464-467; and (e) SEQ ID NOs: 68-71 and 468-469.

[0138] In various cases, the amino acid sequences in Table A1 are separated by at least one (for example, at least two, three, four, five, six, seven, eight, nine, ten, or more) intervening amino acids. In various cases, there are approximately 10 to 20 amino acids between the LC CDR1 and LC CDR2 sequences, and approximately 25 to 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are approximately 14 to 16 amino acids between the LC CDR1 and LC CDR2 sequences, and approximately 30 to 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are approximately 10 to 20 amino acids between the HC CDR1 and HC CDR2 sequences, and approximately 25 to 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are approximately 14 to 16 amino acids between the HC CDR1 sequence and the HC CDR2 sequence, and approximately 30 to 35 amino acids between the HC CDR2 sequence and the HC CDR3 sequence.

[0139] In various embodiments, the antigen-binding protein includes (a) a heavy chain variable region amino acid sequence listed in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 478, 480, 482, 486, and 488, or a variant sequence thereof, which differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (b) a light chain variable region amino acid sequence listed in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 479, 481, 483, 487, and 489, or a variant sequence thereof, which differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b). [Table 5]

[0140] In various embodiments, the antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 478 and 479; (b) SEQ ID NOs: 480 and 481; (c) SEQ ID NOs: 482 and 483; (d) SEQ ID NOs: 486 and 487; and (e) SEQ ID NOs: 488 and 489. In various embodiments, the antigen-binding protein comprises a variant sequence of the sequence having the SEQ ID NOs listed in Table B1, which differs by only one or two amino acids, or has at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, where the differing amino acid(s) are located at the positions described in “Humanized Antibodies” below.

[0141] Humanized antibodies

[0142] In various embodiments, the antigen-binding protein is a humanized version of the antigen-binding protein described in Table A, Table A1, Table B, or Table B1.

[0143] Humanized AB1

[0144] In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, having one or more amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 20, 31, 33, 35, 38, 40, 48, 50, 55, 57, 59, 61, 65, 66, 67, 68, 70, 72, 74, 76, 79, 80, 82, 87, 90, 91, 98, 101, and 116) at one or more of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) within the heavy chain variable region. In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 428. In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 20, 31, 35, 48, 50, 59, 67, 70, 74, 79, 98, 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 429. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 6]

[0145] In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, with the following positions within the light chain variable region: 1, 3, 4, 9, 10, 11, 15, 17, 21, 24, 27, 29, 32, 34, 35, 43, 44, 48, 51, 52, 53, 54, 55, 56, 61, 67, 71, 72, 73, 79, 80, 81, 84, 90, 92, 9 It has one or more amino acid substitutions in one or more of 3, 94, 95, 96, 101, 107 (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 430. In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 4, 21, 32, 34, 48, 51, 53, 61, 67, 79, 84, 91, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 431. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 7]

[0146] Humanized AB3

[0147] In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 3, 5, 18, 19, 23, 31, 33, 35, 40, 42, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 76, 79, 80, 81, 87, 94, 95, 99, 106, 112, 114 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 432. In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 31, 35, 50, 55, 79, 99, 106 (e.g., one, two, three, four, five, six, or seven). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 433. In various embodiments, the amino acids at the positions listed above are selected from the amino acids in the table below. [Table 8]

[0148] In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 9, 17, 18, 25, 27, 28, 30, 34, 40, 43, 45, 48, 50, 52, 53, 55, 56, 70, 72, 74, 76, 84, 85, 90, 91, 93, 94, 97, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 434. In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 25, 34, 48, 53, 55, 84, 85, 90, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 435. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 9]

[0149] Humanized AB4

[0150] In various embodiments, the antigen-binding protein is a humanized version of AB4, as described in Table B or B1, with the following positions within the heavy chain variable region: 5, 11, 12, 13, 20, 29, 31, 33, 37, 38, 40, 45, 48, 50, 55, 56, 57, 59, 61, 62, 65, 66, 67, 68, 70, 72, 74, 76, 79, 82 , has one or more amino acid substitutions in one or more of 84, 87, 91, 97, 101, 117 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 436. In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 20, 29, 31, 37, 45, 48, 56, 59, 61, 62, 65, 66, 68, 70, 74, 79, 84, 97, and 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 437. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 10]

[0151] In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 7, 14, 17, 18, 31, 33, 39, 41, 42, 44, 50, 51, 55, 57, 60, 81, 88, 92, 94, 95, 96, 99, 100, 105 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 438. In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 33, 39, 55, 57, 81, 95, and 96 (e.g., 1, 2, 3, 4, 5, 6, or 7). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 439. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 11]

[0152] Humanized AB9

[0153] In various embodiments, the antigen-binding protein is a humanized version of AB9 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 1, 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 63, 65, 67, 69, 70, 72, 73, 74, 76, 79, 84, 87, 91, 93, 112, and 113 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 444. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the table below. [Table 12]

[0154] In various embodiments, the antigen-binding protein is a humanized version of AB9 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 9, 11, 15, 17, 18, 43, 45, 70, 72, 73, 74, 80, 84, 85, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 445. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 13]

[0155] Humanized AB11

[0156] In various embodiments, the antigen-binding protein is a humanized version of AB11 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 1, 15, 18, 19, 42, 49, 63, 75, 76, 78, 80, 84, 88, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 446. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 14]

[0157] In various embodiments, the antigen-binding protein is a humanized version of AB11 as described in Table B or B1, and has one or more amino acid substitutions at the following positions within the light chain variable region: 4, 9, 17, 22, 64, 78, 80, 81, 82, 83, 84, 87, 89, 104, and 110 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), and optionally, one or more amino acid substitutions at one or more of the following positions: 4, 82, and 110. In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 447 or 448. In various embodiments, the amino acids at the positions listed above are selected from the amino acids in the table below. [Table 15]

[0158] In various embodiments, the antigen-binding protein is (a) a heavy chain variable region amino acid sequence as described in Table C, or a sequence selected from the group consisting of 376-379, 384-387, 391-396, 403-408, 412, 413, and 422-427, or a variant thereof, which differs by only one or two amino acids, or has at least 70%, approximately 70%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% sequence identity. (b) A sequence; or (b) A light chain variable region amino acid sequence as described in Table C, or a sequence selected from the group consisting of 380-383, 388-390, 397-402, 409-411, 414, 415, or a variant thereof which differs by only one or two amino acids, or which has at least 70% or about 70%, or about 80%, or about 85%, or about 90%, or about 95% sequence identity, or (c) Both (a) and (b). [Table 16]

[0159] In various embodiments, the humanized antigen-binding protein comprises pairs of amino acid sequences shown in Table D. Further embodiments include a CLDN6-specific antigen-binding protein comprising CDR1-3 derived from the heavy chain variable region shown in Table D; and CDR1-3 derived from the light chain variable region shown in Table D. In preferred embodiments, this CLDN6-specific antigen-binding protein comprises (i) CDR1-3 derived from the heavy chain variable region comprising the amino acid sequence described in SEQ ID NO: 387; and (ii) CDR1-3 derived from the light chain variable region comprising the amino acid sequence described in SEQ ID NO: 389 in Table D. [Table 17-1] [Table 17-2]

[0160] In various embodiments, the antigen-binding protein comprises a pair of variant sequences, each having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with the sequence numbers listed in Table C. In various embodiments, the antigen-binding protein comprises a pair of sequences: one sequence selected from the sequence numbers listed in Table C and a sequence having the sequence number listed in Table D, and another sequence which is a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with the sequence having the sequence number listed in Table C.

[0161] In various embodiments, the antigen-binding protein comprises a pair of sequences: one sequence selected from the sequence numbers listed in Table D, and another sequence which is a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with the sequence having the sequence numbers listed in Table D. For example, in various embodiments, the antigen-binding protein comprises the sequence of sequence number 387, and the antigen-binding protein further comprises a variant sequence having at least 70% or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with sequence number 389.

[0162] In various cases, the antigen-binding protein is a humanized antigen-binding protein listed in Table D, having one or more amino acid substitutions in the heavy chain (HC) variable region, the light chain (LC) variable region, or both (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). In an exemplary embodiment, this antigen-binding protein is a humanized antigen-binding protein named AB1-11, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, this antigen-binding protein contains the HC of SEQ ID NO: 379 and has one, two, three, four, or five amino acid substitutions. In exemplary embodiments, this antigen-binding protein includes HC CDR1 of SEQ ID NO: 504, HC CDR2 of SEQ ID NO: 505, HC CDR3 of SEQ ID NO: 506, or a combination thereof. In exemplary cases, this antigen-binding protein includes the HC of SEQ ID NO: 503. In some embodiments, this antigen-binding protein includes one of the HCs from SEQ ID NOs: 496-501. In various cases, the light chain variable region includes LC CDR1 of SEQ ID NO: 449, LC CDR2 of SEQ ID NO: 450, LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, this antigen-binding protein includes one of the LCs from SEQ ID NOs: 380-383 and 479. In exemplary cases, this antigen-binding protein includes the LC of SEQ ID NO: 383. In exemplary embodiments, this antigen-binding protein is a humanized antigen-binding protein AB3-7 having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, this antigen-binding protein contains the HC of SEQ ID NO: 387 and has one, two, three, four, five, or six amino acid substitutions. In exemplary embodiments, this antigen-binding protein contains HC CDR1 of SEQ ID NO: 507, HC CDR2 of SEQ ID NO: 508, HC CDR3 of SEQ ID NO: 509, or a combination thereof. In exemplary cases, this antigen-binding protein contains the HC of SEQ ID NO: 502. In some embodiments, this antigen-binding protein contains one of the HCs of SEQ ID NOs: 490-495.In various cases, the light chain variable region includes LC CDR1 of SEQ ID NO: 476, LC CDR2 of SEQ ID NO: 477, LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, this antigen-binding protein includes one of the LCs of SEQ ID NOs: 388-390 and 481. In an exemplary embodiment, this antigen-binding protein includes the LC of SEQ ID NO: 389. In an exemplary embodiment, this antigen-binding protein is a humanized antigen-binding protein of AB3 and has one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, this antigen-binding protein includes the HC of SEQ ID NO: 139 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, this antigen-binding protein includes one of the HCs of SEQ ID NO: 510. In an exemplary embodiment, the antigen-binding protein includes the HC of SEQ ID NO: 138 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, this antigen-binding protein contains any one of the HC sequences of SEQ ID NO: 511. In exemplary embodiments, this antigen-binding protein is a humanized antigen-binding protein of AB1, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, this antigen-binding protein contains the HC sequence of SEQ ID NO: 135 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, this antigen-binding protein contains any one of the HC sequences of SEQ ID NO: 513. In exemplary embodiments, this antigen-binding protein contains the HC sequence of SEQ ID NO: 134 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, this antigen-binding protein contains any one of the HC sequences of SEQ ID NO: 512. In some embodiments, this antigen-binding protein contains the HC sequence of SEQ ID NO: 512 and has one, two, three, four, or five (or more) amino acid substitutions.

[0163] Compositions, pharmaceutical compositions and formulations

[0164] Compositions comprising the antigen-binding proteins of this disclosure are provided herein.

[0165] In some embodiments, the composition includes an agent that enhances the chemical and physical properties of an antigen-binding protein by, for example, stabilizing the antigen-binding protein at a specific temperature, such as room temperature; extending its shelf life; inhibiting degradation, such as oxidative protease-mediated degradation; or extending the half-life of the antigen-binding protein. In some embodiments, the composition includes one of the agents disclosed herein as a heterogeneous portion, which is optionally mixed with or conjugated to the antigen-binding protein of the Disclosure.

[0166] In various aspects of this disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the conjugate of this disclosure (hereinafter referred to as the “Activator”) is formulated into a pharmaceutical composition comprising the Activator together with a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, this disclosure further provides pharmaceutical compositions comprising an Activator intended for administration to a subject, e.g., a mammal.

[0167] In some embodiments, the activator is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the activator has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains the activator at a concentration of about 0.001 to about 30.0 mg / ml.

[0168] In various embodiments, this pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The term also includes any of the drugs approved by U.S. federal regulatory authorities or listed in the United States Pharmacopeia for use in animals, including humans.

[0169] Pharmaceutical compositions include, for example, acidifying agents, additives, adsorbents, aerosol propellants, air replacement agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, cleaning agents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, pigments, emollients, emulsifiers, emulsion stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, fluidity enhancers, and gels. It may contain any pharmaceutically acceptable ingredients, including humectants, granulators, humectants, lubricants, mucosal adhesives, ointment bases, ointments, oily vehicles, organic bases, lozenge bases, pigments, plasticizers, abrasives, preservatives, metal ion sequestering agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, viscosity enhancers, tonic agents, toxic agents, thickeners, water absorbents, water-miscible cosolvents, hard water softeners, or wetting agents. For example, see Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (the whole is incorporated by reference) and Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (the whole is incorporated by reference).

[0170] In various embodiments, the pharmaceutical composition comprises formulation materials that are non-toxic to the recipient at the dosage and concentration used. In specific embodiments, the pharmaceutical composition comprises an activator and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic regulators, tonic agents, antioxidants, antibiotics, antifungals, bulking agents, lyophilization protectants, defoamers, chelating agents, preservatives, colorants, analgesics, or additional pharmaceutical agents. In various embodiments, the pharmaceutical composition comprises one or more polyols and / or one or more surfactants, optionally in addition to one or more excipients, including but not limited to pharmaceutically acceptable salts, osmotic regulators (tonic agents), antioxidants, antibiotics, antifungals, bulking agents, lyophilization protectants, defoamers, chelating agents, preservatives, colorants, and analgesics.

[0171] In certain embodiments, the pharmaceutical composition may include, for example, formulation materials for altering, maintaining, or preserving the composition's pH, volume molar osmotic pressure concentration, viscosity, clarity, color, isotonicity, aroma, sterility, stability, rate of dissolution or release, adsorption, or osmosis. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, tris-HCl, citrate, phosphate, glutamate, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; and salt-forming counterions (e.g., Examples include sodium; preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic acid, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20 or polysorbate 80 (PS80), polysorbate C, triton, tromethamine, lecithin, cholesterol, tyroxapole); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company.

[0172] This pharmaceutical composition can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, about 4 or about 5 to about 8.0 or about 4.5 to about 7.5 or about 5.0 to about 7.5. In various embodiments, the pH of this pharmaceutical composition is 5.5 to 7.5.

[0173] Route of administration

[0174] With respect to this disclosure, an activator, or a pharmaceutical composition containing the same, may be administered to a subject via any preferred route of administration. For example, the activator may be administered to a subject via parenteral administration, such as intravenous infusion or subcutaneous injection.

[0175] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain an antioxidant, a buffer, a bacteriostatic agent, and a solute that renders the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain a suspending agent, a solubilizing agent, a thickening agent, a stabilizer, and a preservative. The term "parenteral" means by some other route than through the digestive tract, such as subcutaneously, intramuscularly, intraspinally, or intravenously. The active agents of the present disclosure may or may not contain a pharmaceutically acceptable surfactant, such as a soap or detergent, a suspending agent, such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or an emulsifying agent and other pharmaceutical adjuvants, in a pharmaceutical carrier such as water, physiological saline, aqueous dextrose and related sugar solutions, alcohols, such as ethanol or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals, such as 2,2-dimethyl-1,3-dioxolan-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, and may be administered with a physiologically acceptable diluent in a sterile liquid or mixture of liquids.

[0176] Oils that can be used in parenteral formulations include petroleum, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil. Fatty acids suitable for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0177] Suitable soaps for use in parenteral formulations include fatty acid alkali metals, ammonium compounds, and triethanolamine salts, and suitable detergents include (a) cationic detergents, such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ethers, and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents, such as aliphatic amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0178] Parenteral formulations, in some embodiments, contain about 0.5% to about 25% by weight of the activator of the present disclosure in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations may typically be in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide having a hydrophobic base, formed by the condensation of propylene oxide and propylene glycol. Parenteral formulations, in some embodiments, are provided in sealed containers of unit or multiple doses, such as ampoules and vials, and can be stored in a freeze-dried state requiring only the addition of a sterile liquid excipient, such as water, immediately before use for injection. In some embodiments, the injectable solutions and suspensions prepared immediately before use are prepared from the types of sterile powders, granules, and tablets described above.

[0179] Injectable formulations comply with the present disclosure. The requirements for an effective pharmaceutical carrier for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Injectable formulations suitable for subcutaneous administration may be co-formulated with hyaluronidase (U.S. Patent No. 9,084,743).

[0180] Kit

[0181] In some embodiments, the antigen-binding proteins of the present disclosure are provided in a kit. In various aspects, the kit includes the ADC as a unit dose. For the purposes herein, "unit dose" refers to an individual amount dispersed in a suitable carrier. In various aspects, the unit dose is an amount sufficient to provide a desired effect in a subject, such as inhibition of tumor growth, reduction of tumor size, treatment of cancer. Accordingly, kits are provided herein that optionally include the ADCs of the present disclosure in unit doses. In various aspects, the kit includes several unit doses, for example, unit doses for one week or one month, and optionally, each of which is individually packaged or otherwise separated from other unit doses. In some embodiments, the kit / unit dose components are packaged with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, such as needles and syringes. In some aspects, the ADCs of the present disclosure are pre-packaged in a ready-to-use form, such as a syringe, drip bag, etc. In some aspects, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (such as solvents, buffers, diluents, etc.) including any of those described herein.

[0182] Various embodiments In certain embodiments, the present disclosure provides a method for inhibiting a solid tumor expressing claudin-6 in a human subject, comprising administering to a human subject an effective amount of a composition comprising a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous moiety having the following structural formula (I): [ka] During the ceremony, The first group of conjugates is bonded to four heterogeneous parts, including structural formula (I); At least about 95% of the first multiple conjugates are structurally homogeneous; and The effective amount is approximately 1.7 mg / kg to 6 mg / kg; 2.0 mg / kg to 6 mg / kg; or This concerns the method, which falls within the range of 2.4 mg / kg to 6 mg / kg.

[0183] In certain embodiments, this disclosure is any method disclosed herein, including post-administration C max The present invention relates to a method for reducing the toxicity of a target and inhibiting solid tumors by having a percentage of unbound circulating MMAE in serum less than approximately 0.01% (w / v). In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.009%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.008%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.007%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.006%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.005%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.004%. In certain embodiments, the percentage of free MMAE is C maxIt is less than approximately 0.003%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.0000010%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.0000009%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.0000008%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.0000007%. In certain embodiments, the percentage of free MMAE is C max This is less than approximately 0.0000006%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.0000005%. In certain embodiments, the percentage of free MMAE is C max It is less than approximately 0.0000004%. In certain embodiments, the percentage of free MMAE is C max This is less than approximately 0.0000003%. In certain embodiments, the free MMAE is C max The percentage is determined after a single dose in cycle 1. In another embodiment, the C of free MMAE max The percentage is determined in cycle 3 after three separate administrations at 3-week intervals of a composition containing a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous portion. In certain embodiments, the conjugate is an anti-CLDN6-specific antibody conjugated to a chemotherapeutic agent or cytotoxic agent. In certain embodiments, the conjugate is TORL-1-23.

[0184] In certain embodiments, this disclosure is any method disclosed herein, including post-administration C max This invention relates to a method for inhibiting solid tumors by reducing the toxicity of the target by reducing the amount of unbound circulating MMAE in the serum to less than approximately 5 ng / mL. In certain embodiments, free MMAE is C max It is less than approximately 4 ng / mL. In certain embodiments, free MMAE is C max It is less than approximately 3 ng / mL. In certain embodiments, free MMAE is C maxis less than about 2 ng / mL. In certain embodiments, the free MMAE is C max is less than about 1 ng / mL. In certain embodiments, the free MMAE is C max is less than about 0.5 ng / mL. In certain embodiments, the free MMAE is C max is less than about 0.15 ng / mL. In certain embodiments, the free MMAE is C max is less than about 0.12 ng / mL.

[0185] In certain embodiments, the present disclosure relates to any method disclosed herein, wherein the unbound circulating MMAE in serum is less than about 10 ng / mL at C max after administration, thereby reducing the toxicity of the subject and inhibiting solid tumors. In certain embodiments, the free MMAE is C max is less than about 9 ng / mL. In certain embodiments, the free MMAE is C max is less than about 8 ng / mL. In certain embodiments, the free MMAE is C max is less than about 7 ng / mL. In certain embodiments, the free MMAE is C max is less than about 6 ng / mL. In certain embodiments, the free MMAE is C max is less than about 5 ng / mL. In certain embodiments, the free MMAE is C max is less than about 4 ng / mL. In certain embodiments, the free MMAE is C max is less than about 3 ng / mL.

[0186] In certain embodiments, the administration is a single-dose administration of the conjugate at a dose of about 1.7 mg / kg to 6.0 mg / kg (including both ends). In certain embodiments, the administration is a single-dose administration of the conjugate at a dose of about 1.7 mg / kg to 3.0 mg / kg (including both ends). In certain embodiments, the percentage of C max of free MMAE is determined after administration of a single dose in cycle 1. In another embodiment, the C maxThe percentage is determined in cycle 3 after three separate doses at 3-week intervals of a composition containing a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous portion. In certain embodiments, the dose is a single dose of the conjugate of approximately 1.7 mg / kg to 3.0 mg / kg (including both ends). In certain embodiments, the conjugate is an anti-CLDN6-specific antibody conjugated to a chemotherapeutic agent or cytotoxic agent. In certain embodiments, the conjugate is TORL-1-23.

[0187] In certain embodiments, this disclosure relates to any method disclosed herein for dose normalization of unbound (free) circulating MMAEs. max The present invention relates to a method for reducing the toxicity of the target and inhibiting solid tumors, where the free MMAE value after administration of the conjugate is less than approximately 35 ng / mL per 1 mg of conjugate. In certain embodiments, dose-normalized C for free MMAE is used. max The value is less than approximately 30 pg / mL per 1 mg of conjugate. In certain embodiments, dose-normalized C for free MMAE is used. max The value is less than approximately 25 pg / mL per 1 mg of conjugate. In certain embodiments, dose-normalized C for free MMAE is used. max The value is less than approximately 20 pg / mL per 1 mg of conjugate. In certain embodiments, dose-normalized C for free MMAE is used. max The value is less than approximately 15 pg / mL per 1 mg of conjugate. In certain embodiments, dose-normalized C for free MMAE is used. max The value is less than approximately 10 pg / mL per 1 mg of conjugate. In certain embodiments, dose-normalized C max This refers to C, measured after a single dose of the conjugate. max Determined from. In certain embodiments, dose normalization C max This refers to the C after administering the same dose three times with a 3-week interval between each dose. max It is determined from the measured values. In certain embodiments, the conjugate is an anti-CLDN6 specific antibody conjugated to a chemotherapeutic agent or cytotoxic agent. In certain embodiments, the conjugate is TORL-1-23.

[0188] In certain embodiments, this disclosure is any method disclosed herein, wherein C after the initial administration of a 3.0 mg / kg conjugate. max This invention relates to a method for inhibiting solid tumors by reducing the toxicity of the target organism and thereby reducing the unbound circulating MMAE in the serum to less than approximately 2.6 ng / mL.

[0189] In certain embodiments, this disclosure is any method disclosed herein, wherein C after the initial administration of a 2.4 mg / kg conjugate. max In this case, the amount of unbound circulating MMAE in serum was approximately 4.1 + This invention relates to a method of inhibiting solid tumors by reducing the toxicity of the target by using a concentration of 2.1 ng / mL.

[0190] In certain embodiments, this disclosure is any method disclosed herein, wherein C after the initial administration of a 2.0 mg / kg conjugate. max In this case, the amount of unbound circulating MMAE in serum was approximately 3.7 + This invention relates to a method of inhibiting solid tumors by reducing the toxicity of the target substance at a concentration of 1.6 ng / mL.

[0191] In certain embodiments, this disclosure is any method disclosed herein, wherein C after the initial administration of 1.7 mg / kg of the conjugate. max In this case, the amount of unbound circulating MMAE in serum was approximately 2.7 + This invention relates to a method for inhibiting solid tumors by reducing the toxicity of the target, using a concentration of 1.9 ng / mL. In certain embodiments, the conjugate is an anti-CLDN6 specific antibody conjugated to a chemotherapeutic agent or cytotoxic agent. In certain embodiments, the conjugate is TORL-1-23.

[0192] In certain embodiments, this disclosure is any method disclosed herein, wherein the C of Cycle 3 after three administrations of the 1.7 mg / kg conjugate. max In this case, the amount of unbound circulating MMAE in serum was approximately 1.4 +It is 0.8 ng / mL, where this conjugate is administered once every three weeks, and cycle 3 starts with the third administration of the conjugate, thereby reducing the toxicity of the subject and inhibiting solid tumors. In certain embodiments, this conjugate is an anti-CLDN6 specific antibody conjugated to a chemotherapeutic agent or a cytotoxic agent. In certain embodiments, this conjugate is TORL-1-23.

[0193] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is an ovarian tumor.

[0194] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is a bladder tumor.

[0195] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is a testicular tumor.

[0196] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is an endometrial tumor.

[0197] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is a non-small cell lung cancer.

[0198] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is a primary peritoneal cancer.

[0199] In certain embodiments, the present disclosure relates to any method disclosed herein where the solid tumor is a fallopian tube cancer.

[0200] In certain embodiments, this disclosure relates to any method disclosed herein, wherein, after administration, human subjects do not experience peripheral neuropathy of grade 3 or higher severity, alopecia of grade 3 or higher severity, fatigue of grade 3 or higher severity, nausea, vomiting, or loss of appetite of grade 3 or higher severity, or constipation of grade 3 or higher severity, or any combination of two or more of these adverse events. In certain embodiments, this disclosure relates to any method disclosed herein, wherein, after administration, human subjects do not experience any adverse events of grade 3 or higher severity.

[0201] In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is approximately 1.7 mg / kg to 5 mg / kg, 1.7 mg / kg to 4 mg / kg, or 1.7 mg / kg to 3 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is approximately 2.0 mg / kg to 5 mg / kg, 2.0 mg / kg to 4 mg / kg, or 2.0 mg / kg to 3 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is approximately 2.4 mg / kg to 5 mg / kg, 2.4 mg / kg to 4 mg / kg, or 2.4 mg / kg to 3 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is approximately 1.7 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is approximately 2.0 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is about 2.4 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is about 3.0 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is about 4 mg / kg. In certain embodiments, this disclosure relates to any method disclosed herein, wherein the effective dose is about 5 mg / kg.

[0202] In certain embodiments, the disclosure relates to any method disclosed herein, wherein an effective amount of the composition is administered once every 1 to 4 weeks, for example, once every 2 to 4 weeks, preferably once every 3 weeks.

[0203] In certain embodiments, this disclosure relates to any method disclosed herein by which the composition is administered intravenously.

[0204] In certain embodiments, the disclosure relates to any method disclosed herein, wherein an effective amount of the composition is administered over a period of about 20 to about 40 minutes, preferably over a period of about 30 minutes.

[0205] In certain embodiments, this disclosure relates to any method disclosed herein for a human subject to achieve a partial response.

[0206] In certain embodiments, this disclosure relates to any method disclosed herein for a human subject to achieve a complete response.

[0207] In certain embodiments, the disclosure relates to any method disclosed herein, wherein the composition further comprises glutamate-sodium hydroxide buffer, sucrose, and polysorbate 80 (PS80).

[0208] In certain embodiments, the disclosure relates to any method disclosed herein, wherein the composition further comprises dextrose and water.

[0209] In certain embodiments, the disclosure relates to any method disclosed herein, wherein the composition further comprises sodium chloride and water.

[0210] In certain embodiments, the disclosure relates to any method disclosed herein in which the average number of heterologous moieties per CLDN6-specific antigen-binding protein in the composition is about 3.5 to about 4, for example, about 3.6 to about 3.9, preferably about 3.7 to about 3.8. In certain embodiments, the disclosure relates to any method disclosed herein in which the average number of heterologous moieties per CLDN6-specific antigen-binding protein in the composition is about 3.8 to about 3.9. In certain embodiments, the disclosure relates to any method disclosed herein in which the average number of heterologous moieties per CLDN6-specific antigen-binding protein in the composition is at least 3.8.

[0211] In certain embodiments, the disclosure relates to any method disclosed herein, wherein in a first plurality of conjugates, heterologous moieties are covalently conjugated at unpaired cysteine ​​residues of a CLDN6-specific antigen-binding protein.

[0212] In certain embodiments, the disclosure relates to any method disclosed herein, wherein in a first plurality of conjugates, heterologous moieties are covalently conjugated at cysteine ​​residues resulting from the cleavage of interchain disulfide bonds between the heavy and light chains of a CLDN6-specific antigen-binding protein.

[0213] In certain embodiments, the disclosure relates to any method disclosed herein, wherein about 95%, about 96%, about 97%, or about 98% of the first plurality of conjugates are structurally homogeneous.

[0214] In certain embodiments, the disclosure relates to any method disclosed herein, wherein a second plurality of conjugates are bonded to eight heterogeneous parts comprising structural formula (I).

[0215] In certain embodiments, the disclosure relates to any method disclosed herein, wherein a third plurality of conjugates are bonded to two heterogeneous parts comprising structural formula (I).

[0216] In certain embodiments, the disclosure relates to any method disclosed herein, wherein a fourth plurality of conjugates are bonded to six heterogeneous parts comprising structural formula (I).

[0217] In certain embodiments, the present disclosure relates to any method disclosed herein, wherein a heterogeneous portion including structural formula (I) has the following structural formula (II): [ka] During the ceremony, [ka] However, this concerns a method involving covalent thiol binding to a CLDN6-specific antigen-binding protein.

[0218] In certain embodiments, this disclosure relates to any method disclosed herein in which the subject has not received prior treatment for a solid tumor.

[0219] In certain embodiments, the disclosure relates to any method disclosed herein, wherein the subject has received at least one prior treatment for a solid tumor. In certain embodiments, the disclosure relates to any method disclosed herein, wherein the subject has received at least two prior treatments for a solid tumor. In certain embodiments, the disclosure relates to any method disclosed herein, wherein the subject has received at least three prior treatments for a solid tumor. In certain embodiments, the disclosure relates to any method disclosed herein, wherein the subject has received at least four prior treatments for a solid tumor.

[0220] In certain embodiments, this disclosure relates to any method disclosed herein, wherein the CLDN6-specific antigen-binding protein comprises the group consisting of: (a) The heavy chain CDR1 amino acid sequence listed in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (b) The heavy chain CDR2 amino acid sequence described in Table A or Table A1, or a variant thereof that differs by only one or two amino acids; (c) The heavy chain CDR3 amino acid sequence listed in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (d) The light chain CDR1 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (e) The light chain CDR2 amino acid sequence listed in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (f) The light chain CDR3 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; or The present invention relates to a method selected from any two or more combinations of (a) to (f).

[0221] In certain embodiments, the present disclosure relates to any method disclosed herein, wherein the CLDN6-specific antigen-binding protein comprises a heavy chain CDR amino sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, 455, 456, and 457, and variant sequences thereof that differ by only one or two amino acids.

[0222] In certain embodiments, the present disclosure relates to any method disclosed herein, wherein the CLDN6-specific antigen-binding protein comprises a light chain CDR amino sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 476, 477, and 454, and variant sequences thereof that differ by only one or two amino acids.

[0223] In certain embodiments, this disclosure relates to any method disclosed herein, wherein the CLDN6-specific antigen-binding protein comprises an antibody or antigen-binding fragment that binds to CLDN6, and this antibody (a) The heavy chain CDR1 amino acid sequence described in SEQ ID NO: 23 or 455, or a variant thereof that differs by only one or two amino acids; (b) The heavy chain CDR2 amino acid sequence described in SEQ ID NO: 24 or 456, or a variant thereof that differs by only one or two amino acids; (c) The heavy chain CDR3 amino acid sequence described in SEQ ID NO: 25 or 457, or a variant thereof that differs by only one or two amino acids; (d) The light chain CDR1 amino acid sequence described in SEQ ID NO: 20 or 476, or a variant thereof that differs by only one or two amino acids; (e) The light chain CDR2 amino acid sequence described in Sequence ID No. 21 or 477, or a variant thereof that differs by only one or two amino acids; (f) The light chain CDR3 amino acid sequence described in SEQ ID NO: 22 or 454, or a variant sequence thereof that differs by only one or two amino acids; This relates to a method that includes any two or more combinations of (a) to (f).

[0224] In certain embodiments, this disclosure relates to any method disclosed herein, wherein the CLDN6-specific antigen-binding protein comprises an antibody or antigen-binding fragment that binds to CLDN6, and this antibody The heavy chain (HC) CDR1-3 amino acid sequences of SEQ ID NOs. 23, 24, and 25, and the light chain (LC) CDR1-3 amino acid sequences of SEQ ID NOs. 20, 21, and 22; or The present invention relates to a method comprising the heavy chain (HC)CDR1-3 amino acid sequences of SEQ ID NOs. 455, 456, and 457, and the light chain (LC)CDR1-3 amino acid sequences of SEQ ID NOs. 476, 477, and 454.

[0225] Furthermore, this disclosure includes a conjugate of a CLDN6-specific antigen-binding protein covalently bonded to a heterologous portion including structural formula (I), [ka] During the ceremony, The first group of conjugates is bonded to four heterogeneous parts, including structural formula (I); At least about 95% of the first multiple conjugates are structurally homogeneous; and The effective amount is approximately 1.7 mg / kg to 6 mg / kg; 2.0 mg / kg to 6 mg / kg; or The dosage range is 2.4 mg / kg to 6 mg / kg (for use in cancer treatment).

[0226] In further embodiments, the present disclosure is any method disclosed herein, wherein the conjugate CLDN6-specific antigen-binding protein comprises an antibody or antigen-binding fragment that binds to CLDN6, and this antibody: (i) HC CDR1 having sequence GFTFSNYW (sequence number 23); (ii) HC CDR2 having sequence IRLKSDNYAT (sequence number 24), (iii) HC CDR3 having sequence XDGPPSGX (sequence number 457) (wherein the formula, the X at position 1 is N and the X at position 8 is S, T, A, C, or Y), (iv) LC CDR1 having sequence ENIYSY (sequence number 20), (v) LC CDR2 having sequence NAK (sequence number 21), and (vi) A method comprising LC CDR3 having sequence QHHYTVPWT (sequence number 22).

[0227] TORL-1-23 (also known as CLDN6-23-ADC) is an antibody-drug conjugate containing an antibody that binds to CLDN6, and this antibody... (i) HC CDR1 having sequence GFTFSNYW (sequence number 23); (ii) HC CDR2 having sequence IRLKSDNYAT (sequence number 24), (iii) HC CDR3 having the sequence XDGPPSGX (sequence number 457) (wherein the formula, the X at position 1 is N and the X at position 8 is S), (iv) LC CDR1 having sequence ENIYSY (sequence number 20), (v) LC CDR2 having sequence NAK (sequence number 21), and (vi) LC CDR3 having sequence QHHYTVPWT (sequence number 22), Here, this antibody is conjugated to approximately four molecules of MMAE, each via a linker containing Val-Cit-PAB.

[0228] Additional exemplary embodiments

[0229] 1. A method for inhibiting solid tumors expressing claudin-6 (CLDN6) in human subjects, comprising administering to a human subject an effective amount of a composition containing a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous portion having the following structural formula (I): [ka] During the ceremony, The first plurality of the aforementioned conjugates are bonded to four heterogeneous parts including structural formula (I); At least about 95% of the first plurality of conjugates are structurally homogeneous; and The effective amount is approximately 1.7 mg / kg to 6 mg / kg; 2.0 mg / kg to 6 mg / kg; or A method within the range of 2.4 mg / kg to 6 mg / kg.

[0230] The method described in 2.1, wherein C after administration max A method in which the percentage of unbound circulating MMAE in serum is less than approximately 0.01% (w / v), thereby reducing the toxicity of the target and inhibiting solid tumors.

[0231] 3. The percentage of the unbound circulating MMAE is C max The method described in 2, which is less than approximately 0.009% (w / v).

[0232] 4. The percentage of the unbound circulating MMAE is C max The method described in 3, which is less than approximately 0.008% (w / v).

[0233] 5. The percentage of the unbound circulating MMAE is C maxThe method described in 4, which is less than approximately 0.007% (w / v).

[0234] 6. The percentage of the unbound circulating MMAE is C max The method described in 5, which is less than approximately 0.006% (w / v).

[0235] 7. The percentage of the unbound circulating MMAE is C max The method described in 6, which is less than approximately 0.005% (w / v).

[0236] 8. The percentage of the unbound circulating MMAE is C max The method described in 7, which is less than approximately 0.004% (w / v).

[0237] 9. The percentage of the unbound circulating MMAE is C max The method described in 8, which is less than approximately 0.003% (w / v).

[0238] 10. Post-administration C max The method according to 1, wherein the percentage of unbound circulating MMAE in the serum is less than approximately 9 ng / mL, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0239] 11. The unbound circulating MMAE is C max The method described in 10, wherein the concentration is less than approximately 8 ng / mL.

[0240] 12. The unbound circulating MMAE is C max The method according to 11, wherein the concentration is less than approximately 7 ng / mL.

[0241] 13. The unbound circulating MMAE is C max The method described in 12, which is less than approximately 6 ng / mL.

[0242] 14. The unbound circulating MMAE is C max The method described in 13, which is less than approximately 5 ng / mL.

[0243] 15. The unbound circulating MMAE is Cmax The method described in 14, which is less than approximately 4 ng / mL.

[0244] 16. The unbound circulating MMAE is C max The method described in 15, which is less than approximately 3 ng / mL.

[0245] 17. Dose-normalized C of the unbound (free) circulating MMAE max The method according to 1, wherein the value is less than approximately 35 ng / mL per 1 mg of conjugate after administration of the conjugate, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0246] 18. Dose-normalized C for the unbound circulating MMAE max The method according to 17, wherein the value is less than approximately 30 pg / mL per 1 mg of conjugate.

[0247] 19. Dose-normalized C for the unbound circulating MMAE max The method according to 18, wherein the value is less than approximately 25 pg / mL per 1 mg of conjugate.

[0248] 20. Dose-normalized C for the unbound circulating MMAE max The method according to 19, wherein the value is less than approximately 20 pg / mL per 1 mg of conjugate.

[0249] 21. Dose-normalized C for the unbound circulating MMAE max The method described in 20, wherein the value is less than approximately 15 pg / mL per 1 mg of conjugate.

[0250] 22. Dose-normalized C for the unbound circulating MMAE max The method according to 21, wherein the value is less than approximately 10 pg / mL per 1 mg of conjugate.

[0251] C after the first dose of the 23.3.0 mg / kg conjugate max The method according to 1, wherein the amount of unbound circulating MMAE in the serum is less than approximately 2.6 ng / mL, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0252] C after the first dose of the 24.2.4 mg / kg conjugate max In this case, the amount of unbound circulating MMAE in serum was approximately 4.1 + The method according to 1, wherein the concentration is 2.1 ng / mL, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0253] C after the first dose of the 25.2.0 mg / kg conjugate max In this case, the amount of unbound circulating MMAE in serum was approximately 3.7 + The method according to 1, wherein the concentration is 1.6 ng / mL, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0254] C after the first dose of the 26.1.7 mg / kg conjugate max In this case, the amount of unbound circulating MMAE in serum was approximately 2.7 + The method according to 1, wherein the concentration is 1.9 ng / mL, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0255] C in cycle 3 after three administrations of the 27.1.7 mg / kg conjugate max In this case, the amount of unbound circulating MMAE in serum is approximately 1.4 + The method according to 1, wherein the concentration is 0.8 ng / mL, and the conjugate is administered once every three weeks, with cycle 3 beginning with the third administration of the conjugate, thereby reducing the toxicity of the target and inhibiting solid tumors.

[0256] 28. The method according to any one of items 1 to 27, wherein the solid tumor is an ovarian tumor.

[0257] 29. The method according to any one of items 1 to 27, wherein the solid tumor is a bladder tumor.

[0258] 30. The method according to any one of items 1 to 27, wherein the solid tumor is a testicular tumor.

[0259] 31. The method according to any one of items 1 to 27, wherein the solid tumor is an endometrial tumor.

[0260] 32. The method according to any one of items 1 to 27, wherein the solid tumor is non-small cell lung cancer.

[0261] 33. The method according to any one of items 1 to 27, wherein the solid tumor is primary peritoneal cancer.

[0262] 34. The method according to any one of items 1 to 27, wherein the solid tumor is fallopian tube cancer.

[0263] A method according to any one of items 35.1 to 34, wherein, after administration, the human subjects do not experience peripheral neuropathy of grade 3 or higher severity, alopecia of grade 3 or higher severity, fatigue of grade 3 or higher severity, nausea, vomiting, or loss of appetite of grade 3 or higher severity, or constipation of grade 3 or higher severity, or any combination of two or more of these adverse events.

[0264] 36. The method according to any one of items 1 to 35, wherein the human subjects do not experience any adverse events of grade 3 or higher severity after administration.

[0265] 37. The method according to any one of items 1 to 36, wherein the effective dose is approximately 1.7 mg / kg to 5 mg / kg, 1.7 mg / kg to 4 mg / kg, or 1.7 mg / kg to 3 mg / kg.

[0266] 38. The method according to any one of items 1 to 36, wherein the effective dose is approximately 2.0 mg / kg to 5 mg / kg, 2.0 mg / kg to 4 mg / kg, or 2.0 mg / kg to 3 mg / kg.

[0267] 39. The method according to any one of items 1 to 36, wherein the effective dose is approximately 2.4 mg / kg to 5 mg / kg, 2.4 mg / kg to 4 mg / kg, or 2.4 mg / kg to 3 mg / kg.

[0268] 40. The method according to any one of items 1 to 36, wherein the effective dose is approximately 1.7 mg / kg.

[0269] 41. The method according to any one of items 1 to 36, wherein the effective dose is approximately 2.0 mg / kg.

[0270] 42. The method according to any one of items 1 to 36, wherein the effective dose is approximately 2.4 mg / kg.

[0271] 43. The method according to any one of items 1 to 36, wherein the effective dose is approximately 3.0 mg / kg.

[0272] 44. The method according to any one of items 1 to 36, wherein the effective dose is approximately 3.6 mg / kg.

[0273] 45. The method according to any one of items 1 to 36, wherein the effective dose is approximately 4 mg / kg.

[0274] 46. ​​The method according to any one of claims 1 to 45, wherein the effective amount of the composition is administered once every 1 to 4 weeks, for example, once every 2 to 4 weeks, preferably once every 3 weeks.

[0275] 47. The method according to any one of items 1 to 46, wherein the composition is administered intravenously.

[0276] 48. The method according to any one of claims 1 to 47, wherein the effective amount of the composition is administered over a period of about 20 minutes to about 40 minutes, preferably over a period of about 30 minutes.

[0277] 49. The method according to any one of items 1 to 48, wherein the human subject has achieved a partial response.

[0278] 50. The method according to any one of items 1 to 48, wherein the human subject has achieved a complete response.

[0279] 51. The method according to any one of claims 1 to 50, wherein the composition further comprises glutamic acid-sodium hydroxide buffer, sucrose, and polysorbate 80 (PS80).

[0280] 52. The method according to any one of claims 1 to 51, wherein the composition further comprises dextrose and water.

[0281] 53. The method according to any one of items 1 to 51, wherein the composition further comprises sodium chloride and water.

[0282] 54. The method according to any one of claims 1 to 53, wherein the average number of heterologous moieties per CLDN6-specific antigen-binding protein in the composition is about 3.5 to about 4, for example, about 3.6 to about 3.9, preferably about 3.7 to about 3.8.

[0283] 55. The method according to any one of items 1 to 54, wherein in the first plurality of conjugates, the heterogeneous portion is covalently conjugated with an unpaired cysteine ​​residue of a CLDN6-specific antigen-binding protein.

[0284] 56. The method according to any one of items 1 to 54, wherein in the first plurality of conjugates, the heterogeneous portion is covalently conjugated by a cysteine ​​residue resulting from the cleavage of the interchain disulfide bond between the heavy chain and light chain of the CLDN6-specific antigen-binding protein.

[0285] 57. The method according to any one of items 1 to 56, wherein about 95%, about 96%, about 97%, or about 98% of the first plurality of conjugates are structurally uniform.

[0286] A method according to any one of items 58.1 to 57, wherein the heterogeneous part including structural formula (I) is structural formula (II): [ka] It has, During the ceremony, [ka] The method is characterized by covalent thiol binding to a CLDN6-specific antigen-binding protein.

[0287] 59. The method according to any one of items 1 to 58, wherein the subject has not received prior treatment for the solid tumor.

[0288] 60. The method according to any one of paragraphs 1 to 58, wherein the subject has received at least one prior treatment for the solid tumor.

[0289] 61. The method according to any one of paragraphs 1 to 58, wherein the subject has received at least two prior treatments for the solid tumor.

[0290] 62. The method according to any one of paragraphs 1 to 58, wherein the subject has received at least three prior treatments for the solid tumor.

[0291] 63. The method according to any one of paragraphs 1 to 58, wherein the subject has received at least four prior treatments for the solid tumor.

[0292] 64. The method according to any one of items 1 to 63, wherein the effective dose is in the range of 3.0 mg / kg to 3.6 mg / kg.

[0293] 65. The method according to any one of claims 1 to 64, wherein the human subject is administered an effective amount of G-CSF before being administered an effective amount of the composition to the human subject.

[0294] A method according to any one of items 66.1 to 65, wherein the CLDN6-specific antigen-binding protein is as follows: (a) The heavy chain CDR1 amino acid sequence listed in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (b) The heavy chain CDR2 amino acid sequence described in Table A or Table A1, or a variant thereof that differs by only one or two amino acids; (c) The heavy chain CDR3 amino acid sequence listed in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (d) The light chain CDR1 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (e) The light chain CDR2 amino acid sequence listed in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (f) The light chain CDR3 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; or The method is selected from the group consisting of any two or more combinations of (a) to (f).

[0295] The method according to 67.66, wherein the CLDN6-specific antigen-binding protein comprises an antibody or antigen-binding fragment that binds to CLDN6, and the antibody is as follows: (a) the heavy chain (HC)CDR1-3 amino acid sequences of SEQ ID NOs. 23, 24, and 25, and the light chain (LC)CDR1-3 amino acid sequences of SEQ ID NOs. 20, 21, and 22; or (b) The method comprising the heavy chain (HC)CDR1-3 amino acid sequences of SEQ ID NOs. 455, 456, and 457, and the light chain (LC)CDR1-3 amino acid sequences of SEQ ID NOs. 476, 477, and 454.

[0296] A method according to any one of 68.1 to 67, wherein the CLDN6-specific antigen-binding protein comprises an antibody that binds to CLDN6 or an antigen-binding fragment thereof, wherein the antibody is as follows: (i) HC CDR1 having sequence GFTFSNYW (sequence number 23); (ii) HC CDR2 having sequence IRLKSDNYAT (sequence number 24), (iii) HC CDR3 having sequence XDGPPSGX (sequence number 457) (wherein the formula, the X at position 1 is N and the X at position 8 is S, T, A, C, or Y), (iv) LC CDR1 having sequence ENIYSY (sequence number 20), (v) LC CDR2 having sequence NAK (sequence number 21), and (vi) The method comprising LC CDR3 having sequence QHHYTVPWT (sequence number 22).

[0297] The method according to 69.68, wherein the CLDN6-specific antigen-binding protein is as follows: The method comprising HC CDR3 having the sequence XDGPPSGX (sequence number 457) (wherein the formula, the X at position 1 is N and the X at position 8 is S).

[0298] A method according to any one of items 70.1 to 67, wherein the CLDN6-specific antigen-binding protein comprises an antibody that binds to CLDN6 or an antigen-binding fragment thereof, wherein the antibody is as follows: (i) CDR1-3 derived from the heavy chain variable region including the amino acid sequence described in SEQ ID NO: 387; and (ii) The method comprising CDR1 to CDR3 derived from a light chain variable region containing the amino acid sequence described in Sequence ID No. 389.

[0299] 71. A conjugate of a CLDN6-specific antigen-binding protein covalently bonded to a heterologous portion containing structural formula (I): [ka] During the ceremony, The first plurality of the aforementioned conjugates are bonded to four heterogeneous parts including structural formula (I); At least about 95% of the first plurality of conjugates are structurally homogeneous; and The effective amount is approximately 1.7 mg / kg to 6 mg / kg; 2.0 mg / kg to 6 mg / kg; or The aforementioned conjugate has a concentration in the range of 2.4 mg / kg to 6 mg / kg (for use in the treatment of cancer).

[0300] array The following sequences were part of the sequence listing filed as Appendix 3 in U.S. Application No. 63 / 468,817, for which this application claims priority. These sequences are less than 4 amino acids or 10 base pairs in length and do not meet the length requirements of ST.26, paragraph 7. Sequence ID 9 Ser Thr Ser Sequence ID 15: Asn Ala Lys Sequence ID 21: Asn Ala Lys Sequence ID 27 Lys Val Ser Sequence ID 33 Lys Val Ser Sequence ID 39 Trp Ala Ser Sequence ID 45 Ser Thr Ser Sequence ID 51 Lys Val Ser Sequence ID 57 Ala Ala Ala Sequence ID 63 Lys Val Ser Sequence ID 69 Leu Ala Ser Sequence ID 75 Ser Thr Ser Sequence ID 81 Arg Thr Ser Sequence ID 87 Ala Ala Ser Sequence ID 93 Trp Ala Ser Sequence ID 99 Ala Ala Ser Sequence ID 105 Ser Thr Ser Sequence ID 111 Trp Ala Ser Sequence ID 117 Trp Ala Ser Sequence ID 123 Lys Val Ser Sequence ID 129 Ser Thr Ser Sequence ID 209 agcacatcc Sequence ID 215 aatgcaaaa Sequence ID 221 aatgcaaaa Sequence ID 227 aaagtttcc Sequence ID 233 aaagtttcc Sequence ID 239 tgggcatcc Sequence ID 245 agcacatcc Sequence ID 251 aaagtttcc Sequence ID 257 gctgcagcc Sequence ID 263 aaagtttcc Sequence ID 269 cttgcatcc Sequence ID 275 tccacatcc Sequence ID 281 aggacatcc Sequence ID 287 gctgcatcc Sequence ID 293 tgggcatcc Sequence ID 299 gctgcatcc Sequence ID 305 agcacatcc Sequence ID 311 tgggcatcc Sequence ID 317 tgggcatcc Sequence ID 323 aaagtttcc Sequence ID 329 tccacatcc Sequence ID 450 Xaa Thr Xaa (The first Xaa is S, T, Q, or A, and the third Xaa is S, T, D, or Q) Sequence ID 459: Xaa Val Xaa (The first Xaa is K, Q, or R, and the third Xaa is S, T, or V) Sequence ID 471: Xaa Ala Ser (The Xaa at position 1 can be H, Y, F, or W) Sequence ID 477 Xaa Ala Lys (the 1st position Xaa can be Q, S, A, D, or N) [Examples] The following embodiments are provided solely to illustrate the present disclosure and are not intended to limit its scope.

[0301] Example 1

[0302] This embodiment describes the administration of CLDN6 ADC for the treatment of cancer in humans.

[0303] Participant Selection Criteria 1. A woman or man aged 18 or older who is willing and capable of providing informed consent. 2. Types of diseases:

[0304] For Part 1: • Histologically or cytologically confirmed diagnosis of advanced (unresectable) or metastatic solid malignant tumor that has not responded to accepted standard treatment, or for which no standard treatment exists.

[0305] For Part 2 Cohort 2A: • Histologically or cytologically confirmed diagnosis of advanced (unresectable) or metastatic ovarian cancer, primary peritoneal cancer, or fallopian tube cancer. • The patient's tumor must be positive for claudin 6 expression, as defined by the claudin 6 reference assay, which is an IHC assay. • Patients must have platinum-resistant disease (defined as progression within 6 months of completion of at least 4 cycles of platinum-containing therapy). Note: This must be calculated from the date of the last dose of platinum therapy to the date of radiography showing progression. Patients who are platinum-resistant during front-line treatment are excluded. • Patients must have received at least one (but no more than four) prior line of systemic anti-cancer therapy, and monotherapy is appropriate as the next step in treatment: a. Adjuvant ± neoadjuvant considered as first-line treatment b. Maintenance therapy (e.g., bevacizumab, PARP inhibitors) is considered as part of a preceding series of treatments (i.e., not counted independently). c. Treatments modified due to toxicity when there is no progression are considered part of the same line (i.e., not counted independently). d. Hormone therapy is not considered a separate treatment method. Regarding Cohort 2B in Part 2: Histologically or cytologically confirmed diagnosis of a solid malignancy that is unresponsive to accepted standard treatment or for which no standard treatment exists. • The patient's tumor must be positive for claudin 6 expression, as defined by the claudin 6 reference assay.

[0306] Regarding Cohort 2C in Part 2: Histologically confirmed diagnosis of progressive (unresectable) or metastatic NSCLC that progresses during or after treatment with platinum-based regimens, PD1 or PDL1 inhibitor-containing regimens, EGFR TKI-containing regimens (for EGFRmu NSCLC), ALK inhibitor regimens (for ALK translocation NSCLC), and KRAS G12C inhibitors (for KRASG12Cmu NSCLC). • The patient's tumor must be positive for claudin 6 expression, as defined by the claudin 6 reference assay.

[0307] 3. Measurable diseases according to RECIST v1.1 Eastern Cooperative Oncology Group (ECOG) Performance Status 0-1 5. Can withstand intravenous blood sampling for PK, has no known intolerance or hypersensitivity to IMP or excipients, and can comply with test requirements.

[0308] 6. Appropriate organ function as shown in Table 2, based on the following test values. [Table 18]

[0309] 7. Female participants who may become pregnant must have a negative urine or serum pregnancy test within 72 hours prior to starting the study drug treatment. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. For a participant to be eligible, they must have a negative serum pregnancy test and agree to use a highly effective method of contraception or to agree that they are not likely to become pregnant from the time of the first administration of the study drug until 90 days after the last administration. Highly effective methods of contraception are defined as combination (containing estrogen and progestogen) hormonal contraception (e.g., oral, vaginal, transdermal), progestin-only hormonal contraception associated with ovulation inhibition (e.g., oral, injectable, implantable, intrauterine device [IUD], intrauterine hormone-releasing system [IUS]), bilateral fallopian tube occlusion, partner vasectomy, or avoidance of sexual intercourse. Abstinence refers to "true abstinence" and means that it is consistent with the patient's desired and normal lifestyle. Regular abstinence (e.g., calendar, ovulation, symptomatic, post-ovulation methods), declarations of contraindication during exposure to experimental treatments, and withdrawal are not acceptable methods of contraception. The possibility of non-pregnancy is defined as follows: a. You are 45 years of age or older and have not had a menstrual period for more than one year. b. Participants with no history of hysterectomy or oophorectomy and who have had amenorrhea for less than two years must have follicle-stimulating hormone (FSH) levels within the postmenopausal range at the time of screening. c. After hysterectomy, bilateral oophorectomy, or tubal ligation. A documented hysterectomy or oophorectomy must be confirmed by the medical records of the actual procedure or by ultrasound. Tubal ligation must be confirmed by the medical records of the actual procedure.

[0310] 8. Female and male participants must agree not to provide eggs or semen, respectively, from the first treatment with the investigational drug until 90 days after the last treatment with the investigational drug.

[0311] 9. Female participants must agree not to breastfeed from the first dose of the study treatment until 90 days after the final dose of the study treatment.

[0312] 10. Male participants must use condoms when having sexual intercourse with pregnant women or women who may become pregnant, from the time of the first treatment with the study drug until four months after the last treatment with the study drug. Withdrawal (cessation of sexual intercourse) and / or use of spermicide without a condom is neither an acceptable method of contraception nor a method of fetal protection. For partners of non-pregnant women who may become pregnant, effective contraception should be considered. Male participants are advised to freeze semen samples before initiating TORL-1-23 administration, considering the potential risks to male fertility.

[0313] Participant exclusion criteria 1. Not recovered from acute toxicity of previous treatment [recovery is defined as NCI CTCAE, version 5.0, grade ≤ 1] (excluding treatment-related alopecia or abnormal clinical laboratory values ​​that meet the eligibility requirements).

[0314] 2. In the case of Cohort 2A a. Patients with low-grade, borderline ovarian tumors or non-epithelial ovarian cancer. b. Patients with primary platinum-refractory ovarian cancer, primary abdominal cancer, or fallopian tube cancer (defined as disease that did not respond or had progressed within 3 months of the final dose of first-line platinum-containing chemotherapy).

[0315] 3. Prior to the first dose of TORL-1-23, the patient received prior chemotherapy, clinical trials, radiotherapy, or other therapies for cancer treatment within 14 days of receiving a small molecule drug or within 28 days of receiving a biologic drug. There is no waiting period required for stereotactic radiosurgery (SRS).

[0316] 4. Progressive or symptomatic brain metastases. Irradiated, asymptomatic brain metastases treated with stable or reduced doses of steroids are acceptable. Pyromal disease is excluded.

[0317] 5. Peripheral neuropathy of grade 2 or higher.

[0318] 6. Participants must not be considered at high medical risk due to a serious, uncontrolled medical condition, a malignant systemic disease, or an active, uncontrolled infection. Examples include, but are not limited to, uncontrolled grand seizure disorder, unstable spinal cord compression, superior vena cava syndrome, or any mental disorder that would prevent informed consent from being obtained.

[0319] 7. History of serious heart disease: • Congestive heart failure > New York Heart Association (NYHA) Grade 2 within the past year • Unstable angina (angina symptoms at rest), new-onset angina (started within the past 3 months) • Myocardial infarction within 6 months prior to the start of the investigational drug treatment • Antiarrhythmic therapy (beta-blockers are permitted) • Any unstable ischemic disease or untreated arrhythmia

[0320] 8. Known history of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML)

[0321] 9. History of another cancer within 3 years prior to day 1 of the study dose (excluding basal cell carcinoma or squamous cell carcinoma of the skin that has been definitively treated). Participants with malignancies at low risk of recurrence, including appropriately treated ductal carcinoma in situ (DCIS) of the breast and prostate cancer with a Gleason score of 6 or less, are not excluded.

[0322] 10. Uncontrolled infection; active and clinically severe infection (>CTCAE Grade 2)

[0323] 11. Participants with paroxysmal disorders requiring medication

[0324] 12. Participants must not be pregnant or breastfeeding.

[0325] 13. Known hypersensitivity or intolerance to any of the test drug, test drug class, or excipients contained in the formulation.

[0326] 14. History of allogeneic bone marrow or organ transplantation

[0327] 15. Any condition (comorbidity, infection, or other comorbidity) that, in the opinion of the principal investigator, impedes participation in the study, poses an excessive risk, or complicates the interpretation of safety data.

[0328] 16. Participants taking any drug that is a potent inducer and / or potent inhibitor of the CYP3A4 enzyme.

[0329] 17. Participants taking any medication that is a P-glycoprotein (P-gp) inhibitor.

[0330] Administration and monitoring protocol

[0331] The TORL-1-23 drug was a sterile, colorless to slightly yellow, clear to slightly milky liquid, essentially free of visible particles, supplied in 10 mL glass vials fitted with a flip-off seal (cap) on a 20 mm rubber stopper. Each vial contained 40 mg of TORL-1-23. The drug was formulated at a target concentration of 10 mg / mL in 20 mM glutamate-NaOH buffer, 8% (w / v) sucrose, 0.02% (w / v) PS80, pH 5.2. Transfer to an intravenous (IV) bag and dilution were required before IV infusion.

[0332] An appropriate volume of TORL-1-23 for injection was transferred to a non-PVC IV infusion bag containing USP-grade 5% dextrose injection or 0.9% sodium chloride injection. The final concentration of the prepared TORL-1-23 dosing solution in 5% dextrose was ≥0.1 mg / mL and ≤4 mg / mL. When diluted with 0.9% sodium chloride, the final concentration of the prepared TORL-1-23 was ≥0.2 mg / mL and ≤4 mg / mL.

[0333] Medication was administered based on the participant's body weight. The investigational drug was administered in mg / kg doses based on the participant's actual body weight at baseline. If a participant's body weight changed by more than 10% from their baseline body weight, the dose was adjusted.

[0334] The investigational drug was administered via a dedicated IV line equipped with a 0.22-micron in-line filter.

[0335] TORL-1-23 was administered as a 30-minute intravenous infusion once every three weeks in a 21-day cycle (±3 days from cycle 1). For details on specific treatment groups, please refer to the explanations in Parts 1 and 2 below.

[0336] Participants were continuously monitored for adverse events (AEs) throughout the study period. A complete safety assessment (physical examination, vital signs measurement, 12-lead ECG, and clinical laboratory tests) was performed regularly at baseline, during treatment, and at follow-up. Widely accepted criteria (i.e., the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0) were used for the documentation and classification of adverse events.

[0337] Participants who experienced a Grade 3 potentially relevant toxicity or an unacceptable Grade 2 toxicity despite optimal supportive care discontinued treatment until their condition improved to Grade 1 or lower (excluding alopecia, fatigue, or peripheral neuropathy). Once fully recovered, treatment was then resumed at a lower dose level.

[0338] Tumor evaluation using the Response Assessment Criteria for Solid Tumors (RECIST) v1.1 was performed at screening, every 6–9 weeks after day 1 of cycle 1 for the first year, and at least every 12 weeks (±4 weeks) thereafter, or if clinically indicated, until disease progression or withdrawal from the study. PK parameters were determined with respect to TORL-1-23 during cycle 1.

[0339] Safety follow-up period: Participants were followed for ongoing and new adverse events for 28 days after the last administration of the investigational drug (IMP), or until all drug-related toxicities were resolved or deemed stable, whichever came first.

[0340] Survival follow-up period: All participants will be followed up for a maximum of two years, at least every three months (±1 month), to monitor their survival and any new systemic anti-cancer treatments. Participants who do not return to the facility will be contacted by phone every three months as an alternative.

[0341] Part 1: Dosage Graduation

[0342] The initial dose level was 0.2 mg / kg intravenously once every three weeks.

[0343] To reduce the number of participants treated at sub-therapeutic dose levels while ensuring a risk-reduced starting dose and dose escalation, dose setting was performed according to the accelerated titration design by Simon et al. (1997). In the accelerated phase, consecutive cohorts of one participant each, using 100% dose increments, were evaluated for 21 days or until the first instance of DLT or the first course of potentially associated toxicity of CTCAE grade 2 or higher. At dose levels above 0.8 mg / kg (two doses above the starting dose), or in cases of DLT or first course grade 2 or higher potentially associated AE, the accelerated phase of dose setting reverted to a standard 3+3 dose escalation design using dose increments of 33% or less across all subsequent dose cohorts.

[0344] At each dose level of the standard 3+3 dose escalation phase, at least three participants have been treated or will be treated (unless a DLT is observed in the first two participants at a given dose level). If a DLT is observed in one of the first three treated participants at a dose level in the standard 3+3 dose escalation phase, an additional three participants are enrolled and treated at the same dose level, or are enrolled and will be treated at the same dose level. If no further DLTs are observed, the next dose level may be initiated using a dose increment of approximately 33% or less. Subsequent dose levels may be initiated until all participants enrolled at the current dose level have been treated and observed for at least one complete 21-day cycle and the number of participants with DLTs in a 4-week cycle has been determined. Dose escalation is continued within acceptable limits up to 6.0 mg / kg, until a DLT is observed in at least two of the 3-6 participants treated at that dose level, or, if MTD has been reached, to a potentially lower dose.

[0345] Based on this information, the treatment groups for the cohort are listed in Table 3.

[0346] [Table 19]

[0347] DLT was classified based on NCI CTCAE, version 5.0, and was defined as one of the following events occurring during the first 21 days of Cycle 1, which is not clearly determined to be due to an underlying disease or external factor (regardless of the assessor's causal determination): • Any death that cannot be clearly determined to be due to an underlying medical condition or external factors • Grade 4 or higher hematological toxicity • Febrile neutropenia of grade 3 or higher; • Grade 3 or higher thrombocytopenia accompanied by bleeding • Grade 3 or higher non-hematological toxicity (except as follows): - DLT is only awarded if an AE persists for more than 72 hours despite appropriate treatment, in cases of untreated diarrhea, nausea, vomiting, constipation, pain, and rash. - Fatigue (This is considered DLT only if it persists for more than one week) -Electrolyte abnormalities (only considered DLT if they persist for more than 72 hours); however, if the patient has no clinical symptoms, then all grade 3+ electrolyte abnormalities should be counted as DLT, regardless of duration. - Elevated amylase or lipase (DLT only occurs if associated with symptoms or clinical symptoms of pancreatitis) - In participants with liver metastases or elevated aspartate aminotransferase (AST) or alanine aminotransferase (ALT), DLT is determined only if AST or ALT exceeds 8 times the upper limit of normal (ULN) for 14 days or more, or if AST or ALT exceeds 5x ULN. • Examples of the Hy rule include the absence of initial signs of AE (e.g., ALT or AST > 3x ULN and total bilirubin [TBILI] > 2x ULN, and cholestatic congestion [elevated serum alkaline phosphatase (ALP)]). • Cycle 2 cannot be started for more than 14 days due to TORL-1-23 related toxicity.

[0348] result

[0349] To date, 25 participants have been enrolled in this study and are being treated at eight dose levels ranging from 0.2 to 2.4 mg / kg IV every three weeks. 95% of the patients have a metastatic environment and have received at least three lines of prior treatment. Table 4 shows the demographics of the 25 participants.

[0350] [Table 20]

[0351] No dose-limiting toxicities (DLTs) were reported. The most common treatment-related adverse events, as shown in Table 5, were grade 1 peripheral neuropathy (n=3), grade 1 alopecia (n=3), grade 1 / 2 fatigue (n=5), grade 1 anemia (n=2), grade 3 anemia (n=2), and grade 1 / 2 nausea (n=3).

[0352] [Table 21]

[0353] No dose reduction or delay in administration due to toxicity was necessary. Preliminary PK data showed sustained exposure to TORL-1-23 and low levels of free MMAEs over the dosing interval.

[0354] Confirmed partial responses (PRs) were observed in 7 out of 25 evaluable participants, including 6 out of 19 with platinum-resistant / refractory ovarian cancer at all dose levels, with 3 out of 4 patients achieving a PR at a dose level of 2.4 mg / kg. Separately, 1 / 3 of patients with metastatic CLDN6-positive testicular cancer achieved a PR; this patient had previously been treated with four different lines of therapy. This participant initially received 1 mg / kg and was subsequently escalated to 1.3 mg / kg; the participant continued to respond through cycle 11 and is currently being treated at 1.7 mg / kg.

[0355] The change in tumor size as a function of dose is shown in Figure 1, with the dashed line labeled "PD" indicating the threshold for disease progression and the dashed line labeled "PR" indicating partial response. TBD indicates the CLDN6 status to be determined. The change in tumor size over time is shown in Figure 2.

[0356] Blood samples were collected from all participants for serum PK evaluation in TORL-1-23. Pre-administration PK samples were collected within one hour before infusion on day 1 of cycle 1, and within 30 minutes before each subsequent administration.

[0357] Using effective analytical methods, serum concentrations of TORL-1-23 total antibody (conjugated and unconjugated), MMAE-conjugated TORL-1-23 antibody, and unconjugated (free) circulating MMAE were analyzed.

[0358] Dose exploration is underway to identify the maximum tolerated dose (MTD) and the evaluation dose in the expanded cohort of the target population. Exposure response modeling using available safety and serum MMAE data indicates that doses up to 6.0 mg / kg may be acceptable.

[0359] Part 2: Recommended Phase 2 Dose Expansion Cohort

[0360] Once a dose is identified as a potential RP2D in Part 1, an expanded cohort of up to 20 participants will be enrolled in each potential RP2D to characterize safety, tolerability, and pharmacokinetics (PK) in participants with advanced claudin-6 IHC-positive ovarian cancer (Cohort 2A), participants with claudin-6-positive advanced solid tumors (Cohort 2B), and participants with claudin-6-positive advanced NSCLC (Cohort 2C). In Cohort 2A, participants must have platinum-resistant disease, defined as having completed at least four cycles of platinum-based therapy and having progressed within six months of the last platinum-based therapy. Participants must have received at least one (but no more than four) prior line of systemic anticancer therapy, and monotherapy is appropriate as the next step in treatment. In Cohort 2B, participants must have advanced (unresectable) or metastatic solid malignancies that have not responded to accepted standard treatment or for which no standard treatment exists. Progressive (unresectable) or metastatic NSCLC that progressed during or after treatment with platinum-based regimens, PD1 or PDL1 inhibitor-containing regimens, EGFR TKI-containing regimens (for EGFRmu NSCLC), ALK inhibitor regimens (for ALK translocation NSCLC), and KRAS G12C inhibitors (for KRASG12Cmu NSCLC) in Cohort 2C.

[0361] Example 2

[0362] This embodiment describes the preclinical efficacy of the conjugate used in the claimed method.

[0363] CLDN6-23-ADC selectively binds to CLDN6, in contrast to other members of the CLDN family, inhibits the proliferation of CLDN6+ cancer cells in vitro, and rapidly translocates into CLDN6+ cells. Robust tumor regression was observed in multiple CLDN6+ xenograft models, and tumor inhibition significantly enhanced the survival of mice with CLDN6+ PDX tumors or CLDN6+ cancer cells (derived from established human cancer cell lines) after treatment with CLDN6-23-ADC.

[0364] IHC evaluation of cancer tissue microarrays shows elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and approximately 11% of endometrial carcinomas are positive for the target (see McDermott et al. (2023) Clin. Cancer Res. 29(11):2131-2143) for additional preclinical efficacy data of CLDN6-23-ADC).

[0365] The antitumor activity of TORL-1-23 is shown in Figure 3. Weekly intravenous administration of TORL-1-23 (upward arrow) for three weeks to mice with CLDN6-positive human cancer xenografts derived from ovarian cancer (OV90) or bladder cancer (UMUC4) at 2.5 mg / kg, or endometrial cancer (ARC2) at 5.0 mg / kg, resulted in sustained inhibition of tumor growth and reduction of tumor volume substantially beyond the final dose (e.g., beyond 83 days in the case of the ARC2 xenograft in Figure 3). The specificity of TORL-1-23's antitumor activity against CLDN6-positive cancers is demonstrated by the lack of antitumor activity on CLDN6-negative human melanoma cancer xenografts (M202) similarly treated with 2.5 mg / kg TORL-1-23. Human IgG1 (Hu-IgG1) administered intravenously once weekly for three weeks at 2.5 mg / kg served as a negative control.

[0366] Example 3

[0367] This embodiment compares the PK properties of the conjugate used in the claimed method with those of other ADCs having the same linker heterogeneity.

[0368] The anti-CLDN6 antibody-drug conjugate TORL-1-23 was administered to cancer patients participating in a Phase 1 clinical trial at doses of 0.2 mg / kg to 3.0 mg / kg every three weeks. Safety, including dose escalation and cohort expansion, was assessed to characterize and guide potential recommended Phase 2 doses (RP2D).

[0369] The results of a preliminary analysis of the measured PK values ​​are presented herein.

[0370] TORL-1-23 Total Antibody Preliminary Analysis

[0371] Figure 4 shows graphs of measured TORL-1-23 total antibody concentrations (conjugated and unconjugated), TORL-1-23 concentration, and unconjugated (free) MMAE concentrations after administration of TORL-1-23 preparations in participating cancer patients who participated in a Phase 1 clinical trial and received TORL-1-23 at doses of 0.2–2.4 mg / kg (0.2, 0.4, 0.8, 1.0, 1.3, 1.7, 2.0, or 2.4 mg / kg) during Cycle 1 (0–504 hours). TORL-1-23 is an anti-CLDN6 antibody conjugated to four MMAEs per antibody. As can be seen in the graphs, serum concentrations of TORL-1-23 total antibody (combining both ADCs and free antibody) were higher than serum concentrations of TORL-1-23.

[0372] In the preliminary analysis, the total antibody C of TORL-1-23 was max The AUC of the TORL-1-23 total antibody was shown to increase almost proportionally to the dose between 0.2 mg / kg and 3.0 mg / kg. 0-168h and AUC 0-504h The dose increased almost proportionally between doses of 0.2 mg / kg and 2.4 mg / kg, and slightly less proportionally between 2.4 and 3.0 mg.

[0373] For TORL-1-23 total antibody, doses of 0.2, 1, 1.3, 1.7, 2.0, 2.4, and 3 mg / kg were used. max AUC 0-168h , and AUC 0-504h Accumulation was observed ranging from minimal to none. However, AUC 0-168h Significant accumulation in C max or AUC 0-504h In the n=1 cohort of cycle 3, this was observed only at a dose of 0.8 mg / kg.

[0374] C max AUC 0-168h , and AUC 0~504h A preliminary assessment of TORL-1-23 total antibody accumulation based on the values ​​showed that, based on a 3-week dosing interval, total antibody accumulation was generally minimal to none in cycle 3 compared to cycle 1. max and C of Cycle 1 max No significant difference was observed in the ratio for doses of 0.2 to 3.0 mg. max The ratio ranged from 0.87 to 1.19. Similarly, AUC 0-168h and AUC 0-504h When analyzing total antibody accumulation based on AUC, 0-168h and AUC 0-504h Accumulation ranged from minimal to absent in cycle 3 compared to cycle 1, and was observed for total TORL-1-23 antibody at doses of 0.2, 1, 1.3, 1.7, 2.0, 2.4, and 3 mg / kg. However, at a dose of 0.8 mg / kg and in a cohort of n=1, the AUC in cycle 3 was low. 0-168h Despite the observation of significant accumulation, C was observed at a dose of 0.8 mg / kg. max or AUC 0-504h No significant accumulation was observed. Therefore, generally speaking, there is no significant accumulation of TORL-1-23 total antibody with a 3-week dosing interval at doses ranging from 0.2 to 3 mg / kg every 3 weeks.

[0375] The average half-life of the TORL-1-23 total antibody in Cycle 1 was in the range of 136–290 hours, while in Cycle 3, the average half-life was slightly shorter, ranging from 121–280 hours.

[0376] TORL-1-23 Preliminary Analysis

[0377] Preliminary analysis of TORL-1-23 concentrations showed that the MMAE conjugate anti-CLDN6 antibody had lower serum concentrations than the total TORL-1-23 antibody (see Figure 4; the solid line corresponds to the total antibody titer, and the nearest dashed line below the solid line corresponds to the ADC).

[0378] Similar to the total antibody for TORL-1-23, TORL-1-23 C max The AUC level increased almost proportionally to the dose between 0.2 mg / kg and 3.0 mg / kg. TORL-1-23 AUC 0-506h The AUC increased almost proportionally with the dose between 0.2 and 2.4 mg / kg. Data for 3.0 mg / kg (n=1) was only available up to 168 hours after administration, resulting in a limited AUC. 0~504h Please note that this is an extrapolation.

[0379] C in Cycle 1 and Cycle 3 max and AUC 0-504h In preliminary assessments of the values, generally, for TORL-1-23, doses of 0.2, 0.8, 1.0, 1.3, and 1.7 mg / kg yielded C max or AUC 0-504h Accumulation was shown ranging from minimal to none. Data for cycle 3 were not yet available for other dose levels at the time of analysis.

[0380] The average half-life of the TORL-1-23 total antibody in Cycle 1 ranged from 127 to 387 hours.

[0381] Unconjugated MMAE preliminary analysis

[0382] Preliminary analysis of unconjugated or free MMAEs in serum showed a nearly dose-proportional increase between doses of 0.2 mg / kg and 2.4 mg / kg (see Figure 4, white symbols connected by dashed lines). Similarly, the AUC of unconjugated MMAEs 0-504h The amount increased almost proportionally to the dose between 0.2 mg / kg and 2.4 mg / kg. For unconjugated or free MMAEs, the amount increased at doses of 0.2, 0.8, 1.3, and 1.7 mg / kg. max or AUC 0-504h Accumulation was observed ranging from minimal to none. At a dose of 1.0 mg / kg, C max and AUC 0-504h A significant accumulation was observed.

[0383] The mean half-lives of unconjugated or free MMAEs in cycle 1 ranged from 68.1 to 131 hours.

[0384] Unexpected advantages of TORL-1-23 over other ADCs with MMAE linked by a valine-citrulline linker

[0385] Compared to eight other ADCs, ADC1-ADC8, that underwent Phase 1 clinical trials (mean HAR = approximately 3.5; Li et al., 2020, MAbs12(1):1699768), the systemic exposure to TORL-1-23, with dose-normalized free MMAE concentration after a single dose, was approximately one-third lower than expected compared to other clinically tested MMAE-containing ADCs, as shown in Figure 5. The therapeutic antibody is a humanized IgG1 ADC conjugated to MMAE via a valine-citrulline linker with four MMAE conjugates per antibody. inf Percent (=(MMAE AUC) inf Total Antibody AUC inf (×100) is the 0.011% MMAE AUC of TORL 1-23 ADC (0.011%). inf This is lower than what was observed in ADC1-ADC8 as explained by Li et al. (2020), and MMAE AUCinf The percentage ranged from 0.012% to 0.039% (mean 0.025% for ADC1-ADC8, median 0.025% for ADC1-ADC8). Similar to the findings from dose-normalized free MMAE concentration analysis, the AUC of MMAE after a single dose of 2.4 mg / kg ADC was measured. inf The percentage for TORL-1-23 is approximately 2.3 times lower than expected compared to other clinically tested MMAE-containing ADCs. TORL-1-23 provides unexpected benefits over the average ADC, as exemplified by the analysis of eight major ADCs conjugated to four MMAEs via a valine-citrulline linker (Li et al., 2020), reducing total exposure to unconjugated MMAEs by approximately 2-3 times. The risk of adverse events associated with general exposure to circulating unconjugated MMAEs is significantly lower than with other antibodies similarly conjugated to MMAEs via a valine-citrulline linker.

[0386] Embedding by reference

[0387] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if they were included herein in whole, with each reference being individually and specifically indicated as being incorporated by reference.

[0388] Incorporation by reference to materials submitted in the priority application

[0389] McDermott et al. Preclinical Efficacy of the Antibody-Drug Conjugate CLDN6-23-ADC for the Treatment of CLDN6-Positive Solid Tumors. Clin Cancer Res. 2023 Jun 1;29(11):2131-2143 (doi:10.1158 / 1078-0432.CCR-22-2981. PMID:36884217;PMCID:PMC10233360 (filed as Appendix 1 of U.S. Application No. 63 / 468,817, which claims priority) is incorporated herein by reference in its entirety.

[0390] Variation

[0391] In the context describing this disclosure (in particular, in the context of the following claims), the terms “a,” “an,” and “the,” as well as similar demonstrative pronouns, should be interpreted as encompassing both singular and plural, unless otherwise indicated herein and unless there is a clear contradiction with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-exclusive terms (i.e., “including but not limited to”), unless otherwise noted.

[0392] Unless otherwise indicated herein, the enumeration of value ranges is intended simply as an abbreviation for each distinct value that falls within a range and each endpoint, and each distinct value and endpoint is incorporated herein as if it were enumerated separately herein.

[0393] All methods described herein may be carried out in any preferred order, unless otherwise indicated herein and unless otherwise clearly contradicted by the context. The use of any examples or various terms provided herein (e.g., "such as") is intended solely to better illustrate the disclosure and does not imply any limitation to the scope of the disclosure unless specifically claimed. No terms herein should be construed as indicating any unclaimed element as essential to the practice of the disclosure.

[0394] Preferred embodiments of the Disclosure, including the best mode for carrying out the Disclosure which is known to the inventors, are described herein. Modifications of these preferred embodiments may become apparent to those skilled in the art when reading the foregoing description. The inventors anticipate that those skilled in the art will use such modifications as appropriate, and intend that the Disclosure may be carried out in ways different from those specifically described herein. Accordingly, the Disclosure includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless it is clearly inconsistent with the context, any combination of the above elements in all conceivable variations of the above elements is incorporated herein.

Claims

1. A method for inhibiting solid tumors expressing claudin-6 (CLDN6) in human subjects, comprising administering to a human subject an effective amount of a composition containing a conjugate of a CLDN6-specific antigen-binding protein covalently bound to a heterologous portion having the following structural formula (I): 【Chemistry 1】 During the ceremony, The first plurality of the aforementioned conjugates are bonded to four heterogeneous parts including structural formula (I); At least about 95% of the first plurality of conjugates are structurally homogeneous; and The effective amount is approximately 1.7mg / kg to 6mg / kg; 2.0 mg / kg to 6 mg / kg; or The method described above, wherein the dose is within the range of 2.4 mg / kg to 6 mg / kg.

2. Post-administration C max The method according to claim 1, wherein the percentage of unbound circulating MMAE in serum is less than approximately 0.01% (w / v), thereby reducing the toxicity of the target and inhibiting solid tumors.

3. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 2, wherein the concentration is less than approximately 0.009% (w / v).

4. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 3, wherein the concentration is less than approximately 0.008% (w / v).

5. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 4, wherein the amount is less than approximately 0.007% (w / v).

6. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 5, wherein the concentration is less than approximately 0.006% (w / v).

7. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 6, wherein the concentration is less than approximately 0.005% (w / v).

8. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 7, wherein the concentration is less than approximately 0.004% (w / v).

9. The percentage of the aforementioned unbound circulating MMAE is C max The method according to claim 8, wherein the concentration is less than approximately 0.003% (w / v).

10. C after administration max The method of claim 1, wherein in the serum, the percentage of unbound circulating MMAE is less than about 9 ng / mL, thereby reducing toxicity in the subject and inhibiting the solid tumor.

11. The aforementioned unbound circulating MMAE is C max The method according to claim 10, wherein the concentration is less than approximately 8 ng g / mL.

12. The aforementioned unbound circulating MMAE is C max The method according to claim 11, wherein the concentration is less than approximately 7 ng g / mL.

13. The aforementioned unbound circulating MMAE is C max The method according to claim 12, wherein the concentration is less than approximately 6 ng / mL.

14. The aforementioned unbound circulating MMAE is C max The method according to claim 13, wherein the concentration is less than approximately 5 ng / mL.

15. The aforementioned unbound circulating MMAE is C max The method according to claim 14, wherein the concentration is less than approximately 4 ng / mL.

16. The aforementioned unbound circulating MMAE is C max The method according to claim 15, wherein the concentration is less than approximately 3 ng / mL.

17. The dose-normalized C of the unbound (free) circulating MMAE max The method according to claim 1, wherein the value is less than approximately 35 ng / mL per 1 mg of conjugate after administration of the conjugate, thereby reducing the toxicity of the target and inhibiting solid tumors.

18. The dose-normalized C for unbound circulating MMAE max The method according to claim 17, wherein the value is less than approximately 30 pg / mL per 1 mg of conjugate.

19. The dose-normalized C for unbound circulating MMAE max The method according to claim 18, wherein the value is less than approximately 25 pg / mL per 1 mg of conjugate.

20. The dose-normalized C for unbound circulating MMAE max The method according to claim 19, wherein the value is less than approximately 20 pg / mL per 1 mg of conjugate.

21. The dose-normalized C for unbound circulating MMAE max The method according to claim 20, wherein the value is less than approximately 15 pg / mL per 1 mg of conjugate.

22. The dose-normalized C for unbound circulating MMAE max The method according to claim 21, wherein the value is less than approximately 10 pg / mL per 1 mg of conjugate.

23. C after the first dose of the 3.0 mg / kg conjugate max The method according to claim 1, wherein the percentage of unbound circulating MMAE in serum is less than approximately 2.6 ng / mL, thereby reducing the toxicity of the target and inhibiting the solid tumor.

24. 2.4 mg / kg of the conjugate after the first administration of C max The method according to claim 1, wherein the unbound circulating MMAE in the serum is approximately 4.1 + 2.1 ng / mL, thereby reducing the toxicity of the target and inhibiting the solid tumor.

25. C after the first dose of the 2.0 mg / kg conjugate max The method according to claim 1, wherein the percentage of unbound circulating MMAE in serum is less than approximately 3.7 + 1.6 ng / mL, thereby reducing the toxicity of the target and inhibiting the solid tumor.

26. C after the first administration of the conjugate at 1.7 mg / kg max The method according to claim 1, wherein the unbound circulating MMAE in the serum is approximately 2.7 + 1.9 ng / mL, thereby reducing the toxicity of the target and inhibiting the solid tumor.

27. C in cycle 3 after three administrations of the 1.7 mg / kg conjugate max The method according to claim 1, wherein the unbound circulating MMAE in serum is approximately 1.4 + 0.8 ng / mL, and the conjugate is administered once every three weeks, with cycle 3 beginning with the third administration of the conjugate, thereby reducing the toxicity of the target and inhibiting the solid tumor.

28. The method according to any one of claims 1 to 27, wherein the solid tumor is an ovarian tumor.

29. The method according to any one of claims 1 to 27, wherein the solid tumor is a bladder tumor.

30. The method according to any one of claims 1 to 27, wherein the solid tumor is a testicular tumor.

31. The method according to any one of claims 1 to 27, wherein the solid tumor is an endometrial tumor.

32. The method according to any one of claims 1 to 27, wherein the solid tumor is non-small cell lung cancer.

33. The method according to any one of claims 1 to 27, wherein the solid tumor is primary peritoneal cancer.

34. The method according to any one of claims 1 to 27, wherein the solid tumor is fallopian tube cancer.

35. The method according to any one of claims 1 to 34, wherein, after administration, the human subject does not experience peripheral neuropathy of grade 3 or higher severity, alopecia of grade 3 or higher severity, fatigue of grade 3 or higher severity, nausea, vomiting, or loss of appetite of grade 3 or higher severity, or constipation of grade 3 or higher severity.

36. The method according to any one of claims 1 to 35, wherein, after administration, the human subject does not experience any adverse events of grade 3 or higher severity.

37. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 1.7 mg / kg to 5 mg / kg, 1.7 mg / kg to 4 mg / kg, or 1.7 mg / kg to 3 mg / kg.

38. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 2.0 mg / kg to 5 mg / kg, 2.0 mg / kg to 4 mg / kg, or 2.0 mg / kg to 3 mg / kg.

39. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 2.4 mg / kg to 5 mg / kg, 2.4 mg / kg to 4 mg / kg, or 2.4 mg / kg to 3 mg / kg.

40. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 1.7 mg / kg.

41. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 2.0 mg / kg.

42. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 2.4 mg / kg.

43. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 3.0 mg / kg.

44. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 3.6 mg / kg.

45. The method according to any one of claims 1 to 36, wherein the effective amount is approximately 4 mg / kg.

46. The method according to any one of claims 1 to 45, wherein the effective amount of the composition is administered once every 1 to 4 weeks, for example, once every 2 to 4 weeks, preferably once every 3 weeks.

47. The method according to any one of claims 1 to 46, wherein the composition is administered intravenously.

48. The method according to any one of claims 1 to 47, wherein the effective amount of the composition is administered over a period of about 20 minutes to about 40 minutes, preferably over a period of about 30 minutes.

49. The method according to any one of claims 1 to 48, wherein the human subject achieves a partial response.

50. The method according to any one of claims 1 to 48, wherein the human subject achieves a complete response.

51. The method according to any one of claims 1 to 50, wherein the composition further comprises glutamic acid-sodium hydroxide buffer, sucrose, and polysorbate 80 (PS80).

52. The method according to any one of claims 1 to 51, wherein the composition further comprises dextrose and water.

53. The method according to any one of claims 1 to 51, wherein the composition further comprises sodium chloride and water.

54. The method according to any one of claims 1 to 53, wherein the average number of heterologous moieties per CLDN6-specific antigen-binding protein in the composition is about 3.5 to about 4, for example, about 3.6 to about 3.9, preferably about 3.7 to about 3.

8.

55. The method according to any one of claims 1 to 54, wherein in the first plurality of conjugates, the heterogeneous portion is covalently conjugated with an unpaired cysteine ​​residue of a CLDN6-specific antigen-binding protein.

56. The method according to any one of claims 1 to 54, wherein in the first plurality of conjugates, the heterogeneous portion is covalently conjugated by a cysteine ​​residue resulting from the cleavage of the interchain disulfide bond between the heavy chain and light chain of the CLDN6-specific antigen-binding protein.

57. The method according to any one of claims 1 to 56, wherein about 95%, about 96%, about 97%, or about 98% of the first plurality of conjugates are structurally uniform.

58. A method according to any one of claims 1 to 57, wherein the heterogeneous portion including structural formula (I) is structural formula (II): 【Chemistry 2】 It has, During the ceremony, 【Transformation 3】 The method involves covalent thiol binding to a CLDN6-specific antigen-binding protein.

59. The method according to any one of claims 1 to 58, wherein the subject has not received prior treatment for the solid tumor.

60. The method according to any one of claims 1 to 58, wherein the subject has received at least one prior treatment for the solid tumor.

61. The method according to any one of claims 1 to 58, wherein the subject has received at least two prior treatments for the solid tumor.

62. The method according to any one of claims 1 to 58, wherein the subject has received at least three prior treatments for the solid tumor.

63. The method according to any one of claims 1 to 58, wherein the subject has received at least four prior treatments for the solid tumor.

64. The method according to any one of claims 1 to 63, wherein the effective amount is in the range of 3.0 mg / kg to 3.6 mg / kg.

65. The method according to any one of claims 1 to 64, wherein the human subject is administered an effective amount of G-CSF before being administered an effective amount of the composition to the human subject.

66. A method according to any one of claims 1 to 65, wherein the CLDN6-specific antigen-binding protein is: (g) The heavy chain CDR1 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (h) The heavy chain CDR2 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (i) The heavy chain CDR3 amino acid sequence described in Table A or Table A1, or a variant thereof that differs by only one or two amino acids; (j) The light chain CDR1 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (k) The light chain CDR2 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; (l) The light chain CDR3 amino acid sequence described in Table A or Table A1, or a variant sequence thereof that differs by only one or two amino acids; or The method is selected from the group consisting of any two or more combinations of (a) to (f).

67. A method according to claim 66, wherein the CLDN6-specific antigen-binding protein comprises an antibody that binds to CLDN6 or an antigen-binding fragment thereof, wherein the antibody is as follows: (a) the heavy chain (HC) CDR 1-3 amino acid sequences of SEQ ID NOs. 23, 24, and 25, and the light chain (LC) CDR 1-3 amino acid sequences of SEQ ID NOs. 20, 21, and 22; or (b) The method comprising the heavy chain (HC)CDR1-3 amino acid sequences of SEQ ID NOs. 455, 456, and 457, and the light chain (LC)CDR1-3 amino acid sequences of SEQ ID NOs. 476, 477, and 454.

68. A method according to any one of claims 1 to 67, wherein the CLDN6-specific antigen-binding protein comprises an antibody that binds to CLDN6 or an antigen-binding fragment thereof, wherein the antibody is as follows: (i) HC CDR1 having sequence GFTFSNYW (SEQ ID NO: 23); (ii) HC CDR2 having sequence IRLKSDNYAT (SEQ ID NO: 24), (iii) HC CDR3 having sequence XDGPPSGX (sequence number 457) (wherein the formula, the first X is N and the eighth X is S, T, A, C, or Y), (iv) LC CDR1 having sequence ENIYSY (sequence number 20), (v) LC CDR2 having sequence NAK (SEQ ID NO: 21), (vi) The method comprising LC CDR3 having sequence QHHYTVPWT (sequence number 22).

69. The method according to claim 68, wherein the CLDN6-specific antigen-binding protein is as follows: The method comprising HC CDR3 having the sequence XDGPPSGX (sequence number 457) (wherein the formula, the X at position 1 is N and the X at position 8 is S).

70. A method according to any one of claims 1 to 67, wherein the CLDN6-specific antigen-binding protein comprises an antibody that binds to CLDN6 or an antigen-binding fragment thereof, wherein the antibody is as follows: (i) CDR1-3 derived from the heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 387; and (ii) The method comprising CDR1 to CDR3 derived from a light chain variable region containing the amino acid sequence described in Sequence ID No.

389.

71. A conjugate of a CLDN6-specific antigen-binding protein covalently bonded to a heterologous portion containing structural formula (I): 【Chemistry 4】 During the ceremony, The first plurality of the aforementioned conjugates are bonded to four heterogeneous parts including structural formula (I); At least about 95% of the first plurality of conjugates are structurally homogeneous; and The effective amount is approximately 1.7mg / kg to 6mg / kg; 2.0 mg / kg to 6 mg / kg; or The aforementioned conjugate has a concentration in the range of 2.4 mg / kg to 6 mg / kg (for use in the treatment of cancer).

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