Antibodies targeting claudin-6 and related multi-specific therapeutic agents

Abstract

Antibodies binding to CLDN6 are disclosed. The antibodies are effective at differentiating between other CLDN-family proteins. Bispecific antibodies targeting CLDN6 and CD3 are also disclosed. These antibodies can be used to diagnose and treat various cancers.

Description

[0001] Title: Antibodies Targeting Claudin-6 and Related Multi- Specific Therapeutic Agents Inventors: Siret Tahk, Paule Hermet, Kai Virumae, Tanel Mahlakoiv, Joan Teyra, Sebastian Zoll, Anu Ustav, Mart Ustav Jr.

[0002] Field of the invention

[0003] The present invention is related to anti-claudin 6 (CLDN6) monoclonal antibodies, to the use of said compounds as single agents or in composition as a multi -specific antibody based modality that enables the simultaneous engagement of other cell surface or soluble proteins as a pharmaceutical agent for the treatment or prophylaxis of diseases such as cancer, autoimmune diseases, inflammation, infectious diseases, and metabolic diseases, as well as to use of said compounds as intermediates for generation of new modified anti-CLDN6 monoclonal antibodies.

[0004] Background of the invention

[0005] Claudin 6 (CLDN6) is a tight junction protein that is primarily expressed during embryonic development and is largely absent in normal adult tissues. However, it has been identified as an oncofetal antigen that is re-expressed in various cancers, particularly in solid tumors such as ovarian cancer, testicular cancer, and lung adenocarcinoma (Kwon et al., 2021; Wang et al., 2022). Due to its restricted expression in normal adult tissues and aberrant expression in malignancies, CLDN6 represents a promising tumor-associated antigen (TAA) for targeted cancer therapies. Preclinical studies have demonstrated that CLDN6 is involved in tumorigenesis by contributing to tumor cell proliferation, migration, and invasion. Functional analyses have indicated that CLDN6 expression alters cellular adhesion and enhances oncogenic signaling pathways such as PI3K / AKT and Wnt / p-catenin, which promote tumor progression (Saito et al., 2020). Several preclinical models, including patient-derived xenografts (PDX) and syngeneic mouse models, have validated CLDN6 as a viable therapeutic target, with promising results using monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor (CAR) T-cell therapies (Ning et al., 2023). CLDN6-targeted therapies are currently being evaluated in clinical trials for solid tumors. A notable example includes CLDN6-specific CAR-T cells, which have demonstrated safety and preliminary efficacy in early-phase clinical trials (Qin et al., 2023). Additionally, antibody-drug conjugates (ADCs) and bispecific T-cell engagers (BiTEs) targeting CLDN6 are underinvestigation, leveraging its tumor-specific expression to minimize off-targettoxicities. Biomarker studies suggest that CLDN6-positive tumors exhibit distinct molecular signatures that could be used for patient stratification and personalized therapeutic approaches (Gao et al., 2021).

[0006] Given its selective tumor expression and its functional role in cancer progression, CLDN6 represents an attractive target for novel immunotherapies and precision medicine strategies. Ongoing research aims to further elucidate the molecular mechanisms underlying CLDN6-mediated oncogenesis and to optimize targeted therapeutic interventions.

[0007] Summary of the invention

[0008] The invention provides multitude of antibodies binding to CLDN6. These antibodies can be engineered into bi-specific antibody-based modalities binding also to CD3 on cytotoxic T-cells that can specifically induce the killing of CLDN6 positive cancer cells.

[0009] The invention provides multitude of antibodies binding to Claudin-6 that can be formatted in a variety of multi-specific antibody modalities in combination for example antibodies binding to CD3 located on the surface of T-cells. These multi-specific T-cell engaging molecules that are targeting simultaneously Claudin-6 and CD3

[0010] Disclosed herein are novel homodimer and heterodimeric bispecific antibodies targeting CD3 and CLDN6. These bispecific antibodies and their applications in cancer treatment are described in detail. Specifically, various formats of anti-CD3 and anti-CLDN6 bispecific antibodies are provided, designed to facilitate the targeting of CD3 positive effector T cells to CLDN6 positive tumors. By engaging CD3 positive T cells, these antibodies enhance immune-mediated tumor cell lysis, making them particularly valuable for treatment of solid tumors with elevated CLDN6 expression, for example such as certain ovarian, lung, renal and testicular carcinomas.

[0011] Furthermore, and importantly, these bispecific antibodies are engineered to differentiate CLDN6 from other claudin family members. While CLDN6 is primarily expressed in tumors, CLDN9 is predominantly found in healthy tissues (e.g., cervix and esophagus), and crossreactivity with CLDN9 could result in off-target toxicity, and if used in diagnostics this feature would provide false positives. Given that CLDN6 shares 96% sequence identity with CLDN9 - differing by only three residues in the extracellular loops, developing an antibodythat selectively binds CLDN6 over CLDN9 presents a significant challenge. The invention therefore includes antibodies and antigen-binding domains with preferential binding to CLDN6 over CLDN9, as well as bispecific antibodies incorporating these antigen-binding domains to enhance specificity.

[0012] These CLDN6-binding domains and associated antibodies have potential applications in the treatment and diagnosis of CLDN6-associated cancers.

[0013] Accordingly, in one aspect, this disclosure provides homodimer and heterodimer antibodies that recognize and bind two distinct antigens. These "bispecific" antibodies are designed to target both CLDN6 and CD3, as described herein. The binding mode of these designed bispecific antibodies can be either monovalent — where a single antigen-binding domain (such as a variable heavy and variable light domain pair) interacts with its target — or in a bivalent binding mode, where two independent antigen-binding domains engage the antigen.

[0014] In some embodiments, the bispecific antibody consists of one CD3-binding domain and one CLDN6-binding domain, such as those in the "CM" format described herein and demonstrated in FIG 4B. In other embodiments, the bi-specific homodimeric antibody includes two CD3-binding domains and two CLDN6-binding domains, exemplified by the "Db-Fc-Fab" format demonstrated in FIG 4A. In other embodiments, the heterodimeric antibody that includes one CD3-binding domain and two CLDN6-binding domains is exemplified by the “TCB” format demonstrated in FIG 4C.

[0015] In the cases of bi-specific antibodies that require heterodimerization in order to ensure the preferential formation of heterodimers over homodimers, the antibodies described herein utilize distinct monomers chains engineered with specific amino acid substitutions that promote heterodimerization.

[0016] According to certain aspects antibodies capable of efficiently binding to CLDN6 protein are disclosed.

[0017] According to certain aspects the antibodies are capable of efficiently binding to CLDN6 protein with low EC50 values.According to certain aspects the antibodies are capable of discriminating between CLDN6 and CLDN9.

[0018] According to certain aspects the antibodies have EC50 values for CLDN6 protein are lower than lOnM and EC50 values for CLDN9 are higher than lOnM.

[0019] According to certain aspects the antibodies may be bispecific for CLDN6 and CD3 proteins. According to certain aspects the bispecific antibodies consists of one CD3-binding domain and one CLDN6-binding domain.

[0020] According to certain aspects the bi-specific antibodies may be homodimeric and include two CD3-binding domains and two CLDN6-binding domains.

[0021] According to certain aspects CLDN6 binding antibodies or fragments thereof comprise a heavy chain variable region (VH) comprising VH CDR 1 selected from SEQ ID NOs: 46-54, VH CDR2 selected from SEQ ID NOs: 55-63, and VH CDR3 selected from SEQ ID NOs: 64-72, respectively, and a light chain variable region (VL) comprising the VL CDR1 selected from SEQ ID NOs: 19-27, VL CDR2 selected from SEQ ID NOs: 28-36, VL CDR 3 selected from SEQ ID NOs: 37-45, respectively.

[0022] According to certain aspects the CLDN6 binding antibodies are bispecific for CD3 and comprise VH CDR1 according to SEQ ID NO:93, or 94; VH CDR2 according to SEQ ID NO: 95 or 96, VH CDR3 according to SEQ ID NO: 97 or 98; and VL CDR1 according to SEQ ID NO: 87 or 88, VL CDR2 according to SEQ ID NO: 89 or 90, and VL CDR3 according to SEQ ID NO: 91 or 92, respectively.

[0023] According to certain aspects the CLDN6 binding antibodies or fragments thereof bind to CLDN6 with EC50 values of less than lOnM, preferably less than 8nM, more preferably less than 6nM and most preferably less than InM.

[0024] According to certain aspects the CLDN6 binding antibodies are selected from the group consisting of ONK0277, ONK0378, ONK0263, ONK0581, ONK0582, ONK0583, ONK0600, ONK0601, ONK0602, ONK1174, ONK1152, ONK0993, ONK0999, DBla ONK0813, DB31a ONK0816, DB2 ONK0815, DB4 ONK0818, ONK1097, ONK1102, ONK1157,ONK1095, ONKllOO, ONK1171, ONK1322, ONK1323, ONK1182, ONK1324, ONK1325, ONK1326, ONK0801 CMla, ONK0804 CM2a, ONK1327, ONK1328,ONK0828 TCB3a, ONK1329, ONK0801 CMla, ONK0804 CM2a, ONK1327, ONK1328,ONK0828 TCB3a, and ONK1329.

[0025] According to certain aspects the antibodies are humanized.

[0026] According to certain aspects bispecific antibodies capable of binding to CLDN6 and CD3 are selected from the group consisting of DBla ONK0813, DB3a ONK0816, DB2 ONK815, DB4 ONK0818, ONK1097, ONK1102, ONK1157, ONK1095, ONKllOO, 0NK1171, ONK1322, ONK1323, ONK1182, ONK1324, ONK1325, ONK1326 ONK0801 CMla, ONK801 CM2a, ONK1327, ONK0828 TCB3a, and ONK1329.

[0027] According to certain aspects the bispecific antibodies binding to CLDN6 and CD3, comprise a linker-sequence selected from SEQ ID NOs 131-135, 161.

[0028] According to certain aspects the CLDN6 binding antibodies contain the Fc mutation of SEQ ID: 136 or 162.

[0029] According to certain aspects an in vitro method is disclosed to distinguish between cells expressing CLDN6 and those expressing CLDN9, the method comprising contacting the cells with CLDN6 binding antibodies disclosed.

[0030] According to certain aspects a method to eliminate false positives from tissue samples is disclosed where the method comprising a step of contacting a tissue sample with a CLDN6 binding antibody provided here.

[0031] According to certain aspects a method to reduce size of a tumor is disclosed where the method comprises a step of administering an CLDN6 binding antibody provided here.

[0032] According to certain aspects a method to treat an ovarian, lung, renal, or testicular tumor is disclosed.

[0033] According to certain aspects antibody drug conjugates comprising the disclosed CLDN6 antibodies is disclosed.

[0034] According to certain aspects pharmaceutical compositions to treat cancer by one or more CLDN6 antibodies or antigen binding fragments is disclosed.

[0035] According to certain aspects a method to diagnose cancerous cells in vitro, wherein the method comprises contacting a tissue from a patient suspected to have cancer with a CLDN6 antibody or fragments thereof provided here, and determining the presence of overexpression of CLDN6.According to certain aspects, use of an antibody or a fragment thereof disclosed here for diagnosing presence of CLDN6 expressing cancerous cells in a human tissue sample is provided.

[0036] According to certain aspects use of an antibody or a fragment thereof disclosed here for treating CLDN6 expressing cancerous cells is provided.

[0037] Brief description of the drawings

[0038] Fig. 1. ELISA-based binding assays to estimate the binding affinities of anti-CLDN6 antibodies to CLDN3, CLDN4, CLDN6, CLDN9 and CD19 pseudotyped VLPs. The figure shows binding of nine different antibodies and their respective effective concentration 50 (EC50) values. All antibodies bind to CLDN6 on VLPs with low nanomolar EC50.

[0039] Fig. 2A. Anti-CLDN6 antibody ONK0378 and ONK0582 binding to PAI cell line (human ovarian teratocarcinoma cell line) at various concentrations evaluated by flow cytometry. Fig 2B. Flow cytometry histograms representing anti-CLDN6 antibody ONK0378 and ONK0582 binding to PAI cell lines at 400 nM concentration.

[0040] Fig 2C. Anti-CLDN6 antibody ONK0378 and ONK0582 binding to OV90 cell line (epithelial like cell isolated from the ovary of a female patient with malignant papillary serous adenocarcinoma) (McDermott MSJ, O'Brien NA, Hoffstrom B, et al. 2023) at various concentrations evaluated by flow cytometry.

[0041] Fig 2D. Flow cytometry histograms representing anti-CLDN6 antibody ONK0378 and ONK0582 binding to OV90 cell line at 400 nM concentration.

[0042] Fig. 3A. Anti-CLDN6 antibody ONK0378 and ONK0582 binding to NCIH1693 (cells isolated from lungs of a female patient with stage 3B non-small cell lung cancer adenocarcinoma) cell line at various concentrations evaluated by flow cytometry.

[0043] Fig 3B. Flow cytometry histograms representing anti-CLDN6 antibody ONK0378 and ONK0582 binding to NCIH1693 cell line at 200 nM concentration.

[0044] Fig 4. Schematic representation of the designed and tested formats of bi-specific CLDN6 and CD3 targeting antibodies Diabody-Fc-Fab (DB), CM and 2: 1 TCB (TCB) that bind to CLDN6 and CD3 with different valencies. Linkers used in designing the bi-specific antibodies are shown in Table 7 and Table 8Fig 5. A, B, and C Flow cytometry binding of the designed CLDN6 and CD3 targeting bispecific antibodies to Jurkat (human T lymphocyte cell leukemia) cell line in a concentration dependent manner. All tested bi-specific antibodies are binding to CD3 positive Jurkat cells. Fig 6.A, B, and C Flow cytometry binding of the designed CLDN6 and CD3 targeting bispecific antibodies to PAI cell line in a concentration dependent manner. All tested bi-specific antibodies are binding to CLDN6 positive PAI cells.

[0045] Fig 7. Anti-CLDN6 antibodies ONK0378, ONK0582 and humanized clones ONK1174, ONK1152 and bi-specific CLDN6 and CD3 targeting antibody ONK1102 binding to PA-1 cells at various antibody concentrations evaluated by flow cytometry. Humanized clones and bi-specific antibody 0NK1102 retain their binding to PAI cells.

[0046] Fig 8. A,B Flow cytometry binding of the CLDN6 and CD3 targeting bi-specific DB and CM molecules to PBMCs (Peripheral Blood Mononuclear Cells) in comparison to vixtimotamab CD3 binding antibody.

[0047] Fig 9. Incucyte -based CLDN6 positive PAI and CLDN6 negative MCF7 (McDermott MSJ, O'Brien NA, Hoffstrom B, et al. 2023) (human breast cancer cell line derived from a patient with metastatic breast cancer) cell line killing assay using the CLDN6 and CD3 targeting bi-specific DB molecules. PAI and MCF7 cells and PBMCs are cultivated in co-culture in the presence of bi-specific antibodies at various concentrations and residual live cells are measured at 84 h. CLDN6 and CD3 targeting bi-specific DB molecules kill CLDN6 positive PAI cells but do not kill CLDN6 negative MCF7 cells.

[0048] Fig 10. A, B. Flow cytometry binding of the CLDN6 and CD3 targeting bi-specific DB molecules to two CLDN6 negative cell lines: PAI CLDN6 KO and MCF7.

[0049] Fig 11. Incucyte-based CLDN6 positive PAI and CLDN6 negative MCF7 cell line killing assay using the CLDN6 and CD3 targeting bi-specific CM molecules. PAI and MCF7 cells and PBMCs are cultivated in co-culture in the presence of bi-specific antibodies at various concentrations and residual live cells are measured at 84 h. CLDN6 and CD3 targeting bi-specific CM molecules kill CLDN6 positive PAI cells and at high concentrations also CLDN6 negative MCF7 cells.Fig 12. Incucyte-based CLDN6 positive PAI and CLDN6 negative MCF7 cell line killing assay using the CLDN6 and CD3 targeting bi-specific TCB molecules. PAI and MCF7 cells and PBMCs are cultivated in co-culture in the presence of bi-specific antibodies at various concentrations and residual live cells are measured at 84 h. CLDN6 and CD3 targeting bi-specific TCB molecules kill CLDN6 positive PAI cells and at high concentrations also CLDN6 negative MCF7 cells.

[0050] Fig 13 A, B. Incucyte-based CLDN6 negative PAI CLDN6 KO cell line killing assay using the CLDN6 and CD3 targeting bi-specific CM and TCB molecules. PAI CLDN6 KO cells and PBMCs are cultivated in co-culture in the presence of bi-specific antibodies at various concentrations and residual live cells are measured at 80 h. CLDN6 and CD3 targeting bi-specific CM and TCB molecules kill CLDN6 negative PAI CLDN6 KO cells and at high concentrations.

[0051] Fig 14. Incucyte-based CLDN6 negative PAI CLDN6 KO cell line killing assay using the CLDN6 and CD3 targeting bi-specific ONK1102 molecule. PAI CLDN6 KO cells and PBMCs are cultivated in co-culture in the presence of bi-specific antibody at various concentrations and residual live cells are measured at 80 h.

[0052] Fig 15.A, B, and C Incucyte-based OV90 killing assay using the CLDN6 and CD3 targeting bi-specific DB, CM and TCB molecules. OV90 cells and PBMCs are cultivated in co-culture in the presence of bi-specific antibodies at various concentrations and residual live cells are measured at 96 h. CLDN6 and CD3 targeting bi-specific molecules kill CLDN6 positive OV90 cells.

[0053] Fig 16. Bio-Layer Interferometry-based binding assay of ONK1174 IgG and ONK1102 DB antibodies to biotinylated CLDN6-VLPs loaded on streptavidin sensors.

[0054] Fig 17. Schematic overview of OV90-based xenograft model experiment for evaluating efficacy of ONK1102. NXG mice were injected subcutaneously (sc) with lxl0e6 cells of OV90 cells. 7 days later human 10xl0e6 PBMCs were engrafted intraperitoneally (IP). 4 days later IP injections of ONK1102 at various concentrations and control PBS started at defined timepoints.

[0055] Fig 18. Tumor volumes of the OV90 xenograft followed by treatment of ONK1102 (+ / - SD). ONK1102 inhibited tumor growth at 0.2 and 2 mg / kg concentrations.Fig 19. Peptide coverage of CLDN6 (expression construct). Lines above the sequence indicate peptides. The positions of extracellular loops and transmembrane domains are marked.

[0056] Fig 20. Isotope envelopes of representative peptide 75-78 showing reduced deuterium uptake in the antibody-bound CLDN6 for 120s and 300s. The vertical grey bar indicated the position of the first mono-isotopic peak in the non-deuterated sample. Percentages of relative deuterium incorporation are shown above the spectra.

[0057] Fig 21. Isotope envelopes of representative peptide 182-199 showing reduced deuterium uptake in the antibody-bound CLDN6 for 120s and 300s. The vertical grey bar indicated the position of the first mono-isotopic peak in the non-deuterated sample. Percentages of relative deuterium incorporation are shown above the spectra.

[0058] Fig 22. Deuterium uptake plots of representative peptides showing the percentage of deuterium incorporation for CLDN6 bound and unbound state overtime. Symbols represent technical replicates. Lines are drawn as connect the averages of the measurement points

[0059] Fig 23. Residues with the highest protection from deuterium uptake in 0NK1174-bound CLDN6 are mapped onto the AlphaFold based structure and marked in black. The positions of extracellular loops and transmembrane domains are indicated

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] Definitions

[0062] As used here, “antibody” is a polypeptide that specifically binds and recognizes an antigen or an antigenic fragment thereof.

[0063] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope.

[0064] As used herein, a "bispecific antibody" refers to an antibody or an antibody-derived molecule that is capable of binding to two different antigenic targets or two distinct epitopes within thesame antigen. A bispecific antibody may be engineered through recombinant DNA technology, chemical conjugation, or other molecular biology based methods. The bispecific antibody may exist in various formats, including but not limited to full-length immunoglobulin structures, fragment-based designs such as single-chain variable fragments (scFv), diabodies, tandem scFvs, dual-variable domain antibodies (DVD-Ig), or other formats optimized for therapeutic or diagnostic applications. Bispecific antibodies can function by mediating interactions between different cellular components, facilitating targeted immune responses, or enhancing specificity and efficacy in therapeutic interventions.

[0065] The term "disease" refers to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and / or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted orthose in contact with a person. Cancer or tumor is within the definition of disease.

[0066] As used herein, the terms "treat," "treatment," "treating," refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a disease, including cancer.

[0067] A treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality.

[0068] As used herein, the term "prevent" or "prevention" refers to stopping, hindering, and / or slowing down a disease. In one embodiment, "prevent" is synonymous with "inhibit".

[0069] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. As used herein, a "subject" means a mammal, preferably a human. The terms, "individual," "patient" and "subject" are used interchangeably herein.The terms "increased", "increase", or "enhance" are all used herein to generally mean an increase by a statically significant amount; the terms "increased", "increase", or "enhance", mean an increase of at least 10% as compared to a 20 reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about 25 a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0070] The terms "decrease", "reduce", "reduction", or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. For example, "decrease", 30 "reduce", "reduction", or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an 5 individual without a given disease.

[0071] As used here, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0072] As used herein, the term "in combination" refers to the use of more than one prophylactic and / or therapeutic agent simultaneously or sequentially and in a manner that their respective effects are additive or synergistic.

[0073] “Composition” as used herein may be a single or a combination of antibodies or antigen binding fragments thereof disclosed herein, which can be the same or different, in order to prophylactic or therapeutic treatment. Such combinations can be selected according to the desired immunity or effect. The antibody-based pharmaceutical composition of the present invention may beformulated by any number of strategies known in the art (e.g., see McGoff and Scher, 2000, Solution Formulation of Proteins / Peptides: In McNally, E. J., ed. Protein Formulation and Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158; Akers and Defdippis, 2000, Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, Pa.: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127).

[0074] Combination therapies: The compounds of this invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects. The present invention relates also to such combinations. Possible combinatorial approaches of the compounds include standard chemotherapy options, but also existing and novel targeted therapy and immunotherapy combinations (for example angiogenesis inhibitors, receptor tyrosine kinase inhibitors, CTLA4 and PD-1 / PD-L1 inhibitors). Additionally, compounds affecting other pathways in driving cancer proliferation can be added to the composition.

[0075] Generally, the use of systemic oncologic agents in combination with a compound or composition of the present invention will serve to:

[0076] (1) yield better efficacy in reducing the growth of a tumor or even eliminate the tumor as compared to administration of either agent alone,

[0077] (2) provide for the administration of lesser amounts of the administered chemotherapeutic / targeted / immunogenic agents,

[0078] (3) provide for a treatment that is well tolerated in the patient with fewer deleterious pharmacological complications than observed with single agent therapies and certain other combined therapies,

[0079] (4) provide for treating a broader spectrum of different cancer types in mammals, especially humans,

[0080] (5) provide for a higher response rate among treated patients,

[0081] (6) provide for a longer survival time among treated patients compared to standard treatments, (7) provide a longer time for tumor progression, and / or(8) yield efficacy and tolerability results at least as good as those of the agents used alone, compared to known instances where other cancer agent combinations produce antagonistic effects.

[0082] The compounds and compositions of this disclosure are expected to be effective as therapeutic agents in treating various tumor types. In vitro and in vivo experiments will be conducted with breast cancer, colorectal cancer, and pancreatic cancer all of which are expected to have response to the compounds and compositions. Further the compositions and compounds may be used in treating a number of other cancer types, such as, but not limited to ovarian, testicular, colorectal, lung cancer, hepatocarcinoma, biliary cancer, cervical cancer.

[0083] Immunization of chickens, discovery and isolation of CLDN6 binding monoclonal antibody using Hybrifree technology

[0084] A total of four chickens (C5, C6, Cl 7, Cl 8) were immunized. Each chicken received five injections of CLDN6 DNA expression vectors through electroporation (EP). The final boost, the sixth injection, was administered using CLDN6-VLPs. The chickens were immunized intramuscularly and intradermally with DNA every two weeks. For boosting with VLPs, the injection routes were intramuscular, intradermal, and intravenous.

[0085] Approximately two weeks after the 5th immunization, all chickens were boosted with CLDN6-VLPs. Three days after the boost, their spleens were isolated, homogenized, and cryopreserved in liquid nitrogen.

[0086] The panning experiments were performed on CLDN6-VLP (10 pg / ml) immobilized to Nunc™ MaxiSorp™ (Thermo Scientific™) coated 96-well plates respectively. Splenocytes (2 x 104or 4 x 104cells per panning reaction) from all immunized test animals (chicken C5, C6, C17, Cl 8) were used for B-cell enrichment. For negative selection splenocytes were first incubated 15 min on off-target (CD19-VLP; 50 pg / ml in panning solution and 10 pg / ml on the plate), subsequently, the unbound cells were transferred to specific on-target coated 96-well plates.

[0087] After 45-60 min incubation, wells were washed with PBS to remove the unbound cells. RNA was isolated and cDNA was synthesized using SuperScript® IV First-Strand Synthesis System for RT-PCR (Invitrogen) and used for VH and VL amplification.The proprietary VH and VL primers were designed using chicken sequences stored at IMGT® [http: / / www.imgt.org / IMGTrepertoire / Accessed Aug 2015] and literature data (Kivi G. et al.

[0088] 2016).

[0089] Amplified VH and VL were purified and cloning reactions were performed for cloning into hlgGl-X encoding two-cassette expression vector by using the Ligase Independent Cloning (LIC) method. The resulting antibody library pools were grown in E. coli strain DH5a. Plasmid DNA was purified and transfected into CHOEBNALT85-1E9 cells for transient production of hlgGl antibodies in serum-free media for 48 - 72 hours in a 24-well format. Cell culture supernatants were tested for CLDN6-VLP binding in ELISA (VLP 10 pg / ml in PBS). Cell culture supernatants were also tested for cross-reactivity on CD19-VLP and CLDN9-VLP. Single clones from 57 ELISA positive mini-pool libraries were selected in LB-Amp solid medium, colonies were grown in liquid medium o / n on a shaker at 37°C in 96-well microtiter plates (total 14 x 96-well plates), plasmid DNA was isolated and transfected into CHOEBNALT85-1E9 cells for antibody transient production. 48-72 hours after transfection, supernatants were analyzed by ELISA on CLDN6-coated plates. Identified ELISA-positive hlgGl clones were analyzed by sequencing.

[0090] Unique anti-CLDN6 antibody clones were identified from chickens C6 and C18. No positive clones were identified from chickens C5 and C17. The unique anti-CLDN6 antibody clones were subjected to the next step. Plasmid miniprep DNA was isolated, analyzed and transfected into CHOEBNALT85-1E9 cells (Icosagen Cell Factory OU) for antibody transient production in a 6-well format (2 ml of media). Antibody expression was confirmed by ELISA 48-72h after transfection. Full heavy and light chain sequences were confirmed by sequencing and found to be accurate and unique. Approximately ten days after transfection, cells were removed by centrifugation (300 ref for 10 minutes) and 2 ml of supernatants were purified by affinity chromatography using Mag Sepharose PrismA beads (Cytiva) followed by buffer exchange into PBS with PD MiniTrap G25 columns (Cytiva). Concentrations of purified anti-CLDN6 antibodies were determined by Nanodrop2000 (A280, IgG protocol).

[0091] ELISA-based VLP binding assay

[0092] An ELISA-based VLP binding assay was carried out to compare the EC50 values of antibody clones to CLND3, CLDN4, CLDN6, CLDN9, CD 19 pseudotyped VLPs (Figure 1). MaxiSorpTM high-capacity binding ELISA plates (Thermo Fisher Scientific, US) were coatedwith 5 pg / mL CLND3, CLDN4, CLDN6, CLDN9 or CD 19 pseudotyped VLPs in PBS overnight. The plate was washed and blocked for Ih with 0,05% Tween 20, 2% BSA, 0,2% Proclin 300 in PBS. Serial dilutions of antibodies were incubated on a washed plate for 1 hour, followed by a 1 hour incubation with goat anti-human IgG horse radish peroxidase (HRP) conjugated (Invitrogen) antibody. TMB (3,3',5,5'-Tetramethylbenzidine) Solution VII (Biopanda Diagnostics) was used as a substrate for HRP and 0.5 M H2SO4 was used as stop solution. After every step, the wells were washed with 0.05 % Tween 20, 0,1% Proclin 300 PBS solution. Absorption at 450 nm was measured with Thermo Scientific™ Multiskan™ FC Microplate Photometer. Exemplary wise used antibodies ONK0277, ONK0378, ONK0263, ONK0581, ONK0582, ONK0583, GNK0600, ONK0601 and ONK0602 bound preferentially to CLDN6 VLPs with low nanomolar EC50 values (Figure l).EC50 values for the antibodies are shown in the figure.

[0093] Flow cytometry-based cell binding assays

[0094] Flow cytometry-based PAI, OV90 and NCIH1693 cell line (all from ATCC) binding assay was used to demonstrate binding of the anti-CLDN6 antibodies to CLDN6 positive cell lines (FIG.l, FIG. 2, FIG. 7)

[0095] Flow cytometry-based PAI and Jurkat cell line (ATCC) binding assay was used to demonstrate binding of the CLDN6 and CD3 targeting bispecific antibodies to CD3 and CLDN6 positive cell lines (FIG. 5 and FIG. 6, respectively). Human healthy donor-derived PBMC binding by CLDN6 and CD3 targeting bispecific antibodies is demonstrated in FIG. 8.

[0096] For flow cytometry assays, 0.1 x 106cells were incubated in the presence of various concentrations of anti-CLDN6 antibody solutions in flow cytometry buffer (1% FBS, ImM EDTA in PBS) on ice for 30 minutes. This was followed by an incubation with a secondary goat anti-human IgG AF488 antibody (Jackson Immuno Research) for 30 minutes. The mean fluorescent intensity (MFI) was measured using CytoFLEX System B5-R3-V5 (Beckman Coulter, Inc.) flow cytometer and median fluorescence intensity (MFI) was extracted for anti-CLDN6 antibodies and for anti-HEL isotype (Icosagen Cell Factory) control. When applicable, MFI ratio was calculated by dividing the MFI of each antibody to secondary only control condition MFI.The exemplary wise used antibodies ONK0378 and ONK0582 demonstrate binding to PAI, OV90 and NCIH1693 cell lines in comparison to the isotype control.

[0097] The exemplary wise used CLDN6 and CD3 targeting bispecific antibodies ONK0813, ONK0816, ONK0804 and ONK0801 demonstrate binding to Jurkat cells and human PBMCs. ONK0828 binds to Jurkat cells. CLDN6 and CD3 targeting bispecific antibodies ONK0816, ONK0804, ONK0801, ONK0828, ONK1102 demonstrate binding to CDN6 positive PAI cells.

[0098] Evaluation of binding kinetics using Bio-Layer interferometry

[0099] For evaluation of the binding properties of the obtained 0NK1174 and 0NK1102 molecules to Claudin 6 we performed bio-layer interferometry assay.

[0100] Bio-layer interferometry studies were carried out with Octet ForteBio Red96e instrument. Octet Streptavidin (SA) sensor tips (lot: 2410003211) were equilibrated off-line in analysis buffer (lx PBS + 1 mg / ml BSA) for 10 minutes. The sensors were preconditioned in 0.1 M Glycine (pH = 1.7) for 3 x 5 seconds and placed in analysis buffer for baseline measurement for 120 seconds. CLDN6 pseudotyped, biotinylated VLP-s were diluted in analysis buffer to 30 pg / ml and immobilised. Ligand loading was carried out until a response of 2.0 run was reached. Subsequently, unbound material was removed in analysis buffer for 120 seconds and a dilution series (0.4-300 nM) of the analyte (ONK1102, ONK1174) was titrated for 600 seconds in the association phase. For dissociation measurement, the sensor tips were transferred to analysis buffer for 1800 seconds. Data treatment was carried out in Octet Analysis Studio 13.0.3.52. The obtained data was referenced for buffer signal (sensors loaded with ligand and transferred to a well with analysis buffer only), the Y axis was aligned to the average of the second baseline step. Inter-step correction was applied for the dissocation step and Savitzky-Golay filtering was applied. The kinetics were global-fitted with a 1:2 binding model to obtain kinetic parameters. For 0NK1174 the binding parameters were: KD = 75.7 nM; on-rate ka= 3.00 x 1051 / M s, off-rate k&= 221 x 1021 / s. For 0NK1102 the KD= 9.5 nM; on-rate k32.96 x 1041 / M s, off-rate k = 2.82 x |()41 / s.

[0101] Cell killing assaysIncucyte S3 Live cell analysis instrument (Sartorius) was used to evaluate the effect of CLDN6 and CD3 targeting bispecific antibodies on cancer cells in co-culture with human healthy donor (HD) PBMCs (FIGs 9-12). PAI, OV90 and MCF7 cells were transduced with Incucyte Nuclight Red Lentivirus (Sartorius) and stable PAl-Red, OV90-Red and MCF7-Red cell lines were generated. For killing assay, PAl-Red, OV90-Red or MCF7-Red cells and HD-derived PBMCs were co-cultured at 1:10 ratio in the presence of with different concentrations of CLDN6 and CD3 targeting bispecific antibodies for 84 or 96 hours in RPMI1640 medium supplemented with 10 % of FBS on flat bottom tissue culture 96- or 384-well plates. Red Integrated intensity from PAl-Red, OV90-Red or MCF7-Red cells was measured using Incucyte. Red Integrated intensity was normalized to 0 h timepoint for each well and the obtained ratios (cell growth) was normalized to untreated condition. Exemplary bispecific antibodies ONK0813, ONK0815, ONK0816, ONK0818, ONK1102, ONK0801, ONK0804 and ONK0828 were used in killing assays. CLDN6 and CD3 targeting bispecific antibodies ONK0815, ONK0816, ONK0818, ONK1102 induce an efficient and highly specific CLDN6 positive PAI and OV90 cancer cell killing and do not induce killing of CLDN6 negative MCF7 cell line. ONK0801, ONK0804 and ONK0828 molecules induce the killing of CLDN6 positive cells but killing activity is also observed for CLDN6 negative MCF7 cells at high concentrations of the antibodies.

[0102] Antitumor efficacy against OV90 xenograph tumors

[0103] NXGmice (Janvier Labs) (15 per group) were inoculated with lxl0e6 of OV90 cells sc. 7 days later, 10xl0e6 human PBMCs were engrafted IP. IP treatments of ONK1102 or control antibodies were started as illustrated in FIG. 15. FIG. 16 shows the results of measurements of the tumor volume over time (+ / - SD). A statistically significant tumor growth inhibition was observed for 0NK1102 at 0.2 mg.kg and 2mg / kg doses as compared to PBS control (Ordinary one-way ANOVA).

[0104] HDX-MS for determining the binding epitope of ONK1174

[0105] In order to determine the epitope of CLDN6, which is recognised by CLDN6-specific antibody ONK1102, hydrogen-deuterium -exchange mass spectrometry (HDX-MS) was carried out. For peptide mapping, detergent solubilised and peptidisc-reconstituted CLDN6 was utilised. Digestion parameters were optimised for high coverage of the extracellular part of the protein which contains the antibody binding site. Consequently, low coverage was obtained for transmembrane domains of CLDN6. After establishing digestion conditions,deuterium uptake experiments were set up with CLDN6 alone (unbound) and in complex with 0NK1174 (bound), the parental IgG-type molecule of 0NK1102 which harbours identical CDRs. CLDN6 was used in 2-fold molar excess to fully saturate the bivalent antibody, thus avoiding the presence of free, unbound CLDN6 during deuteration experiments. Within the extracellular region of CLDN6, 2 segments with reduced deuterium uptake of >5% compared to the unbound state were identified in the antibody-bound state. The 2 segments cover residues 70-78 in the construct used (Fig 17) (corresponding to residues 35-43 in wt CLDN6) and 185-195 (residues 150-160 in wt CLDN6) (Fig 18). Both segments were found protected from deuterium uptake in the bound state after 120s and 300s and are represented by several overlapping peptides. In the Alphafold3 generated structure of CLDN6, peptides of the 2 segments that were found protected by antibody binding locate to adjacent positions and constitute a continuum. Segment 185-195 includes Q191 (Q156 in wt CLDN6) which is one out of three residues that are different in CLDN6 in comparison to CLDN9 (Fig 20). No differences in deuterium uptake protection could be identified for peptides containing the other 2 residues that are different between CLDN6 and CLDN9, R180 (R145 in wtCLDN6) and M64 (M29 in wtCLDN6).

[0106] Characterization of antibody clones

[0107] The antibody clones obtained are described in the table below in terms of VH and VL sequences, as well as heavy chain and light chain CDRs.

[0108] Antibodies VH and VL Sequences:

[0109] Table 1: Antibodies VH and VL Sequences

[0110]

[0111] Table 2: Antibodies VH and VL Sequences

[0112]

[0113] Table 3: Antibodies VH and VL Sequences

[0114]

[0115] Antibodies HC and LC Sequences Table 4: Antibodies HC and LC Sequences

[0116]

[0117] Table 5: Antibodies HC and LC Sequences

[0118]

[0119] Table 5 (cont.)

[0120]

[0121] 10Table 6: Antibodies HC and LC Sequences

[0122]

[0123] 10Table 6 (cont.)

[0124]

[0125] Linkers and FC Mutations:

[0126] Table 7: Linkers and FC Mutations

[0127]

[0128] Table 8: Linkers and FC Mutations

[0129]

[0130] Antigens

[0131] Table 9: Antigens

[0132]

[0133] CDR Sequences:

[0134] Table 10: IgG Antibodies Hybrifree

[0135]

[0136] Table 11 : CD3 Binders

[0137]

[0138] The specific binding to CLDN6 in comparison to other related Claudin family members was demonstrated in an ELISA-based CLDN3, CLDN4, CLDN6, CLDN9 VLP binding assay (FIG.1). Furthermore, the antibodies bind to PAI, OV90, NCIH1693 tumor cells that express CLDN6 endogenously (FIG. 1, FIG. 3).

[0139] The antibodies ONK0378 and ONK0582 were selected as candidates for humanization that resulted in the antibodies 16VL-KSPG+73VH and 42 GM VL + BSM_IGLV3_19_1_VH and were further optimized to ONK1174 and ONK1152 antibodies, respectively that retained highly specific binding to PAI cells expressing endogenous CLDN6 (FIG. 7).

[0140] Based on 16VL-KSPG+73VH and ONK0582 CLDN6 binding arms, various bispecific antibody formats were designed (FIG. 4). To compare the DB format with other well-established bispecific T cell engager formats, CM and 2: 1 TCB bispecific molecules were generated (FIG 4). For initial screening purposes variable heavy (VH) and variable light (VL) chain sequences of Vixtimotamab (VH SEQ ID: 81, VL SEQ ID: 82) antibody were used as the CD3 binding arm.

[0141] The designed bi-specific antibodies were evaluated for binding to CD3 positive Jurkat cell line (FIG. 5), human HD PBMCs (FIG. 8.) and CLDN6 positive PAI cells (FIG. 6). After initial screening of the lead antibodies, the CD3 binding arm was substituted to VH and VL chain sequences of SP34-derived antibodies ONK0993 (VH SEQ ID: 83, VL SEQ ID:84) and ONK0999 (VH SEQ ID: 85, VL SEQ ID:86). The CLDN6 binding arm was substituted to VH and VL chain sequences of ONK1152 (VH SEQ ID: 76, VL SEQ ID: 80) or ONK1174 (VH SEQ ID: 74, VL SEQ ID: 78)

[0142] Based on functional CLDN6 positive cancer cell line killing assays (FIGs. 9-12) the diabody-Fc-Fab based modality was confirmed as the preferred candidate. By combining the 0NK1174 (VH SEQ ID: 74, VL SEQ ID: 78)-based CLDN6 binding arm in the C-terminal Fab arm and the optimized SP34-based CD3 binding arm from ONK0999 (VH SEQ ID: 85, VL SEQ ID:86) in the diabody-Fc-Fab based modality we obtained the molecule 0NK1102 (VH SEQ ID: 108, VL SEQ ID: 120) that was used for further characterization in binding assays (FIG. 7) and cell killing assays (FIG. 9).

[0143] In an in vivo experimental system, where immunocompromised mice were xenotransplanted with OV90 ovarian cancer cells, ONK1102 antibodies at high (2mg / kg) or medium (0.2mg / kg) dose had an impact on tumor growth (Fig. 16.) No changes in animal body mass were observed during the treatments.References:

[0144] McDermott MSJ, O'Brien NA, Hoffstrom B, et al. Preclinical Efficacy of the Antibody-Drug Conjugate CLDN6-23-ADC for the Treatment of CLDN6-Positive Solid Tumors. Clin Cancer Res. 2023;29(l 1):2131-2143. doi:10.1158 / 1078-0432.CCR-22-2981

Claims

CLAIMSWhat is claimed is:

1. An antibody capable of efficiently binding to CLDN6 protein.

2. An antibody capable of efficiently binding to CLDN6 protein with low EC50 values.

3. The antibody of claim 1, wherein the antibody exhibits low affinity to CLDN9.

4. The antibody of claim 3, wherein the EC50 values for CLDN6 protein are lower than lOnM and EC50 values for CLDN9 are higher than lOnM.

5. The antibody of any one of the claims 1-4, wherein the antibody is bispecific for CLDN6 and CD3 proteins.

6. A bispecific antibody of claim 5, wherein the bispecific antibody consists of one CD3-binding domain and one CLDN6-binding domain.

7. A bispecific antibody of claim 4, wherein the bi-specific antibody is homodimeric antibody and includes two CD3-binding domains and two CLDN6-binding domains.

8. A CLDN6 binding antibody or fragment thereof of any of the previous claims, wherein the antibody comprises a heavy chain variable region (VH) comprising VH CDR 1 selected from SEQ ID NOs: 46-54, VH CDR2 selected from SEQ ID NOs: 55-63, and VH CDR3 selected from SEQ ID NOs: 64-72, respectively, and a light chain variable region (VL) comprising the VL CDR1 selected from SEQ ID NOs: 19- 27, VL CDR2 selected from SEQ ID NOs: 28-36, VL CDR 3 selected from SEQ ID NOs: 37-45, respectively.

9. The CLDN6 binding antibody or fragment thereof of claim 8, wherein the antibody is bispecific for CD3 and comprises VH CDR1 according to SEQ ID NO:93, or 94; VH CDR2 according to SEQ ID NO: 95 or 96, VH CDR3 according to SEQ ID NO: 97 or 98; and VL CDR1 according to SEQ ID NO: 87 or 88, VL CDR2 according to SEQ ID NO: 89 or 90, and VL CDR3 according to SEQ ID NO: 91 or 92, respectively.

10. The CLDN6 binding antibody or fragment thereof according to any of claims 1 to 9, wherein the antibody or the fragment binds to CLDN6 with EC50 values of less than lOnM, preferably less than 8nM, more preferably less than 6nM and most preferably less than InM.

11. The CLDN6 binding antibody or fragment thereof according to claim 1, wherein the antibody is selected from the group consisting of ONK0277, ONK0378, ONK0263, ONK0581, ONK0582, ONK0583, ONK0600, ONK0601, ONK0602, ONK1174, ONK1152, ONK0993, ONK0999, DBlaONK0813, DB31a ONK0816, DB2 ONK0815, DB4 ONK0818, ONK1097, ONK1102, ONK1157,ONK1095, ONKllOO, ONK1171, ONK1322, ONK1323, ONK1182, ONK1324, ONK1325, ONK1326, ONK0801 CMla, ONK0804 CM2a, ONK1327, ONK1328, ONK0828 TCB3a, ONK1329, ONK0801 CMla, ONK0804 CM2a, ONK1327, ONK1328,ONK0828 TCB3a, andONK1329.

12. A humanized CLDN6 binding antibody of claim 11.

13. A bispecific antibody or fragment thereof selected from the group consisting of DBla ONK0813, DB3a ONK0816, DB2 ONK815, DB4 ONK0818, ONK1097, ONK1102, ONK1157, ONK1095, ONKllOO, ONK1171, ONK1322, ONK1323, ONK1182, ONK1324, ONK1325, ONK1326 ONK0801 CMla, ONK801 CM2a, ONK1327, ONK0828 TCB3a, and ONK1329.

14. A bispecific antibody binding to CLDN6 and CD3 and comprising a linker-sequence selected from SEQ IDNOs 131-135, 161.

15. An antibody according to any one of the previous claims containing the Fc mutation of SEQ ID: 136 or 162.

16. An in vitro method to distinguish between cells expressing CLDN6 and those expressing CLDN9, the method comprising contacting the cells with CLDN6 binding antibody of any one of the claims 1 to 15.

17. A method to eliminate false positives from tissue samples, the method comprising a step of contacting a tissue sample with a CLDN6 binding antibody of any one of the claim 1 to 15.

18. A method to reduce size of a tumor, said method comprising a step of administering an antibody of any one of the claims 1 to 15 to the subject.

19. The method of claim 18 wherein the tumor is ovarian, lung, renal, or testicular tumor.

20. An antibody drug conjugate, wherein the antibody is according to any one of claims 1 to 15.

21. A pharmaceutical composition to treat cancer, the composition comprising one or more CLDN6 antibodies or antigen binding fragments thereof according to any of claims 1 to 15.

22. A method to diagnose cancerous cells in vitro, wherein the method comprises contacting a tissue from a patient suspected to have the cancer with a CLDN6 antibody or fragment thereof of any of the claims 1 to 15, and determining the presence of over-expression of CLDN6.

23. Use of an antibody or a fragment thereof of any one of the claims 1 to 15 for diagnosing presence of CLDN6 expressing cancerous cells in a human tissue sample.

24. Use of an antibody or a fragment thereof of any one of the claims 1 to 15 for treating CUDN6 expressing cancerous cells.

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