Anti-CLDN6 antibody and method of use

Abstract

This disclosure provides an antibody that binds to human CLDN6 and its antigen-binding fragment, a pharmaceutical composition comprising the antibody or its antigen-binding fragment, and the use of the antibody or its antigen-binding fragment or the composition for treating diseases such as cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to International Application PCT / CN2023 / 079815, filed on 6 March 2023. The entire contents of said application are incorporated herein by reference.

[0002] Disclosed herein are antibodies or antigen-binding fragments that bind to human claudin 6 (CLDN6), multispecific antibodies or antigen-binding fragments that bind to CLDN6 and human surface antigen classification 3 (human CD3), and methods for producing them. Specifically, this disclosure provides, in particular, pharmaceutical compositions comprising such antibodies or antigen-binding fragments, and methods for treating cancer. [Background technology]

[0003] The following background explanation of this technology is provided solely to aid in understanding this technology and does not constitute a description of prior art or a component of prior art.

[0004] The claudin (CLDN) gene family encodes endogenous membrane proteins that are crucial structural and functional components of tight junctions (TJs). CLDNs exhibit a four-transmembrane topology, with two extracellular loops and both their N-terminus and C-terminus located in the cytoplasm (Krause et al., Biochimica et Biophysica Acta (BBA)-Biomembranes. 2008). There are 26 human CLDNs that are tissue-specifically expressed in epithelial and endothelial cells (Gunzel et al., Physiol Rev. 2013). By interacting with each other in both cis (intracellular) and trans (intercellular) interactions, CLDNs play a vital role in regulating paracellular permeability and maintaining cell polarity (Tsukita et al., Trends in Biochemical Sciences. 2019). Furthermore, CLDN can function as a scaffold protein for assembling complexes in intercellular junctions and can transmit signals that regulate gene expression and cellular behavior into the cell (Matter et al., Nat Rev Mol Cell Biol. 2003, Singh et al., Pflugers Arch. 2017).

[0005] CLDN6 was first identified and characterized in 2001 (Turksen et al., Developmental Dynamics. 2001). CLDN6 expression is dynamically regulated by various factors and mechanisms (Du et al., Mol Med Rep. 2021). CLDN6 is one of the earliest proteins expressed as a cell surface-specific marker in embryonic stem cells with an epithelial fate and in human pluripotent stem cells (hPSCs) (Ben-David et al., Nat Commun. 2013). Interestingly, CLDN6 expression could be detected in embryonic tissues, including the stomach, pancreas, lung, and kidney, but not in corresponding adult tissue samples (Reinhard et al., Science. 2020, Abuazza et al., Am J Physiol Renal Physiol. 2006, Hashizume et al., Dev Dyn. 2004). In particular, while CLDN6 is silenced at the transcriptional level in normal adult tissues, upregulation of CLDN6 has been reported in several cancer types, including ovarian cancer, endometrial cancer, testicular cancer, lung cancer, and gastric cancer (Kohmoto et al., Gastric Cancer.2020, Kojima et al., Cancers (Basel).2020, Micke et al., Int J Cancer.2014, Sullivan et al., Am J Surg Pathol.2012, Ushiku et al., Histopathology.2012). Differential CLDN6 expression and membrane localization between cancer tissue and normal tissue make it an attractive target for cancer immunotherapy.

[0006] A key consideration regarding CLDN6 targeting is that many members of the CLDN family share high sequence identity, with claudin 9 (CLDN9) being the most similar to CLDN6. The extracellular loops of CLDN6 and CLDN9 differ by only three of their 76 residues. Given that CLDN9 is highly expressed in some normal tissues, achieving higher selectivity for CLDN6 than for CLDN9 is crucial for any CLDN6-targeted antibody-based therapy. [Overview of the project]

[0007] This disclosure provides an anti-CLDN6 antibody and its antigen-binding fragment. This disclosure encompasses the following embodiments.

[0008] In some embodiments, the disclosure provides an antibody or an antigen-binding fragment thereof comprising an antigen-binding domain that specifically binds to human claudin 6 (CLDN6).

[0009] In some embodiments, the antigen-binding domain does not bind to other members of the claudin (CLDN) protein family.

[0010] In some embodiments, the antigen-binding domain does not bind to human claudin 9 (CLDN9).

[0011] In some embodiments, the antigen-binding domain has higher selectivity for human CLDN6 than for human CLDN9.

[0012] In some embodiments, the antigen-binding domain that specifically binds to human CLDN6 includes: (a) a heavy chain variable region including (i) heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 2, (iii) HCDR3 of SEQ ID NO: 3, and (iv) a light chain variable region including light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 4, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6; (b) a heavy chain variable region including (i) HCDR1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 23, (iii) HCDR3 of SEQ ID NO: 3, and (iv) LCDR1 of SEQ ID NO: 4, (v) LCD of SEQ ID NO: 5 (i) a light chain variable region including R2 and (vi) LCDR3 of SEQ ID NO: 6, (c) a heavy chain variable region including (i) HCDR1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 39, (iii) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (iv) LCDR1 of SEQ ID NO: 40, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6, or (d) a heavy chain variable region including (i) HCDR1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 45, (iii) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (iv) LCDR1 of SEQ ID NO: 40, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6.

[0013] In some embodiments, the antigen-binding domain includes: (a) a heavy chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 7, and a light chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 8; and (b) a sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 24 (c) A heavy chain variable region containing an amino acid sequence having (c) a heavy chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 12, (c) a heavy chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 41, and an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 42 (d) A light chain variable region containing the sequence, a heavy chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 43, and a light chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 44, and a heavy chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 46. (f) A variable region and a light chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 47, or (f) a heavy chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 46, and a light chain variable region containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 42.

[0014] In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten amino acids in sequence numbers 7, 8, 12, 24, 41, 42, 43, 44, 46, or 47 are inserted, deleted, or substituted.

[0015] In some embodiments, the antigen-binding domain includes: (a) the heavy chain variable region having an amino acid sequence including SEQ ID NO: 7 and the light chain variable region having an amino acid sequence including SEQ ID NO: 8; (b) the heavy chain variable region having an amino acid sequence including SEQ ID NO: 24 and the light chain variable region having an amino acid sequence including SEQ ID NO: 12; (c) the heavy chain variable region having an amino acid sequence including SEQ ID NO: 41 and the light chain variable region having an amino acid sequence including SEQ ID NO: 42; (d) the heavy chain variable region having an amino acid sequence including SEQ ID NO: 43 and the light chain variable region having an amino acid sequence including SEQ ID NO: 44; (e) the heavy chain variable region having an amino acid sequence including SEQ ID NO: 46 and the light chain variable region having an amino acid sequence including SEQ ID NO: 47; or (f) the heavy chain variable region having an amino acid sequence including SEQ ID NO: 46 and the light chain variable region having an amino acid sequence including SEQ ID NO: 42.

[0016] In some embodiments, the antibody or antigen-binding fragment disclosed herein is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

[0017] In some embodiments, the antibody is a multispecific antibody.

[0018] In some embodiments, the antibody is a bispecific antibody.

[0019] In some embodiments, the antibodies or antigen-binding fragments disclosed herein have antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC).

[0020] In some embodiments, the antibodies or antigen-binding fragments disclosed herein have reduced glycosylation, no glycosylation, or are hypofucosylated.

[0021] In some embodiments, the antibodies or antigen-binding fragments disclosed herein contain an increased bisecting GlcNac structure.

[0022] In some embodiments, the Fc domain of the antibodies or antigen-binding fragments disclosed herein is IgG1.

[0023] In some embodiments, the Fc domain is IgG1 with reduced effector function.

[0024] In some embodiments, the Fc domain is IgG4.

[0025] In some aspects, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment disclosed herein.

[0026] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0027] In some embodiments, the pharmaceutical composition further comprises histidine / histidine HCl, trehalose dihydrate, and / or polysorbate 20.

[0028] In some aspects, the disclosure provides a method of treating cancer, comprising administering to a patient having a need for treatment of cancer an effective amount of an antibody or antigen-binding fragment disclosed herein.

[0029] In some embodiments, the cancer is a solid cancer.

[0030] In some embodiments, the cancer is selected from gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor, and / or glioma.

[0031] In some embodiments, the antibody or antigen-binding fragment is administered in combination with one or more additional therapeutic agents.

[0032] In some embodiments, the one or more therapeutic agents are selected from paclitaxel or paclitaxel preparations, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacitidine.

[0033] In some embodiments, the one or more therapeutic agents are paclitaxel, lenalidomide, or 5-azacitidine.

[0034] In some embodiments, the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

[0035] In some embodiments, the anti-PD-1 antibody is tislerizumab.

[0036] In some embodiments, the Disclosure provides isolated nucleic acids encoding antibody or antigen-binding fragments disclosed herein.

[0037] In some embodiments, the disclosure provides a vector comprising the nucleic acid.

[0038] In some embodiments, the present disclosure provides a host cell containing the nucleic acid or the vector.

[0039] In some embodiments, the present disclosure provides a process for producing an antibody or antigen-binding fragment as disclosed herein, comprising culturing a host cell as disclosed herein and recovering the antibody or antigen-binding fragment from the culture.

[0040] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are used in methods for treating cancer.

[0041] In some embodiments, the present disclosure provides the use of antibodies or antigen-binding fragments disclosed herein in the manufacture of agents for the treatment of cancer.

[0042] In some embodiments, the pharmaceutical compositions disclosed herein are used in methods for treating cancer.

[0043] An antibody or its antigen-binding fragment containing an antigen-binding domain that specifically binds to human CLDN6.

[0044] The antibody or antigen-binding fragment wherein the antigen-binding domain does not bind to other members of the CLDN family.

[0045] The antibody or antigen-binding fragment wherein the antigen-binding domain does not bind to human CLDN9.

[0046] The antibody or antigen-binding fragment wherein the antigen-binding domain has higher selectivity than human CLDN9.

[0047] The antibody or antigen-binding fragment comprises the following antigen-binding domain that specifically binds to the human CLDN6: (i) A heavy chain variable region including (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6. (ii) A heavy chain variable region including (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 23, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6. (iii) A heavy chain variable region including (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 39, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 40, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6, or (iv) A heavy chain variable region including (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 45, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 40, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0048] The antibody or antigen-binding fragment of the present invention comprises the following antigen-binding domains: (i) A heavy chain variable region (VH) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 7, and a light chain variable region (VL) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 8, (ii) A heavy chain variable region (VH) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 24, and a light chain variable region (VL) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 12, (iii) A heavy chain variable region (VH) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 41, and a light chain variable region (VL) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 42, (iv) A heavy chain variable region (VH) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 43, and a light chain variable region (VL) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 44, (v) A heavy chain variable region (VH) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 46, and a light chain variable region (VL) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 47, or (vi) A heavy chain variable region (VH) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 46, and a light chain variable region (VL) containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 42.

[0049] The antibody or antigen-binding fragment of the present invention, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NOs. 7, 8, 12, 24, 41, 42, 43, 44, 46, or 47 are inserted, deleted, or substituted.

[0050] The antibody or antigen-binding fragment of the present invention comprises the following antigen-binding domains: (i) Heavy chain variable region (VH) containing SEQ ID NO: 7, and light chain variable region (VL) containing SEQ ID NO: 8, (ii) Heavy chain variable region (VH) containing SEQ ID NO: 24, and light chain variable region (VL) containing SEQ ID NO: 12, (iii) Heavy chain variable region (VH) containing SEQ ID NO: 41, and light chain variable region (VL) containing SEQ ID NO: 42, (iv) Heavy chain variable region (VH) containing SEQ ID NO: 43, and light chain variable region (VL) containing SEQ ID NO: 44, (v) A heavy chain variable region (VH) containing SEQ ID NO: 46, and a light chain variable region (VL) containing SEQ ID NO: 47, or (vi) Heavy chain variable region (VH) containing sequence number 46, and light chain variable region (VL) containing sequence number 42.

[0051] The antibody or antigen-binding fragment of the present invention is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

[0052] The antibody of the present invention is a multispecific antibody.

[0053] The antibody of the present invention is a bispecific antibody.

[0054] The antibody or antigen-binding fragment of the present invention having antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC).

[0055] The antibody or antigen-binding fragment of the present invention, wherein glycosylation is reduced, glycosylation is absent, or low fucosylation is present.

[0056] The antibody or antigen-binding fragment of the present invention, comprising an increased bisected GlcNac structure.

[0057] The antibody or antigen-binding fragment of the present invention, wherein the Fc domain is IgG1.

[0058] The antibody or antigen-binding fragment of the present invention, wherein the Fc domain is IgG1 with reduced effector function.

[0059] The antibody or antigen-binding fragment of the present invention, wherein the Fc domain is IgG4.

[0060] A pharmaceutical composition comprising an antibody or antigen-binding fragment of the present invention, further comprising a pharmaceutically acceptable carrier.

[0061] The pharmaceutical composition further comprises histidine / histidine HCl, trehalose dihydrate, and polysorbate 20.

[0062] A method for treating cancer, comprising administering an effective amount of the antibody or antigen-binding fragment of the present invention to a patient in need of cancer treatment.

[0063] The method wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0064] The method wherein the antibody or the antigen-binding fragment is administered in combination with another therapeutic agent.

[0065] The method wherein the therapeutic agent is paclitaxel or a paclitaxel preparation, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacitidine.

[0066] The method wherein the therapeutic agent is paclitaxel, lenalidomide, or 5-azacitidine.

[0067] The method wherein the therapeutic agent is an anti-PD1 antibody or an anti-PDL1 antibody.

[0068] The method wherein the anti-PD1 antibody is tislerizumab.

[0069] Isolated nucleic acids encoding the antibody or antigen-binding fragment of the present invention.

[0070] A vector containing nucleic acids according to the present invention.

[0071] Host cells containing the nucleic acid or vector of the present invention.

[0072] A process for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cells and recovering the antibody or the antigen-binding fragment from the culture.

[0073] In one embodiment, the antibody or its antigen-binding fragment includes one or more complementarity-determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 23, 39, 40, and 45.

[0074] In another embodiment, the antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region comprising one or more complementarity-determining regions (HCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 23, SEQ ID NO: 39, and SEQ ID NO: 45; and / or (b) a light chain variable region comprising one or more complementarity-determining regions (LCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 40.

[0075] In another embodiment, the antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region comprising three complementarity-determining regions (HCDRs), wherein the HCDRs are HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 45, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and / or (b) a light chain variable region comprising three complementarity-determining regions (LCDRs), wherein the LCDRs are LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 40, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0076] In another embodiment, the antibody or its antigen-binding fragment includes: (a) A heavy chain variable region comprising three complementarity-determining regions (HCDRs), The HCDR is a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3. The heavy chain variable region of the HCDR is HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 23, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3. The HCDR is a heavy chain variable region in which HCDR1 contains the amino acid sequence of SEQ ID NO: 1, HCDR2 contains the amino acid sequence of SEQ ID NO: 39, and HCDR3 contains the amino acid sequence of SEQ ID NO: 3, or The HCDR is a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 15, HCDR2 containing the amino acid sequence of SEQ ID NO: 45, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3. (b) a light chain variable region comprising three complementarity determination regions (LCDRs), The LCDR is a light chain variable region in which LCDR1 contains the amino acid sequence of SEQ ID NO: 4, LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and LCDR3 contains the amino acid sequence of SEQ ID NO: 6, or The light chain variable region is such that the LCDR contains the amino acid sequence of SEQ ID NO: 40 (LCDR1), the amino acid sequence of SEQ ID NO: 5 (LCDR2), and the amino acid sequence of SEQ ID NO: 6 (LCDR3).

[0077] In another embodiment, the antibody or antigen-binding fragment comprises an antigen-binding domain including: (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0078] In another embodiment, the antibody or antigen-binding fragment comprises an antigen-binding domain including: (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 23, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0079] In another embodiment, the antibody or antigen-binding fragment comprises an antigen-binding domain including: (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 39, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 40, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0080] In another embodiment, the antibody or antigen-binding fragment comprises an antigen-binding domain including: (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 45, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region including (d) LCDR1 of SEQ ID NO: 40, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0081] In one embodiment, the antibody or antigen-binding fragment of the Disclosure comprises: (a) a heavy chain variable region having an HCDR or VH amino acid sequence as described in Table 1, and / or (b) a light chain variable region having an LCDR or VL amino acid sequence as described in Table 1.

[0082] In another embodiment, the antibody or antigen-binding fragment of the Disclosure comprises: (a) an amino acid sequence comprising one, two, or three amino acid substitutions in the amino acid sequence of the HCDR or VH listed in Table 1, and / or (b) a light chain variable region comprising an amino acid sequence comprising one, two, three, four, or five amino acid substitutions in the amino acids of the LCDR or VL listed in Table 1. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0083] In one embodiment, the antibody of the Disclosure is an isotype of IgG1, IgG2, IgG3, or IgG4. In a more specific embodiment, the antibody of the Disclosure comprises the Fc domain of wild-type human IgG1 (also known as human IgG1wt or huIgG1) or IgG2.

[0084] In one embodiment, the antibody disclosed herein has a binding affinity (K) to CLDN6. D ) 1x10 -6 M~1x10-10 It binds with M. In another embodiment, the antibody of the present disclosure binds to CLDN6 with a binding affinity (K D ) of about 1×10 -6 M, about 1×10 -7 M, about 1×10 -8 M, about 1×10 -9 M, or about 1×10 -10 M.

[0085] In another embodiment, the anti-human CLDN6 antibody of the present disclosure exhibits interspecies binding activity against cynomolgus monkey CLDN6.

[0086] In one embodiment, the antibody of the present disclosure has a strong Fc-mediated effector function. The antibody mediates antibody-dependent cell cytotoxicity (ADCC) against target cells expressing CLDN6.

Brief Description of the Drawings

[0087] [Figure 1A] Shows the cell binding activity of the chBG87P engineered variant. The cell binding activities of the first-round BG87P humanized revertant variants (BG87P-z0, BG87P-Bz0, BG87P-Bz1, BG87P-Bz2, BG87P-Bz3, BG87P-Bz4, BG87P-Bz5, BG87P-Bz6, BG87P-Bz7, and BG87P-Bz8) against HEK293T / human CLDN6 are shown in comparison with the anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1B] Shows the cell binding activity of the chBG87P engineered variant. The cell binding activities of the combinatorial humanized variants (BG87P-21, BG87P-22, BG87P-23, and BG87P-24) against HEK293T / human CLDN6 are shown in comparison with the anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1C] Shows the cell binding activity of the chBG87P engineered variant. The cell binding activities of the combinatorial humanized variants (BG87P-25, BG87P-26, and BG87P-27) against HEK293T / human CLDN6 are shown in comparison with the anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1D] This section shows the cell binding activity of chBG87P-modified variants. The cell binding activity of combined humanized variants (BG87P-21, BG87P-22, BG87P-23, and BG87P-24) to the cancer cell line PA-1 is shown in comparison to anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1E] This section shows the cell binding activity of chBG87P-modified variants. The cell binding activity of combined humanized variants (BG87P-25, BG87P-26, and BG87P-27) to the cancer cell line PA-1 is shown in comparison to that of anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1F] This section shows the cell binding activity of chBG87P-modified variants. The cell binding activity of post-translational modification (PTM) removal variants (BG87P-m1, BG87P-m2, BG87P-m3, BG87P-m4, BG87P-m5, BG87P-m6, BG87P-m7, and BG87P-m8) to HEK293T / human CLDN6 is shown in comparison with anti-CLDN6 chimeric BG87P (chBG87P) and BG87P-Bz0. [Figure 1G] This section shows the cell binding activity of chBG87P-modified variants. The cell binding activity of BG87P-soluble variants (BG87P-21, BG87P-34, and BG87P-33) to HEK293T / human CLDN6 is shown in comparison to anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1H] This shows the cell binding activity of the chBG87P manipulated variants. The nonspecific binding activity of the soluble manipulated variants (BG87P-21, BG87P-34, and BG87P-33) to HEK293T-human CLDN9 is shown in comparison to anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1I] This shows the cell binding activity of chBG87P-modified variants. The cross-reactivity of humanized variants (BG87P-21, BG87P-34, and BG87P-33) to CHOK1-cyno CLDN6 is shown in comparison to anti-CLDN6 chimeric BG87P (chBG87P). [Figure 1J]This shows the cell binding activity of chBG87P-modified variants. The cross-reactivity of humanized variants (BG87P-21, BG87P-34, and BG87P-33) to CHOK1-mouse CLDN6 is shown compared to anti-CLDN6 chimeric BG87P (chBG87P). [Figure 2] The expected hydrophobic patch in Schroedinger's homology model of chimeric BG87P is illustrated. I97-Y98-Y100-V100a of HCDR3, together with Y49-W50 of HCDR2 (Y49 is the last residue of FR2 in the variable region of the light chain, and W50 is the first residue of HCDR2), is expected to form an exposed hydrophobic patch. [Figure 3] This shows the hydrophobicity of selected humanized BG87P variants (BG87-33, BG87-34, BG87P-21) after manipulation, as identified by HIC-HPLC. [Figure 4A] This shows a comparison of binding activity between chimeric sp34 and humanized sp34 in Hut78 cells. It also shows a comparison of binding affinity between chimeric sp34 (ch-sp34) and humanized sp34 BG53P (BG53P) measured by melt-flow index (MFI) in Hut78 cells. [Figure 4B] This shows a comparison of binding activity between chimeric sp34 and humanized sp34 in Hut78 cells. It also shows a comparison of binding affinity between chimeric sp34 (ch-sp34), humanized sp34 BG53P (BG53P), and BG56P. [Figure 5] This shows a comparison of the binding activity between humanized sp34 BG56P (BG56P) and humanized sp34 scFv BG561p (BG561P). [Figure 6A] This shows a comparison of binding affinities between humanized sp34 scFv cells in Hut78 cells. This shows a comparison of binding affinities between humanized sp34 scFv BG561p (BG561P) and humanized scFv BG562P (BG562P) in Hut78 cells. [Figure 6B]This shows a comparison of binding affinities between humanized sp34 scFv in Hut78 cells. This also shows a comparison of binding affinities between humanized scFv BG562P (BG562P) and humanized scFv BG563P (BG563P) in Hut78 cells. [Figure 6C] This shows a comparison of binding affinities between humanized sp34 scFv in Hut78 cells. This also shows a comparison of binding affinities between humanized scFv BG563P (BG563P) and humanized scFv BG564P (BG564P) in Hut78 cells. [Figure 7] A schematic diagram of CLDN6×CD3 BsAb BG143P is shown. [Figure 8A] This shows the target binding activity of CLDN6 × CD3 BsAb BG143P. This shows the CD3 binding activity of BG143P in CD3-expressing Jurkat cells. [Figure 8B] This shows the target binding activity of CLDN6 × CD3 BsAb BG143P. This shows the CLDN6 binding activity of BG143P in CLDN6-expressing PA-1 cells. [Figure 9A-1] This study demonstrates the on-target functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with differing CLDN6 expression. Cell lysis assays show the cytotoxicity of BG143P-redirected T cells in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells. [Figure 9A-2] This study demonstrates the on-target functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with differing CLDN6 expression. Cell lysis assays show the cytotoxicity of BG143P-redirected T cells in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells. [Figure 9B-1] This study demonstrates the on-target functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with differing CLDN6 expression. It also shows the IFN-γ induction activity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells. [Figure 9B-2]This study demonstrates the on-target functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with differing CLDN6 expression. It also shows the IFN-γ induction activity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells. [Figure 9C-1] This study demonstrates the on-target functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with differing CLDN6 expression. It also shows the IL-2 inducing activity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells. [Figure 9C-2] This study demonstrates the on-target functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with differing CLDN6 expression. It also shows the IL-2 inducing activity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells. [Figure 10A] This shows the functional specificity of CLDN6×CD3 BsAb BG143P to human CLDN6 and CLDN9. It also shows the binding specificity of BG143P to human CLDN6 (left graph) and CLDN9 (right graph) in NCI-H1299 cells. [Figure 10B] This shows the functional specificity of CLDN6×CD3 BsAb BG143P against human CLDN6 and CLDN9. It also shows the killing specificity (cytolytic activity) of BG143P against human CLDN6 (left graph) and CLDN9 (right graph) in NCI-H1299 cells. [Figure 10C] This demonstrates the functional specificity of CLDN6×CD3 BsAb BG143P to human CLDN6 and CLDN9. It also shows the cytokine (IFN-γ) induction by BG143P to human CLDN6 (left graph) and CLDN9 (right graph) in NCI-H1299 cells. [Figure 11A]This study demonstrates the in vivo efficacy of CLDN6×CD3 BsAb BG143P in an OV-90 xenograft model of PBMC-humanized mice. Tumor volume over time is shown. Mice were either left untreated (no PBMC), treated with PBS (PBS ip QW), treated with 0.01 mg / kg of BG143P (BG143P-0.01 mg / kg, ip), treated with 0.03 mg / kg of BG143P (BG143P-0.01 mg / kg, ip), or treated with 0.1 mg / kg of BG143P (BG143P-0.1 mg / kg, ip). Treatment was performed once a week and is represented by a triangle on the x-axis. [Figure 11B] This study demonstrates the in vivo efficacy of CLDN6×CD3 BsAb BG143P in an OV-90 xenograft model of PBMC-humanized mice. It shows the percentage of hCD45+ cells showing human PBMC rearrangement in the peripheral blood of mice treated with PBMC injection at days 13, 21, and 27 after PBMC injection. These mice were either left untreated (no PBMCs), treated with PBS (PBS ip QW), treated with 0.01 mg / kg of BG143P (BG143P-0.01 mg / kg, ip), treated with 0.03 mg / kg of BG143P (BG143P-0.01 mg / kg, ip), or treated with 0.1 mg / kg of BG143P (BG143P-0.1 mg / kg, ip). [Figure 12A] This study demonstrates the in vivo efficacy of CLDN6 × CD3 BsAb BG143P in an aB16F10 / human CLDN6 syngeneic model of hCD3EDG transgenic mice. Tumor volume over time is shown. Mice were treated with PBS (PBS ip QW), 0.01 mg / kg BG143P (BG143P-0.01 mg / kg, ip), 0.03 mg / kg BG143P (BG143P-0.01 mg / kg, ip), or 0.1 mg / kg BG143P (BG143P-0.1 mg / kg, ip). Treatment was performed once a week and is represented by a triangle on the x-axis. [Figure 12B]This study demonstrates the in vivo efficacy of CLDN6×CD3 BsAb BG143P in an aB16F10 / human CLDN6 syngeneic model of hCD3EDG transgenic mice. The post-transplant body weight of mice treated with PBS (PBS ip QW), 0.01 mg / kg BG143P (BG143P-0.01 mg / kg, ip), 0.03 mg / kg BG143P (BG143P-0.01 mg / kg, ip), or 0.1 mg / kg BG143P (BG143P-0.1 mg / kg, ip) from day 11 to day 27 is shown as an indicator of mouse tolerance to the antibody.

[0088] definition Unless otherwise defined elsewhere in this document, all other technical and scientific terms used herein have the meanings generally understood by those skilled in the art.

[0089] As used herein, including in the attached claims, singular words such as "a," "an," and "the" include references to their corresponding plural forms unless the context clearly indicates otherwise.

[0090] As used herein, the term “or” means and is used interchangeably with the term “and / or” unless the context clearly indicates otherwise. As used herein similarly, the term “and / or” encompasses all possible combinations of one or more related enumerations, and, if interpreted as an alternative ("or"), the absence of any combination.

[0091] As used herein, "approximately" when used with a number means the stated number, as well as a range of plus or minus 10% of that number. For example, "approximately 10" should be understood as both "10" and "9 to 11".

[0092] As used herein, expressions in the form of "A / B" or "A and / or B" mean (A), (B), or (A and B), and expressions in the form of "at least one of A, B, and C" mean (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0093] As used herein, the term “anticancer agent” refers to any agent that may be used to treat cell proliferation disorders such as cancer, and includes, but is not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, and immunotherapeutic agents.

[0094] The term "Claudin 6" or "CLDN6" refers to a member of the CLDN family. CLDN6 has a molecular weight of 23 kDa. CLDN6 has four transmembrane domains and a PDZ-binding domain at the cytoplasmic carboxyl terminus. The amino acid sequence of human CLDN6 can be found at UniPort ID P56747. An exemplary human CLDN6 sequence is Sequence ID No. 87.

[0095] The term "Claudin 9" or "CLDN9" refers to another member of the CLDN family. CLDN9 has a molecular weight of 23 kDa, and its amino acid sequence is found at UniPort ID O95484. An exemplary human CLDN9 sequence is Sequence ID No. 88.

[0096] As used herein, the terms “Surface Antigen Classification 3” or “CD3” refer to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated, and include, for example, CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 obtained from intracellular processing. The term also encompasses spontaneously occurring variants of CD3, including, for example, splice variants or allele variants. CD3 includes, for example, the human CD3ε protein with a length of 207 amino acids (NCBI RefSeq number NP_000724) and the human CD3γ protein with a length of 182 amino acids (NCBI RefSeq number NP_000064).

[0097] As used herein, the terms “administer,” “administer,” “treat,” and “treat” mean, when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Therapy of cells includes contact of the reagent with the cells and contact of the reagent with the fluid in which the cells are in contact. The terms “administer” and “treat” also mean, for example, the treatment of cells in vitro and ex vivo with a reagent, diagnostic agent, conjugate compound, or another cell. The term “subject” as used herein includes any living organism. A non-limiting example is an animal. In any embodiment, the animal is a mammal (e.g., primates, higher primates, humans, rats, mice, dogs, cats, rabbits). In any embodiment, the mammal is a human. In any embodiment, the subject is a patient who has or is at risk of having the disorders described herein. In any embodiment, treating any disease or disorder means improving the disease or disorder (i.e., delaying, preventing, or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, “treating,” “treating,” or “treatment” means mitigating or improving at least one physical parameter, including one that may not be recognizable to the patient. In yet another embodiment, “treating,” “treating,” or “treatment” means modifying the disease or disorder physically (e.g., stabilizing recognizable symptoms), physiologically (e.g., stabilizing physical parameters), or both. In yet another embodiment, “treating,” “treating,” or “treatment” means preventing or delaying the onset, appearance, or progression of the disease or disorder. In some embodiments, as used herein with respect to cancer, “preventing,” “preventing,” or “prevention” means eliminating or reducing the risk of developing cancer. Prevention may also mean preventing recurrence or secondary cancer after an initial cancer has been treated or cured.

[0098] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to any individual mammalian subject, e.g., a cow, a dog, a cat, a horse, or a human. In certain embodiments, the subject, individual, or patient is a human.

[0099] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen at multiple sites via non-covalent bonds. Generally, the more interactions there are, the stronger the affinity.

[0100] As used herein, the term “antibody” refers to a polypeptide of the immunoglobulin family that can bind noncovalently, reversibly, and specifically to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, namely CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), and interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four framework regions (FRs), arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0101] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies. The antibody may be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0102] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species.

[0103] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to antibodies that have a structure substantially similar to that of a natural antibody or that have a heavy chain containing an Fc region.

[0104] In some embodiments, the anti-CLDN6 antibody comprises at least one antigen-binding site and at least a variable region. In some embodiments, the anti-CLDN6 antibody comprises an antigen-binding fragment from a CLDN6 antibody described herein. In some embodiments, the anti-CLDN6 antibody is isolated or recombinant.

[0105] In this specification, the terms “monoclonal antibody” or “mAb” or “Mab” refer to a substantially homogeneous population of antibodies, i.e., the antibody molecules in the population have identical amino acid sequences, except for spontaneously occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a number of different antibodies with different amino acid sequences within variable domains, particularly complementarity-determining regions (CDRs), which are often specific to different epitopes. The modifier “monoclonal” characterizes the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be interpreted as requiring antibody production by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497, U.S. Patent No. 4,376,110, Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988, and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein may be any immunoglobulin class such as IgG, IgM, IgD, IgE, IgA, and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. High-titer monoclonal antibodies can be obtained through in vivo production. In this case, cells from individual hybridomas are intraperitoneally injected into mice, such as Balb / c mice, stimulated with pristine, to induce the production of ascites fluid containing high concentrations of the desired antibody. From such ascites fluid, or from the culture supernatant, isotype IgM or IgG monoclonal antibodies can be purified using column chromatography methods well known to those skilled in the art.

[0106] Generally, the basic structural unit of an antibody is a tetramer. Each tetramer contains two identical polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The carboxyl-terminus of the heavy chain can define a constant region, primarily involved in effector function. Typically, human light chains are classified into kappa and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and the isotypes of the antibody are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 amino acids.

[0107] The variable region of each light chain / heavy chain (VL / VH) pair forms the antibody binding site. Therefore, generally, an intact antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0108] Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions" or "CDRs," which are located between relatively conserved framework regions (FRs). CDRs are usually aligned by framework regions, enabling binding to specific epitopes. Generally, both the light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4), from the N-terminus to the C-terminus. The location of the CDR and framework region can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001), Chothia and Lesk, J.Mol.Biol., 196:901-917 (1987), Chothia et al., Nature, 342:877-883 (1989), Chothia et al., J.Mol.Biol., 227:799-817 (1992), Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The See Immunologist, 7, 132-136 (1999) and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering procedure).The definition of antigen-binding sites is also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000), and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001), MacCallum et al., J.Mol.Biol., 262:732-745 (1996), and Martin et al., Proc.Natl.Acad.Sci.USA, 86:9268-9272 (1989), Martin et al., Methods Enzymol., 203:121-153 (1991), and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), while the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), while the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the definitions of CDRs by Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.In IMGT, the CDR amino acid residues in the VH region are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), while the CDR amino acid residues in the VL region are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR region of an antibody can be determined using the program IMGT / DomainGap Align.

[0109] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues from the "CDR" (e.g., LCDR1, LCDR2, and LCDR3 of the light chain variable domain, and HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain). See Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (which defines the antibody CDR region by sequence). Also see Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (which defines the antibody CDR region by structure). The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0110] Unless otherwise specified, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which a full-length antibody binds, for example, a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from single-chain Fv(ScFv), nanobodies, and antibody fragments.

[0111] As used herein, "specifically binds" an antibody to a target protein means that such antibody exhibits selective binding to its target compared to binding to other proteins, but this specificity does not necessarily require absolute binding specificity. "Specifically binding" or "selectively binding" of an antibody is used in the context of describing an interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, referring to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biologics, e.g., a biological sample, blood, serum, plasma, or tissue sample. Therefore, under specific, designated immunoassay conditions, the antibody or its antigen-binding fragment binds specifically to a particular antigen at least twice as much as the background level, and does not specifically bind to other antigens present in the sample in significant amounts. In one embodiment, under designated immunoassay conditions, the antibody or its antigen-binding fragment binds specifically to a particular antigen at least ten times as much as the background level of binding, and does not specifically bind to other antigens present in the sample in significant amounts.

[0112] As used herein, the “antigen-binding domain” comprises at least three CDRs and specifically binds to an epitope. The “antigen-binding domain” of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain that specifically binds to a first epitope, and a second antigen-binding domain that specifically binds to a second epitope and similarly consists of at least three CDRs. Multispecific antibodies can be bispecific, triplicate, quadruplicate, etc., with the antigen-binding domains directed to each specific epitope. A multispecific antibody may be polyvalent (e.g., a bispecific quadruple antibody) and contain multiple antigen-binding domains, for example, two, three, four, or more antigen-binding domains that specifically bind to a first epitope, and two, three, four, or more antigen-binding domains that specifically bind to a second epitope.

[0113] In this specification, the term "human antibody" means an antibody containing only human immunoglobulin protein sequences. Human antibodies may contain mouse glycans if they are produced in mice, mouse cells, or mouse cell-derived hybridomas. Similarly, "mouse antibody" or "rat antibody" means an antibody containing only mouse immunoglobulin protein sequences or an antibody containing only rat immunoglobulin protein sequences, respectively.

[0114] The terms "humanized" or "humanized antibody" refer to a form of antibody that contains sequences derived from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all hypervariable loops in the variable domains correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all FR regions are FR regions of the human immunoglobulin sequence. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region (Fc). When it is necessary to distinguish humanized antibodies from rodent-parent antibodies, the prefix "hum," "hu," "Hu," or "h" is added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequence as the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, improve the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0115] The term "epitope" refers to a specific site on an antigen to which an antibody binds. This specific site on the antigen can be determined, for example, by crystal structure analysis. Other methods such as hydroxyl radical protein footprinting and alanine scanning mutagenesis may also be used, but these may have low resolution.

[0116] The term "monospecific antibody" refers to an antibody that specifically binds to only one antigen. A monospecific antibody may bind to only one epitope of an antigen, or it may bind to two or more epitopes of an antigen. A monospecific antibody that binds to two or more epitopes of an antigen is called a monospecific polyepitope antibody.

[0117] The term "multispecific antibody" refers to an antibody that specifically binds to two or more antigens (e.g., a bispecific antibody, a trispecific antibody, etc.). Non-exclusive examples of multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) in which the VH / VL unit has polyepitope specificity, antibodies having two or more VL and VH domains in which each VH / VL unit binds to a different epitope, antibodies having two or more single variable domains in which each single variable domain binds to a different epitope, diabodies, triabodies, etc., as well as full-length antibodies and / or covalently or non-covalently linked antibody fragments.

[0118] The terms "polyepitope antibody" and "antibody having polyepitope specificity" are used interchangeably herein and refer to antibodies that bind to two or more epitopes on the same or different antigens.

[0119] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, including both the Fc region of the natural sequence and the Fc region of variants. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The lysine residue at the C-terminus of the Fc region (residue 447 according to the Eu numbering system) may be removed, for example, during the production or purification of the antibody, or by manipulating the nucleic acid encoding the antibody heavy chain using recombinant techniques. Thus, a composition of intact antibodies may include antibody populations in which the Lys447 residue has been completely removed, antibody populations in which the Lys447 residue has not been removed, and antibody populations having a mixture of antibodies containing and not containing the Lys447 residue.

[0120] A "functional Fc region" possesses effector functions of the Fc region in the natural sequence. Exemplary effector functions include C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays disclosed herein or known in the art. Functional Fc regions may have substantially the same effector functions as wild-type IgG, lower effector functions compared to wild-type IgG, or higher effector functions compared to wild-type IgG. For antibodies containing human Fc regions, the comparison is typically performed against wild-type human IgG1.

[0121] "Natural Fc regions" include amino acid sequences identical to those of naturally occurring Fc regions. Examples of natural human Fc regions include the Fc regions of natural human IgG1 (non-A and A allotypes), natural human IgG2, natural human IgG3, and natural human IgG4, as well as their naturally occurring variants.

[0122] The "variant Fc region" includes at least one amino acid modification (e.g., about 1 to about 10 amino acid modifications, and in some embodiments, about 1 to about 5 amino acid modifications), preferably one or more amino acid substitutions, which result in an amino acid sequence different from the amino acid sequence of the Fc region of the natural sequence. The variant Fc region as used herein preferably has at least about 80% homology to the Fc region of the natural sequence and / or the Fc region of the parent polypeptide, preferably at least about 90% homology, or preferably at least about 95% homology. In some embodiments, the variant Fc region may have lower or higher effector function compared to wild-type IgG. For antibodies containing a human Fc region, the comparison is typically made against wild-type human IgG1.

[0123] As used herein, the term “Fc component” refers to the hinge region, CH2 domain, or CH3 domain of the Fc region.

[0124] The term "hinge region" is generally defined as extending from approximately IgG residues 216–230 (Eu numbering), 226–243 (Kabat numbering), or 1–15 (IMGT-specific numbering).

[0125] The term "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include diabody, Fab, Fab', F(ab')2, and F(ab). cExamples of binding fragments include, but are not limited to, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds-diabodies), triabodies, tetrabodies, single-chain antibodies, scFv, scFv dimers, single-domain antibodies, single-domain antibodies, and multivalent domain antibodies. Typically, binding fragments compete for specific binding with the intact antibody from which they are derived. Binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins.

[0126] The term "Fab" refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) attached by disulfide bonds to the variable and primary constant regions of a single heavy chain.

[0127] The term "Fab" refers to a Fab fragment that includes part of the hinge region.

[0128] The term "F(ab')2" refers to the dimer of Fab'. The F(ab')2 antibody fragment was originally produced as a pair with a Fab' fragment containing a hinged cysteine. Other chemical combinations of antibody fragments are also known.

[0129] The term "Fv" refers to the smallest fragment of an antibody that has a complete antigen-binding site. An Fv fragment consists of a single variable region of a light chain bound to a variable region of a single heavy chain.

[0130] The term "single-chain antibody" refers to an antibody consisting of a heavy-chain variable region and a light-chain variable region linked by a linker. In most, though not all, cases, the linker may be a peptide. The length of the linker varies depending on the type of single-chain antibody. Higher-order forms can be obtained by covalently or non-covalently linking two or more single-chain antibodies together. Examples of single-chain antibodies and their higher-order forms include, but are not limited to, single-domain antibodies, multivalent domain antibodies, single-chain variant fragments (scFv), divalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), diabodies, and triabodies and tetrabodies.

[0131] The terms “single-chain Fv antibody” and “scFv” are used interchangeably herein and refer to a single-chain antibody consisting of a heavy-chain variable region and a light-chain variable region linked by a linker. In most, but not all, cases, the linker may be a peptide. The linker peptide is preferably about 5 to 30 amino acids long, or about 10 to 25 amino acids long. Typically, the linker allows for the stabilization of the variable domain without hindering proper folding and the creation of an active binding site. In preferred embodiments, the linker peptide is rich in glycine and serine or threonine. By covalently or noncovalently linking two or more scFvs together, higher-order forms such as di-scFv, tri-scFv, and tetra-scFv can be obtained. The antigen-binding site of each scFv in a higher-order form may target the same or different antigens or epitopes.

[0132] The terms "single-chain Fv-Fc antibody" or "scFv-Fc" refer to a full-length antibody consisting of scFv attached to an Fc region.

[0133] A "diabody" is a higher-order variant of a single-chain antibody consisting of two single-chain antibodies. For each single-chain antibody, a linker too short to pair two domains on the same chain is used to pair these domains with a complementary domain on the other chain, thereby creating two antigen-binding sites. In most, though not all, cases, the linker can be a peptide. These antigen-binding sites can target the same or different antigens or epitopes. Triabodies (three single-chain antibodies assembled to form three antigen-binding sites), tetrabodies (four single-chain antibodies assembled to form four antigen-binding sites), and higher-order variants can similarly be produced. See, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15;90(14):6444-8 (1993), EP404097, WO93 / 11161.

[0134] A "single-domain antibody" refers to an antibody fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, it may contain two or more V H The domains are covalently linked by a peptide linker, creating a multivalent domain antibody. Two or more Vs of the multivalent domain antibody H The domain may target the same or different antigens or epitopes.

[0135] The term "heavy-chain antibody" refers to an antibody consisting of two heavy chains. Heavy-chain antibodies can be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNARs from cartilaginous fish. See, for example, Riechmann L. and Muyldermans S., J Immunol Methods. December 10;231(1-2):25-38 (1999), Muyldermans S., J Biotechnol. June;74(4):277-302 (2001), WO94 / 04678, WO94 / 25591, or U.S. Patent No. 6,005,079. Heavy-chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Camelized antibodies lack a light chain but possess a true antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8(1993), Nguyen VK et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. April;54(1):39-47(2002), Nguyen VK et al. Immunology. May;109(1):93-101(2003)). The variable domain (VHH domain) of heavy-chain antibodies represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007 Jun.15(2007)).

[0136] The term “corresponding human germline sequence” refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. The corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence may be a sequence or subsequence containing only the framework region, only the complementarity-determining region, the framework and complementarity-determining regions, a variable segment (as defined above), or any other combination of sequences or subsequences containing the variable region. Sequence identity can be determined using the methods described herein, e.g., aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, that constant region also originates from such human sequences, e.g., human germline sequences, or variants of human germline sequences, or from an antibody containing a consensus framework sequence obtained from human framework sequence analysis, as described, for example, Knappik et al., J.Mol.Biol.296:57-86, 2000.

[0137] The term “equilibrium dissociation constant (KD, M)” refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-1). The equilibrium dissociation constant can be measured using any method known in the art. The antibodies of this disclosure generally have an equilibrium dissociation constant less than about 10⁻⁷ or less than 10⁻⁸ M, for example, less than about 10⁻⁹ M or less than 10⁻¹⁰ M, and in some embodiments, less than about 10⁻¹¹ M, less than 10⁻¹² M or less than 10⁻¹³ M.

[0138] In this specification, the terms “cancer” or “tumor” have the broadest meaning as understood in the art and refer to a physiological condition in mammals typically characterized by uncontrolled cell proliferation. In connection with this disclosure, cancer is not limited to any particular type or location.

[0139] In relation to this disclosure, where an amino acid sequence is mentioned, the term “conservative substitution” means the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, such as its binding affinity to CLDN6. In particular, general conservative conversions of amino acids are well known in the art.

[0140] As used herein, the “knob and hole” technique refers to an amino acid that directs the pairing of two polypeptides in vitro or in vivo by introducing a spatial projection (knob) into one polypeptide and a socket or cavity (hole) into the other polypeptide at the interface in which they interact. For example, knobs and holes are introduced at the Fc:Fc binding interface, CL:CHI interface, or VH / VL interface of an antibody (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al, 1997, Protein Science 6:781-788). In some embodiments, knobs and holes ensure the correct pairing of two different heavy chains during the production of multispecific antibodies. For example, a multispecific antibody having knob and hole amino acids within its Fc region may further contain a single variable domain linked to each Fc region, or it may further contain different heavy chain variable domains that pair with similar or different light chain variable domains. The knob and hole technique can also be used in the VH or VL region to ensure correct pairing.

[0141] As used herein in the context of the “knob and hole” technology, the term “knob” refers to an amino acid mutation that introduces a protrusion (knob) into a polypeptide at an interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0142] As used herein in the context of “knobs and holes,” the term “hole” refers to an amino acid change that introduces a socket or cavity (hole) in a polypeptide at an interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0143] Examples of suitable algorithms for determining sequence identity percentage and sequence similarity include the BLAST algorithm, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W within the query sequence, which are words that match or satisfy a threshold score T that has a positive value when aligned with words of the same length in the database sequence. T is called the neighbor word score threshold. These first hit neighbor words serve as a value to initiate a search for longer HSPs that contain them. Word hits are extended toward both ends of each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a matching pair of residues, always >0) and N (penalty score for mismatched residues, always <0). For amino acid sequences, the cumulative score is calculated using a score matrix. Word hits are stopped in each direction if the cumulative alignment score falls by X from the maximum attainable value; if the cumulative score becomes zero or less due to the accumulation of one or more negative score residue alignments; or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses 11 word lengths (W), 10 expected values ​​(E), M=5, N=-4, and double-strand comparison as defaults.For amino acid sequences, the BLAST program uses a word length of 3, an expected value of 10 (E), and a BLOSUM62 score matrix of 50 (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignment (B), expected value of 10 (E), M=5, N=-4, and double-strand comparison as defaults.

[0144] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that a match between two sequences of nucleotides or amino acids occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0145] The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput.Appl.Biosci.4:11-17,(1988). This algorithm is incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch, J.Mol.Biol.48:444-453,(1970), which is incorporated into the GAP program of the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0146] The term “nucleic acid” is used herein in the same sense as the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form and polymers thereof. This term encompasses nucleic acids containing known nucleotide analogs or modified skeletal residues or bonds, which include synthetic nucleic acids, naturally occurring nucleic acids, and naturally occurring nucleic acids, which have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0147] In relation to nucleic acids, the term "operably ligated" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to a functional relationship between a transcriptional regulatory sequence and a transcription sequence. For example, a promoter or enhancer sequence is functionally ligated to a coding sequence if it stimulates or modulates the transcription of that coding sequence in a suitable host cell or other expression system. Generally, promoters and transcriptional regulatory sequences that are operably ligated to a transcription sequence are physically contiguous to the transcription sequence; i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequence in which they enhance transcription.

[0148] In some embodiments, the Disclosure provides compositions, for example, pharmaceutically acceptable compositions comprising an anti-CLDN6 antibody as described herein and formulated with at least one pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically acceptable excipient” includes all physiologically compatible solvents, dispersions, isotonic agents, and absorption retarders, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or dermal administration (e.g., by injection or infusion).

[0149] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and liquid, semi-solid, and solid dosage forms such as suppositories. The appropriate form depends on the intended method of administration and therapeutic use. Typical suitable compositions are in the form of injection and infusion solutions. One suitable method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0150] As used herein, the term “therapeutic dose” refers to the amount of antibody sufficient to produce a treatment for a disease, disorder, or symptom when administered to a subject to treat the disease, disorder, or symptom. The “therapeutic dose” may vary depending on the antibody, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the symptoms of the disease, disorder, and / or the age of the subject being treated, and / or the weight of the subject being treated. An appropriate dose in any given case may be obvious to those skilled in the art and may also be determined by routine experimentation. In the case of combination therapy, the “therapeutic dose” refers to the total amount of the combined subjects to effectively treat the disease, disorder, or condition.

[0151] The term “combination therapy” refers to the administration of two or more therapeutic agents for the treatment of a therapeutic condition or disorder described herein. Such administrations include the simultaneous administration of these therapeutic agents in substantially simultaneous mode. Such administrations also include the simultaneous administration of each active ingredient in multiple containers or in separate containers (e.g., capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to a desired dose before administration. Furthermore, such administrations also include the use of various therapeutic agents in a sequential manner, either at approximately the same time or at different times. In any case, the treatment regimen provides the beneficial effects of drug combination in the treatment of the conditions or disorders described herein.

[0152] As used herein, the expression "in combination with" means that the anti-CLDN6xCD3 multispecific antibody is administered to the subject simultaneously with, immediately before, or immediately after, the administration of further therapeutic agents. In certain embodiments, the anti-CLDN6xCD3 multispecific antibody is administered as a co-formulation with further therapeutic agents. [Modes for carrying out the invention]

[0153] This disclosure provides antibodies, antigen-binding fragments, and anti-CLDN6 antibodies. Furthermore, this disclosure provides antibodies having desirable pharmacokinetic properties and other desirable attributes, which can be used to reduce the likelihood of cancer or to treat cancer. This disclosure further provides pharmaceutical compositions comprising such antibodies for the prevention and treatment of cancer and related disorders, as well as methods for manufacturing and using such pharmaceutical compositions.

[0154] Anti-CLDN6 antibody This disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to CLDN6. The antibody or antigen-binding fragment of this disclosure includes, but is not limited to, antibodies or antigen-binding fragments thereof that are produced as described below.

[0155] This disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antibody fragment (e.g., an antigen-binding fragment) comprises a VH domain having the amino acid sequence listed in Table 1. This disclosure also provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises an HCDR having the amino acid sequence of any one of the HCDRs listed in Table 1. In one embodiment, this disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody comprises (or alternatively consists of) 1, 2, 3, or more HCDRs having the amino acid sequence of any of the HCDRs listed in Table 1.

[0156] This disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises a VL domain having the amino acid sequence described in Table 1. This disclosure also provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises an LCDR having any one of the LCDR amino acid sequences described in Table 1. In particular, this disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises (or alternatively consists of) 1, 2, 3, or more LCDRs having any of the LCDR amino acid sequences described in Table 1.

[0157] Other antibodies or antigen-binding fragments of the present disclosure include modified amino acids that have at least 60%, 70%, 80%, 90%, 95%, or 99% identity in the CDR region with the CDR regions disclosed in Table 1. In some embodiments, the amino acid sequence has at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity. In some embodiments, it includes amino acid modifications in which one, two, three, four, or five or fewer amino acids are changed in the CDR region compared to the CDR region shown in the sequences of Table 1.

[0158] Other antibodies in this disclosure include antibodies in which the amino acids or the nucleic acids encoding the amino acids have been modified, but which have at least 60%, 70%, 80%, 90%, 95%, or 99% identity with the sequences listed in Table 1. In some embodiments, the amino acid sequences have at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity. In some embodiments, it includes an amino acid sequence modification in which, when compared to the variable region shown in the sequences listed in Table 1, one, two, three, four, or five or fewer amino acids have been changed in that variable region, while retaining substantially the same therapeutic activity.

[0159] This disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to CLDN6. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0160] This disclosure provides an antibody and an antigen-binding fragment thereof that bind to an epitope of human CLDN6. In certain embodiments, the antibody and antigen-binding fragment may bind to the same epitope of CLDN6.

[0161] This disclosure also provides antibodies and their antigen-binding fragments that bind to the same epitopes as the anti-CLDN6 antibodies listed in Table 1. Therefore, further antibodies and their antigen-binding fragments may be identified based on their ability to cross-compete with other antibodies in a binding assay (e.g., statistically significantly inhibit binding competitively). The ability of a test antibody to inhibit the binding of the antibodies and their antigen-binding fragments of this disclosure to CLDN6 indicates that the test antibody may compete with the antibody or its antigen-binding fragment for binding to CLDN6. Such antibodies may bind to the same or related (e.g., structurally similar or spatially proximal) epitopes on CLDN6 as the competing antibody or its antigen-binding fragment, although such antibodies are not bound by any single theory. In certain embodiments, antibodies that bind to the same epitopes on CLDN6 as the antibodies or their antigen-binding fragments of this disclosure are human or humanized monoclonal antibodies. Such human or humanized monoclonal antibodies may be prepared and isolated as described herein.

[0162] In one embodiment, the anti-CLDN6 antibody disclosed herein may be an anti-CLDN6 multispecific antibody. A certain antibody molecule is a multispecific antibody molecule, for example, comprising several antigen-binding domains, in which case at least one antigen-binding domain sequence specifically binds to CLDN6 as a first epitope and a second antigen-binding domain sequence specifically binds to the second epitope. In one embodiment, the multispecific antibody comprises a third, fourth, or fifth antigen-binding domain. In one embodiment, the multispecific antibody is a bispecific, triplicate, or quadruplicate antibody. In each example, the multispecific antibody comprises at least one anti-CLDN6 antigen-binding domain and at least one anti-CD3 antigen-binding domain.

[0163] In one embodiment, the multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. The bispecific antibody comprises a first antigen-binding domain that specifically binds to CLDN6, and a second antigen-binding domain that specifically binds to another epitope. This includes a bispecific antibody comprising a heavy-chain variable domain and a light-chain variable domain that specifically binds to CLDN6 as the first epitope, and a heavy-chain variable domain that specifically binds to CD3 as the second epitope. In a bispecific antibody comprising such antigen-binding fragment, the antigen-binding fragment may be Fab, F(ab')2, Fv, or single-chain Fv(ScFv) or scFv.

[0164] Previous experiments (Coloma and Morrison Nature Biotech. 15:159-163 (1997)) described tetravalent bispecific antibodies manipulated by fusing the DNA encoding the Fv (scFv) of a single-chain anti-dansyl antibody after the C-terminus (CH3-scFv) or after the hinge (hinge-scFv) of an IgG3 anti-dansyl antibody. This disclosure provides a polyvalent antibody (e.g., a tetravalent antibody) having at least two antigen-binding domains, which can be readily produced by recombinant expression of the nucleic acid encoding the polypeptide chain of the antibody. The polyvalent antibodies described herein comprise 3 to 8, preferably 4, antigen-binding domains that specifically bind to at least two antigens.

[0165] Linker It is also understood that the domains and / or regions of the polypeptide chain of the bispecific tetravalent antibody may be separated by linker regions of varying lengths. In some embodiments, the antigen-binding domain is separated from each other by linker regions from the entire CL, CH1, hinge, CH2, CH3, or Fc region. For example, VL1-CL-(linker)VH2-CH1, VH-linker-VL. Such linker regions may contain any combination of amino acids or a limited set of amino acids. Such linker regions may be flexible or rigid (see US2009 / 0155275).

[0166] Multispecific antibodies, with or without the use of a flexible linker, can be dimerized via dimerization devices such as leucine zippers (Kostelny et al., J.Immunol. 1992 148:1547-53, de Kruifetal J.Biol.Chem. 1996 271:7630-4) and Ig C / CH1 domains (Muller et al., FEBS Lett. 422:259-64), as well as in diabodies (Holliger et al., (1993) Proc.Nat.Acad.Sci.USA. 1998 90:6444-8, Zhu et al., Bio / Technology (NY) 1996 14:192-6), Fab-scFv fusion (Schoonjans et al., J.Immunol. 2000 165:7050-7), and mini-antibody formats (Packet). According to al., Biochemistry 1992.31:1579-84, Packet al., Bio / Technology 1993 11:1271-7), it is constructed by genetically fusing two single-stranded Fv (scFv) or Fab fragments (Mallender et al., J. Biol. Chem. 1994 269:199-206, Packet al., Proc. Natl. Acad. Sci. USA. 1995 92:7021-5, Zapata et al., Protein Eng. 1995 8.1057-62).

[0167] The bispecific tetravalent antibodies disclosed herein include a linker region between one or more of the antigen-binding domain, CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain, or Fc region, comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues. In some embodiments, the amino acids glycine and serine constitute the amino acids within the linker region. In another embodiment, the linker is GS, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGSGS, GGGGSGS, GGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS, RADAAAAGGPGS, RADAAAA(G4S)4, SAKT It may be TP, SAKTTPKLGG, SAKTTPKLEEGEFSEARV, ADAAP, ADAAPTVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP, AKTTPPSVTPLAP, AKTTAP, AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP, GENKVEYAPALMALS, GPAKELTPLKEAKVS, and GHEAAAVMQVQYPAS, or any combination thereof (see WO2007 / 024715).

[0168] Dimerization-specific amino acids In one embodiment, the polyvalent antibody contains at least one dimerization-specific amino acid change. This dimerization-specific amino acid change creates a “knob-hole” interaction, increasing the correct assembly of the polyvalent antibody. The dimerization-specific amino acid may be within a CH1 domain or a CL domain or a combination thereof. The dimerization-specific amino acid is used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL), and can be found in at least the disclosures WO2014082179, WO2015181805 family and WO2017059551. The dimerization-specific amino acid may be within an Fc domain and may be combined with a dimerization-specific amino acid within a CH1 or CL domain. In one embodiment, the disclosure provides a bispecific antibody containing at least one dimerization-specific amino acid pair.

[0169] Further modifications to the FC domain framework In yet another embodiment, the effector function of an antibody is modified by altering the Fc region by substituting at least one amino acid residue with a different amino acid residue. For example, one or more amino acids can be substituted with different amino acid residues, resulting in an antibody that has a modified affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand with modified affinity may be, for example, the Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0170] In another embodiment, one or more amino acid residues may be substituted with one or more different amino acid residues so that the antibody has modified C1q binding and / or reduced or absent complement-dependent cell-mediated cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0171] In yet another embodiment, one or more amino acid residues are modified to alter the antibody's ability to immobilize complement. This approach is described, for example, in Publication WO94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of the antibody or its antigen-binding fragment of the Disclosure are replaced with one or more allotype amino acid residues for the IgG1 subclass and kappa isotype. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant regions of the light chains of kappa isotypes, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0172] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the antibody's ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or to increase the antibody's affinity for the Fcγ receptor. This approach is described, for example, in Presta's publication WO00 / 42072. Furthermore, the binding sites of FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0173] In yet another embodiment, the glycosylation of the multispecific antibody is modified. For example, a non-glycosylated antibody (i.e., an antibody with no or reduced glycosylation) can be produced. By modifying glycosylation, for example, the antibody's affinity for an "antigen" can be increased. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more variable region framework glycosylation sites can be removed by one or more amino acid substitutions, thereby eliminating glycosylation at that site. Such nonglycosylation can increase the antibody's affinity for an antigen. Such approaches are described, for example, in U.S. Patents 5,714,350 and 6,350,861 by Co et al.

[0174] Additionally or alternatively, antibodies with altered glycosylation types (e.g., low-fucosylated antibodies with reduced fucosyl residue levels, or antibodies with increased bisected GlcNAc structures) can be produced. Such modified glycosylation patterns have been shown to enhance the ADCC activity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with modified glycosylation pathways. Cells with modified glycosylation pathways have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with modified glycosylation. For example, Hang et al. (EP1,176,195) describe cell lines in which the FUT8 gene encoding fucosyltransferase is functionally disrupted so that the antibodies expressed in the cell lines exhibit low fucosylation. Presta's publication WO03 / 035835 describes Lecl3 cells, a mutant CHO cell line with reduced ability to bind fucose to Asn(297)-linked carbohydrates, which also results in decreased fucosylation of antibodies expressed in its host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s WO99 / 54342 describes a cell line engineered to express a glycoprotein-modified glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) so that antibodies expressed in the engineered cell line show increased bisected GlcNac structure, resulting in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0175] In another embodiment, when reduction of ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to possess only moderate ADCC and little CDC effector function (Moore GL, et al., 2010 MAbs, 2:181-189). However, natural IgG4 has been found to be less stable in acidic buffers or under stress conditions such as elevated temperature (Angal, S. 1993 Mol Immunol, 30:105-108, Dall'Acqua, W. et al. 1998 Biochemistry, 37:9266-9273, Aalberse et al. 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably binding the antibody to IgG4 Fc manipulated by a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the less desirable intrinsic properties of IgG4 is its ability to dynamically separate its two heavy chains in solution to form half an antibody, which in vivo generates a bispecific antibody via a process called "Fab arm exchange" (Van der Neut Kolfschoten M, et al., 2007 Science, 317:1554-157). A mutation from serine to proline at position 228 (EU numbering system) appears to inhibit the separation of the IgG4 heavy chain (Angal, S. 1993 Mol Immunol, 30:105-108, Aalberse et al., 2002 Immunol, 105:9-19).Some amino acid residues in the hinge and γFc region have been reported to affect antibody interactions with the Fcγ receptor (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040, Mukherjee, J. et al., 1995 FASEB J, 9:115-119, Armour, K. et al., 1999 Eur J Immunol, 29:2613-2624, Clynes, RA et al, 2000 Nature Medicine, 6:443-446, Arnold JN, 2007 Annu Rev immunol, 25:21-50). Furthermore, some IgG4 isoforms that occur rarely in the human population can also produce different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To produce multispecific antibodies with good stability but low ADCC and CDC, it is possible to modify the hinge and Fc region of human IgG4 and introduce several modifications. These modified IgG4 Fc molecules can be found in U.S. Patent No. 8,735,553 by Li et al., which is incorporated herein by reference.

[0176] Antibody production Antibodies and their antigen-binding fragments can be produced by any means known in the art, including but not limited to recombinant expression of antibody tetramers, chemosynthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression may be derived from any suitable host cells known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0177] This disclosure further provides polynucleotides encoding antibodies as described herein, for example, polynucleotides encoding segments including heavy chain or light chain variable regions or complementarity-determining regions as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 9, 56, 60, and 64. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 10, 57, 61, or 65.

[0178] The polynucleotides of this disclosure can encode the variable region sequence of an anti-CLDN6 antibody. They can also encode both the variable and constant regions of the antibody. Some of the polynucleotide sequences encode polypeptides containing the variable regions of both the heavy and light chains of an exemplified anti-CLDN6 antibody.

[0179] Similarly provided in this disclosure are expression vectors and host cells for producing the anti-CLDN6 antibody. The choice of expression vector depends on the host cell in which the vector is intended to be expressed. Typically, the expression vector comprises a promoter and other regulatory sequences (e.g., enhancers) operably ligated to a polynucleotide encoding the anti-CLDN6 antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent the expression of the insertion sequence outside of the control of induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducible conditions without biasing the population for encoding sequences that are better tolerated by the host cell. In addition to promoters, other regulatory elements may also be required or desired for the efficient expression of the anti-CLDN6 antibody or antigen-binding fragment. These elements typically include the ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by incorporating the appropriate enhancer into the cell line being used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, an SV40 enhancer or a CMV enhancer can be used to increase expression in mammalian host cells.

[0180] The host cells for possessing and expressing the anti-CLDN6 antibody chain may be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of this disclosure. Other suitable microbial hosts include rods, e.g., Bacillus subtilis, and other Enterobacteria, e.g., Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be constructed from these prokaryotic hosts, typically containing expression regulatory sequences (e.g., origins of replication) that are compatible with the host cell. Furthermore, various well-known promoters exist, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or phage-lambda-derived promoter systems. These promoters typically have ribosome-binding sites, etc., for initiating and completing transcription and translation, and optionally control expression with operator sequences. Other microorganisms, such as yeast, can also be used to express anti-CLDN6 antibodies. Insect cells can also be used in combination with baculovirus vectors. In other embodiments, mammalian host cells are used to express and produce the anti-CLDN6 antibody of this disclosure. For example, these may be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines having exogenous expression vectors. These include any normal non-immortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures for polypeptide expression is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987.Mammalian host cell expression vectors may contain expression regulatory sequences such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Appropriate promoters may be constitutive, cell type-specific, stage-specific, and / or tunable or modifiable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (e.g., the human early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0181] Production of bispecific antibodies The current standard for engineered heterodimer antibody Fc domains is a knob and hole (KiH) design that introduces mutations at the core CH3 domain interface. The resulting heterodimers have a lower CH3 melting temperature (below 69°C). In contrast, the ZW heterodimer Fc design has a thermal stability of 81.5°C, which is comparable to that of the wild-type CH3 domain.

[0182] Detection and diagnostic methods The antibodies or antigen-binding fragments of this disclosure are useful in a variety of applications, including but not limited to methods for detecting CLDN6. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of CLDN6 in a biological sample. As used herein, the term “detecting” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissue. In other embodiments, such tissue includes normal tissue and / or cancerous tissue that express CLDN6 at high levels compared to other tissues.

[0183] In one embodiment, the present disclosure provides a method for detecting the presence of CLDN6 in a biological sample. In a particular embodiment, the method includes contacting the biological sample with an anti-CLDN6 antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may be, but is not limited to, urine, tissue, saliva, or blood samples.

[0184] The method also includes a method for diagnosing disorders related to CLDN6 expression. In a particular embodiment, the method includes contacting test cells with an anti-CLDN6 antibody, determining (quantitatively or qualitatively) the level of CLDN6 expression by the test cells by detecting the binding of the anti-CLDN6 antibody to the CLDN6 polypeptide, and comparing the level of expression by the test cells with the level of CLDN6 expression in control cells (e.g., normal cells or non-CLDN6-expressing cells of the same tissue origin as the test cells), wherein a higher level of CLDN6 expression in the test cells compared to the control cells indicates the presence of a disorder related to CLDN6 expression.

[0185] Treatment method The antibodies or antigen-binding fragments of this disclosure are useful in a variety of applications, including but not limited to methods for treating CLDN6-related disorders or diseases. In one embodiment, the CLDN6-related disorder or disease is cancer.

[0186] In one embodiment, the present disclosure provides a method for treating cancer. In a particular embodiment, the method comprises administering an effective amount of anti-CLDN6 antibody or antigen-binding fragment to a patient in need thereof. In some embodiments, the cancer is a solid tumor. Examples of such cancers include, but are not limited to, gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor, and / or glioma.

[0187] The antibodies or antigen-binding fragments disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if local treatment is required, intrafocal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any suitable route, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules, including single or multiple doses, bolus administration, and pulse infusion at various time points, are contemplated herein, but are not limited to these.

[0188] The antibodies or antigen-binding fragments of this disclosure can be formulated, administered, and given in accordance with appropriate medical practice. Factors to consider in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the plan of administration, and other factors known to the healthcare professional. The antibody may, but is not necessarily, be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective dose of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These are generally used in the same doses and routes of administration as described herein, or at about 1–99% of the doses described herein, or in any dose and route that is empirically / clinically determined to be appropriate.

[0189] For the prevention or treatment of a disease, the appropriate dose of the antibody or antigen-binding fragment of this disclosure will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is administered to the patient appropriately, either as a single dose or over a series of treatments. Depending on the type and severity of the disease, approximately 1 μg / kg to 100 mg / kg of antibody may be an initial candidate dose for administration to the patient, for example, by one or more individual doses or by continuous infusion. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. Depending on the condition, for repeated administrations over several days or longer periods, the treatment is generally continued until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to approximately 20, or for example, approximately 6 doses of antibody). A higher loading dose can be administered first, followed by one or more lower doses. However, other drug regimens may be useful. The progression of this treatment can be easily monitored using conventional techniques and assays.

[0190] Combination therapy In one embodiment, the anti-CLDN6 antibody of this disclosure can be used in combination with other therapeutic agents. Other therapeutic agents that may be used in conjunction with the anti-CLDN6 antibody of this disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel formulations, (e.g., Abraxane®), docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacitidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multi-kinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targeted agents (e.g., rituximab, o Fatumumab (RO5072759, LFB-R603), CD52-targeted drugs (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19-targeted drugs (e.g., MEDI-551, MOR2) 08) Examples include, but are not limited to, MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), EGEN-001, and polo-like kinase 1 inhibitors (e.g., BI 672).

[0191] The anti-CLDN6 antibody disclosed herein can be used in combination with other therapeutic agents, such as immune checkpoint antibodies. Examples of such immune checkpoint antibodies include anti-PD1 antibodies. Examples of anti-PD1 antibodies include, but are not limited to, tislerizumab, pembrolizumab, or nivolumab. Tislerizumab is disclosed in US8,735,553. Pembrolizumab (formerly MK-3475), disclosed in US8,354,509 and US8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab is approved for the treatment of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is being clinically studied for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. patent US8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, renal cancer, and Hodgkin lymphoma.

[0192] Other immune checkpoint antibodies for use in combination with anti-CLDN6 antibodies include anti-TIGIT antibodies. Such anti-TIGIT antibodies include, but are not limited to, those disclosed in WO2019 / 129261.

[0193] Other immune checkpoint antibodies for use in combination with anti-CLDN6 antibodies include anti-OX40 antibodies. Such anti-OX40 antibodies include, but are not limited to, those disclosed in WO2019 / 223733.

[0194] Other immune checkpoint antibodies for use in combination with anti-CLDN6 antibodies include anti-TIM3 antibodies. Such anti-TIM3 antibodies include, but are not limited to, those disclosed in WO2018 / 036561.

[0195] Pharmaceutical compositions and preparations Also provided are compositions such as pharmaceutical formulations, comprising an anti-CLDN6 antibody or its antigen-binding fragment, or a polynucleotide comprising a sequence encoding an anti-CLDN6 antibody or antigen-binding fragment. In certain embodiments, the composition comprises one or more anti-CLDN6 antibodies or antigen-binding fragments, or one or more polynucleotides comprising a sequence encoding one or more anti-CLDN6 antibodies or antigen-binding fragments. These compositions may further comprise a suitable carrier, for example, a pharmaceutically acceptable excipient, including a buffer, which is well known in the art.

[0196] The pharmaceutical formulations of anti-CLDN6 antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments of desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include buffers, such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, etc.), low molecular weight (less than about 10 residues) polypeptides, and proteins. Examples of pharmaceutically acceptable carriers herein include, but are not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein include, but are not limited to, interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patents US7,871,607 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more further glycosaminoglycanases, such as chondroitinase.

[0197] In one embodiment, the formulation consists of L-histidine / L-histidine hydrochloride monohydrate, trehalose, and polysorbate 20. In another embodiment, the concentration of the anti-CLDN6 antibody formulation is an isotonic solution with a pH of approximately 5.5, consisting of 10 mg / mL of anti-CLDN6 antibody, 20 mM histidine / histidine HCl, 240 mM trehalose dihydrate, and 0.02% polysorbate 20, after being prepared with sterile water for injection.

[0198] An exemplary lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0199] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of molded articles, such as films or microcapsules.

[0200] Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0201] Sequence List The sequence listings for this disclosure are shown in Tables 1-3 below. [Table 1-1] [Table 1-2] [Table 1-3] Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Examples]

[0202] Example 1. Generation of mouse anti-CLDN6 antibody To generate antibodies against CLDN6, cohorts of 20–25 inbred mice of the BALB / C, SJL strain were immunized with human CLDN6 overexpressing cells (L929 / human CLDN6, in-house prepared). Each cohort was subjected to an immunization strategy including a unique combination of CLDN6 antigen, dose, injection route, adjuvant, and timing of immunization. Four–five cohorts, totaling five animals, were immunized. Animals were immunized over various periods from 0 to 56 days. To monitor the immune response, titrated serum was screened by FACS typically 21–56 days after 2–4 immunizations. Serum was screened for antibodies binding to CLDN6 overexpressing cell CHOK1 / human CLDN6. In each animal, the CLDN6-specific antibody response was measured, and animals with sufficient anti-CLDN6 Ig titers were selected for a 4-day final boost.

[0203] Lymphoid organs, including the spleen and lymph nodes, were isolated from mice immunized as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells derived from SP2 / 0 using PEG-based fusion. The resulting cells were plated into 96-well cell culture plates using standard 1640 medium supplemented with HAT for hybridoma selection.

[0204] Example 2. Screening and selection of anti-CLDN6 antibodies Hybridomas were generated as described in Example 1. After 10–13 days of culture and growth medium changes, the culture supernatant of the hybridomas was collected from individual wells and screened to identify wells that secreted CLDN6-specific antibodies. First, all supernatants were screened against at least two overexpression cell lines, including CHOK1 / human CLDN6 and CHOK1 / human CLDN9 (prepared in-house). Antibody binding in the overexpression cell lines was measured by FACS. Supernatants from over 20,000 culture wells in four hybridoma fusions were screened for CLDN6 antibodies. Briefly, 100 μL of hybridoma culture supernatant was co-incubated with CLDN6-expressing cancer cell lines (e.g., PA-1 or CHOK1 / human CLDN6 stable cell line) or control cells (e.g., parental CHOK1) for 30–60 minutes, washed, and incubated with anti-mouse IgG Fc secondary antibody conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

[0205] Hybridomas from positive wells were transferred to a 24-well plate containing fresh medium and grown for 2-3 days. They were then screened again by flow cytometry to confirm antibody binding to cyno-CLDN6 overexpressing cell lines and human CLDN6-positive cancer cell lines (PA-1). Antibody binding to cyno-CLDN6 overexpressing cell lines and human CLDN6-positive cancer cell lines (PA-1) was measured by flow cytometry. Briefly, 100 μL of hybridoma culture supernatant was co-incubated with CLDN-expressing cancer cell lines (PA-1 or CHOK1 / human CLDN6 and CHOK1 / human CLDN9 stable cell lines, etc.) or control cells (parental CHOK1, etc.) for 30-60 minutes, washed, and incubated with anti-mouse IgG Fc secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

[0206] Example 3. Subcloning of selected CLDN6 Ab secretory hybridomas Selected CLDN6 antibody-secreting hybridomas were subcloned once or twice to ensure monoclonality. Briefly, approximately 80–100 viable hybridoma cells were plated in 3 mL of semi-solid methylcellulose medium (Stem Cell Technologies) in 6-well plates. After 7–10 days, hybridoma colonies that had emerged from single cells as visible clones were collected in 96-well plates and cultured further in fresh medium for 2–4 days. The culture supernatant was screened by ELISA and flow cytometry as described above to confirm human and cyno-CLDN6 binding. Stable hybridoma subclones were cultured in vitro for cell cryopreservation, antibody production, and cloning and sequencing of antibody VH and VL genes.

[0207] Example 4. Identification of the EC50 value of CLDN6 binding for mouse anti-CLDN6 antibody. After subcloning, selected anti-CLDN6 antibody-secreting hybridomas were seeded into T75 flasks containing 40 ml of fresh 1640 medium supplemented with 2% FBS for antibody production. After 7–10 days of incubation, the hybridoma supernatant was collected for antibody purification using a protein A column. The binding activity of the mouse anti-CLDN6 antibody to CLDN6-positive cells was then characterized using flow cytometry. The EC50 values ​​of clone BG87P are shown in Tables 4–6. This data indicates that clone BG87P binds to human CLDN6 but not to human CLDN9. Furthermore, BG87P binds to mouse CLDN6 and cyno-CLDN6. [Table 4] [Table 5] [Table 6]

[0208] Example 5. Cloning and sequencing of antibody VH and VL genes After removing the supernatant, the CLDN6 antibody-secreting hybridomas selected after subcloning were lysed in 100 mL of RLT buffer in a 96-well round-bottom plate. This mRNA-containing lysate was then transferred to a 96-well deep-well plate for mRNA isolation, cDNA synthesis, and DNA sequencing using standard sequencing techniques (Sanger sequencing and next-generation sequencing). In general, total RNA from cell lysates was prepared using a total RNA isolation kit according to the manufacturer's instructions. cDNA was generated by reverse transcription of mRNA using Super Script III first-strand synthesis SuperMix (Invitrogen®) according to the manufacturer's instructions. The nucleic acid and amino acid sequences of BG87P are shown in Table 1 (SEQ ID NOs: 1-10).

[0209] Generation of chimeric BG87P antibody (chBG87P) The ChBG87P antibody was generated by subcloning the variable region (SEQ ID NOs: 7 and 8) of mouse BG87P into an in-house developed expression vector containing human wild-type IgG1 and the constant region of the kappa chain. This antibody was expressed by co-transfection of the two constructs described above into HEK293T cells and purified using a Protein A column (catalog: 17-5438-02, GE Life Sciences®). The purified chimeric antibody was concentrated into PBS containing 0.5–10 mg / ml, aliquoted, and stored in a freezer at -80°C.

[0210] Example 6. Humanization of anti-CLDN6 chimeric BG87P antibody (chBG87P) [Humanization approach] To humanize chBG87P, human germline IgG genes were searched for sequences that share a high degree of homology with the protein sequence of the chBG87P variable region by sequence comparison against the IMGT human immunoglobulin gene database. Human IGHV and IGKV genes, which are frequently present in the human antibody repertoire and have high homology with chBG87P, were selected as templates for humanization.

[0211] Design of humanized variants Humanization was performed using CDR grafting technology, followed by the incorporation of significant revertant mutations. These humanized antibodies were manipulated in human IgG1 wild-type format using in-house developed expression vectors. In the first round of initial humanization, mutations from mouse variable regions to human amino acid residues in the framework region were induced by 3D structural analysis, and structurally important mouse framework residues for maintaining the standard structure of the CDR were preserved in this first round of humanization design. Five revertant mutations on the heavy chain and three mutations on the light chain were selected, and the following single-point mutations were performed to search for significant revertant mutations: BG87P-Bz1 (VH SEQ ID NO: 15 and VL SEQ ID NO: 14), BG87P-Bz2 (VH SEQ ID NO: 16 and VL SEQ ID NO: 14), BG87P-Bz3 (VH SEQ ID NO: 17 and VL SEQ ID NO: 14), BG87P-Bz4 (VH SEQ ID NO: 18 and VL SEQ ID NO: 14), BG87P-Bz5 (VH SEQ ID NO: 19 and VL SEQ ID NO: 14), BG87P-Bz6 (VH SEQ ID NO: 13 and VL SEQ ID NO: 20), BG87P-Bz7 (VH SEQ ID NO: 13 and VL SEQ ID NO: 20), and BG87P-Bz8 (VH SEQ ID NO: 13 and VL SEQ ID NO: 22). BG87P-Bz0 (VH: SEQ ID NO: 13 and VL: SEQ ID NO: 14) is a variant that incorporates all theoretical revertant mutations, and the binding ability of BG87P-Bz0 should be comparable to that of the parent chBG87P. Comparison of binding data will reveal revertant mutations that significantly affect binding. Specifically, the LCDR of chBG87P (SEQ ID NOs: 4-6) was grafted onto the frameworks of human germline variable genes IGKV1-5 and 01-IGKJ4*01 along with the mouse framework residues A43S, L78V, and Y87F (obtained as SEQ ID NO: 14). The HCDR of chBG87P (SEQ ID NOs: 1-3) was grafted onto the frameworks of human germline variable genes IGHV1-3 and 01-JH6c while retaining the mouse framework residues V2I, T28S, I69L, R71V, and Y91F (obtained as SEQ ID NO: 13).BG87P-z0 (VH: SEQ ID NO: 11 and VL: SEQ ID NO: 12) is a humanized variant in which the resulting HCDR and LCDR are grafted, but no back mutations from mouse VH and VL frameworks occur.

[0212] Expression and purification of chBG87P and humanized antibodies All first-round BG87P humanized variants (BG87P-z0, BG87P-Bz0, BG87P-Bz1, BG87P-Bz2, BG87P-Bz3, BG87P-Bz4, BG87P-Bz5, BG87P-Bz6, BG87P-Bz7, and BG87P-Bz8) were each constructed as humanized full-length antibodies using an in-house developed expression vector containing the human wild-type IgG1 and the constant region of the kappa chain with subcloning sites that are easy to adapt. All humanized variants were expressed by co-transfection of the above two constructs into HEK293T cells and purified using a Protein A column (Catalog: 17-5438-02, GE Life Sciences). The purified antibodies were concentrated to PBS containing 0.5 - 10 mg / ml, aliquoted, and stored in a -80°C freezer.

[0213] Identification of cell-binding activities of first-round humanized BG87P variants (hBG87P) and PTM-removed variants To measure affinity, the binding activity of BG87P-related manipulated variants was evaluated using CLDN6-overexpressing HEK293T cells expressing high levels of human CLDN6 and the cancer cell line PA-1. Live cells were seeded in 96-well plates and incubated with a series of dilutions of chBG87P and its manipulated variants. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The EC50 values ​​of dose-dependent binding to CLDN6-expressing cell lines were identified by fitting dose-response data to a 4-parameter logistic model using GraphPad Prism. The cell binding activity of the first round of BG87P humanized variants to HEK293T / human CLDN6 was compared with chBG87P and is shown in Figure 1A. The cell binding affinity (EC50) and Emax (MFI) of the first round of humanized variants were normalized against chBG87P for direct comparison and ranking (Table 7).

[0214] Starting from the chBG87P antibody and BG87P-Bz0, several additional amino acid changes were made in the CDR regions of both VH and VL to further improve the biophysical properties for therapeutic use in humans. The considerations included removing post-translational modifications (PTMs) while maintaining binding activity and improving thermal stability (Tm). The resulting variants are BG87P-m1 (VH SEQ ID NO: 31 and VL SEQ ID NO: 8), BG87P-m2 (VH SEQ ID NO: 32 and VL SEQ ID NO: 8), BG87P-m3 (VH SEQ ID NO: 33 and VL SEQ ID NO: 8), as well as BG87P-m4 (VH SEQ ID NO: 34 and VL SEQ ID NO: 8) and BG87P-m5 (VH SEQ ID NO: 35 and VL SEQ ID NO: 14), BG87P-m6 (VH SEQ ID NO: 36 and VL SEQ ID NO: 14), BG87P-m7 (VH SEQ ID NO: 37 and VL SEQ ID NO: 14), and BG87P-m8 (VH SEQ ID NO: 38 and VL SEQ ID NO: 14). The cell-binding activities of the chBG87P and BG87P-Bz0 related PTM-removed variants against HEK293T / human CLDN6 were compared with chBG87P and BG87-Bz0, respectively (Figure 1F). The cell-binding affinity (EC50) and Emax (MFI) were normalized against chBG87P for direct comparison and ranking (Table 7). These results showed that other substitutions of potentially harmful residues, except for the H33A mutation that gave rise to BG87P-m4 and BG87P-m8, maintained the binding ability of their respective parents.

Table 7

Table 8

[0215] Identification of cell-binding activities of the second round of significant reversion mutations combined with PTM-removal sites Following a comprehensive analysis of EC50 and Emax data from the first round of humanized cell binding (Table 7), four critical reversion sites, VH:V2I, VH:T28S, VH:I69L, and VH:Y91F, were identified and combined with PTM removal sites for the second round of validation and identification of final humanization candidates. The PTM removal mutation VH:V65G showed a high G position in human germline cells (G 62%, V < 1%), indicating a potential advantage in antibody framework stability. It was included in the variant BG87P-m3 and showed improved Emax and EC50 compared to chBG87P, as shown in Figure 1F and Table 7. This was incorporated into the second round of combinations for further validation. Table 1 shows the VH and VL sequences of the second-round humanized variants BG87P-21, BG87P-22, BG87P-23, BG87P-24, BG87P-25, BG87P-26, and BG87P-27 obtained.

[0216] Following the identification of the cell-binding activity of the humanized combination variants shown in Figures 1B and 1C in HEK293T / human CLDN6 cells and Figures 1D and 1E in PA-1 cells, BG87P-21 was selected as a superior humanized candidate for further investigation (the amino acid sequences of VH and VL are SEQ ID NOs. 24 and 12, respectively). BG87P-21 contains the significant revertant site VH:T28S and the PTM site VH:V65G, and it showed comparable cell-binding affinity to chBG87P. Emax was reduced by 22% in HEK293T / human CLDN6 and by 40% in PA-1 (Tables 7 and 8).

[0217] Feasibility assessment of humanized anti-CLDN6 antibodies Biophysical properties were profiled to identify top-performing humanized anti-CLDN6 antibodies. This data shows that BG87P-21 exhibits moderate to high risk in hydrophobicity, followed by a risk of self-interaction in PBS buffer, as indicated by AC-SINS, B22KD, and CIC readings (Tables 9-11). [Table 9]

[0218] For hydrophobicity evaluation, 50 μg of a 1 mg / ml sample was diluted with mobile phase A solution (1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0) to achieve a final ammonium sulfate concentration of approximately 1 M before analysis. A MABPac HIC-10 column was used with a linear gradient of mobile phase A and mobile phase B solutions (50 mM sodium phosphate, pH 7.0) at a flow rate of 0.5 ml / min for 29 minutes. Peak retention time was monitored using A280 absorbance. As shown in Table 9, both chBG87P and BG87P-21 exhibited higher hydrophobicity, exceeding the internal standard of 21.1 minutes in IgG format.

[0219] In the thermal stability evaluation, the thermal stability of BG87P-related operational variants was expressed by the thermal denaturation transition midpoint Tm (°C), which was measured by exogenous fluorescence. Tm was measured using the Applied Biosystems QuantStudio® 6 Flex System. 20 μL of 1 mg / ml sample was mixed with 20 μL of 40XSYPRO orange. The plate was scanned from 25°C to 95°C at a rate of 0.9°C / min. Tm was assigned using the first derivative of the raw data from the QuantStudio® 6 Flex System Analysis software. These results are summarized in Table 9. This shows that both chBG87P and the humanized variant BG87P-21 exhibited good thermal stability.

[0220] To identify the aggregation tendencies of BG87P-related operational variants, static light scattering intensity was measured using the Uncle system (Unchained Labs). During the measurement, approximately 8.8 μL of 1 mg / ml protein sample was loaded into a cuvette. These samples were held at 25°C for 120 seconds and then heated to 95°C at a rate of 0.3°C / min. The scattering data was collected at a 90° angle with a laser wavelength of 266 nm. Tagg (aggregation temperature) was analyzed and calculated using Uncle Analysis software. The results are summarized in Table 9. Both chBG87P and the humanized variant showed acceptable Tagg.

[0221] CIC is a technique for identifying antibody candidates with low solubility or nonspecific binding tendencies. Human serum-derived IgG or other ligands were chemically conjugated to an NHS-activated chromatography resin. To evaluate protein solubility, the retention time of the protein in this resin was tested using HPLC. After conjugating the column with IgG from human serum, the antibody sample and sample buffer were diluted to 0.1 mg / mL with mobile phase (PBS). The diluted sample and buffer were transferred to HPLC vials for LC-MS analysis. The results summarized in Table 9 show that both chBG87P and BG87P-21 exhibit acceptable nonspecific interactions with human IgG.

[0222] Overview of B22 and KD and the purpose of using this test method. This method is used to investigate weak protein-protein interactions, predict aggregation tendencies, clarify the influence of formulation components on intermolecular interactions, and assist in the selection of formulation buffers. The antibody of the buffer-exchanged sample was diluted to 1 mg / mL, centrifuged at 14000 rpm for 30 minutes, and then Tm, Tag, and DLS were checked. The sample was loaded into the Uni. at 9 μL / well. Each sample was set up with a double well. Set the instrument parameters according to Uncle's guidance and run the experiment. In this experiment, B22 and Kd modes were used. Run information: Temperature (°C): 25. Incubation time (seconds): 120. Number of acquisitions: 4. Acquisition time (seconds): 5. Attenuator control: Auto. Laser. Control: Auto run. kd: For the diffusion interaction parameter, if protein interactions increase (they are attracted to each other) as the concentration increases, the proteins behave as if they were larger and the diffusion coefficient (KD) decreased (negative gradient). B22: For the second virial coefficient, if protein interactions increase (they are attracted to each other) as the concentration increases, the proteins behave as if they were larger and 1 / R90 decreased (negative gradient). This data showed that in PBS, both chBG87P and the humanized variant attracted each other and tended to aggregate under these conditions (Table 10).

[0223] AC-SINS is an assay for obtaining self-interactions of a sample and predicting the likelihood of aggregation. It is based on concentrating antibodies from a diluted solution surrounding gold nanoparticles pre-coated with polyclonal capture. Interactions between immobilized antibodies reduce the interparticle distance and increase the plasmon wavelength (wavelength of maximum absorbance), which can be readily measured by optical means. The antibodies were diluted to 0.05 mg / mL each in the supply buffer. After the preparation of the gold nanoparticles, the gold nanoparticle solution and the coating solution were mixed using a 9:1 volume ratio. After incubation at room temperature for 1 hour, empty sites of the AuNPs were blocked using thilolated PEG (final concentration 0.1 μM). Then, incubation was performed at room temperature for another 1 hour. The particle solution was then centrifuged at 15000 rpm for 6 minutes. The upper solution was discarded. The particles were redissolved using 1 / 10 of the starting volume of storage buffer. Ten μL of these concentrated coated particles were incubated with 100 μL of the test antibody solution in a polypropylene plate at room temperature for 2 hours, and then 90 μL of the resulting solution was transferred to a polystyrene UV-transmitting plate. This data showed that both chBG87P and BG87P-21 exhibited suboptimal self-interaction tendencies (Table 9). [Table 10] [Table 11]

[0224] Due to the hydrophobic patch, the HIC retention time for chBG87P exceeded 25 minutes, and for humanized BG87P-21 it was 21.9 minutes, both higher than the acceptable threshold of 21.1 minutes in IgG format. The underlying cause is the hydrophobic patch of HCDR3, particularly the I97-Y98-Y100-V100a portion, and the Y49-W50 (mainly W50) edges of the light chain FR2 (framework region 2) and LCDR2 (Figure 2A). Automated antibody pipeline analysis of BG87P aggregation by Schrodinger also indicated a high risk of aggregation (Table 12).

Table 12

[0225] Example 7. Solubility Engineering of Humanized Anti-CLDN6 Antibody Overall Strategy for Solubility Engineering of BG87P-21 In the foregoing description, chBG87P has been engineered into a humanized antibody, and the inventors have identified BG87P-21 as the final top clone. However, the potential development risk of the hydrophobic patch driven by the HCDR3 of chBG87P has not been resolved (Figure 2). Considering that chBG87P exhibits promising binding activity and excellent CLDN6 selectivity (Figure 1A, Figure 1B, Figure 1C, Figure 1D, Figure 1E, Figure 1F, Figure 1G, and Figure 1H), further engineering of BG87P-21 was performed to remove the hydrophobic patch for optimal manufacturability and reduction of potential ADA risk.

[0226] To engineer the solubility issue of BG87P-21, two major strategies were used, namely, single-point mutation and framework exchange.

Table 13-1

Table 13-2

[0227] Numerous single-point mutations were designed based on two principles: one is to replace hydrophobic amino acids with more hydrophilic ones, and the other is to mutate rare amino acids at the same Kabat position in the human antibody repertoire with more common ones. In the first round of screening, 57 variants were tested, and in the second round, 104 variants were tested. Seven sites could be replaced with other, more affinity amino acids, resulting in binding affinity equivalent to the parent BG87P-21 and a slight improvement in affinity (Table 14). The top variants selected in the first and second rounds of screening were combined to generate 56 variants for further validation. The combined variants BG87P-31 and BG87P-32 (VH and VL amino acid sequences are SEQ ID NOs. 46 and 42, respectively) were selected as top candidates. These compounds exhibited comparable binding affinity to BG87P-21, with HIC retention improved to 17.4 minutes for BG87P-31 and to 18.49 minutes for BG87P-32, both of which were superior to the parent BG87P-21's 22.3 minutes (Table 14 and Figure 3). [Table 14]

[0228] However, despite the reduction in hydrophobicity during the single-point mutation approach for solubility manipulation, the risk of self-association, as identified by AC-SINS, remained moderate to high. The reason for this unresolved problem is the risk of hydrophobicity, which is mainly reflected in the amount of hydrophobic patch actually presented on the antibody surface, but the reasons for inducing self-interaction also include isoelectric point issues, uniform charge distribution, and even unknown specific interactions (Doi.org / 10.1021 / mp200566k). Therefore, the risk of self-interaction caused by the above reasons may not be mitigated simply by substituting hydrophobic residues with hydrophilic residues. Furthermore, we found that parent chBG87P exhibited a lower tendency for self-interaction, with an AC-SINS value of around 12.85 nm in PBS buffer, while having a longer HIC retention time of 25 minutes (Table 14). Another finding was that the calculated net charge of chBG87P was lower than that of BG87P-21, at 2.9 vs. 8.8. Therefore, the inventors hypothesized that the framework or net charge could influence the self-association effect.

[0229] The inventors tested further frameworks for IGHV3-23 and IGKV1-39. Both revertant mutations based on the new paired framework and top point mutations selected in the soluble manipulation of BG87P-21 were incorporated (Table 13). The final top candidate, BG87P-34, did not show any red flags in any of the specified biophysical properties (Table 14; the amino acid sequences of VH and VL are SEQ ID NOs. 43 and 44, respectively).

[0230] Furthermore, after replacing the framework with IGHV3-23 and IGKV1-39, the Emax of BG87P-21, which was lost in the original humanization procedure, was recovered (Figure 1G). The principle for identifying critical revertant mutations used in these two humanization procedures is the same, and therefore, the possibility that any revertant mutation was excluded in a previous round of humanization due to Emax disadvantage is ruled out. One explanation for the recovery of Emax in cell binding by framework replacement is that while the VH-VL angles of different pairs of frameworks may vary, a specific VH-VL angle may contribute to the maintenance of Emax in cell binding. The final lead clone BG87P-34 shows good cross-reactivity in different species (Figures 1I and 1J). Nonspecific binding to human CLDN9 was performed using HEK293T / human CLDN9, and the data showed that BG87P-34 has better selectivity for human CLDN6 than for CLDN9 (Figure 1H).

[0231] Example 8. Humanization and scFv manipulation of anti-human CD3 antibody sp34. The mouse clone sp34 (Blumberg 1990 PNAS 87(18):7220-24), which has been reported in detail, was the optimal clone for developing anti-CD3-based therapeutics due to its cyno-CD3 cross-reactivity. For the humanization of sp34, human germline IgG genes were searched for sequences that share a high degree of homology with the protein sequences of the variable region of sp34 (sequence numbers 48-57) by blasting the human immunoglobulin gene databases on the IMGT (http: / / www(dot)imgt(dot)org / IMGT_vquest / share / textes / index(dot)html) and NCBI (http: / / www(dot)ncbi(dot)nlm(dot)nih(dot)gov / igblast / ) websites. Human IGVH and IGVK genes, which are frequently present in the human antibody repertoire (Glanville 2009 PNAS 106:20216-20221) and homologous to sp34, were selected as templates for humanization.

[0232] Humanization was performed using CDR grafting technology (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press), and the resulting humanized antibody (hu-sp34) was manipulated in human IgG1 format using an expression vector developed in-house. In the first round of humanization, mutations from mouse to human amino acid residues in the framework region were induced by simulated 3D structure, and structurally important mouse framework residues for maintaining the standard structure of the CDR were retained in the first type of this humanized antibody p34. Specifically, the VL CDRs of sp34 (SEQ ID NOs. 51-53) were grafted onto the framework of the human germline variable gene IGVκ3-15 while retaining several mouse framework residues (Q1, A2, V4, V36, E38, L43, F44, T45, G46, G49, L66, D69, A71, I85, and F87). The CDRs of sp34 VH (SEQ ID NOs. 48-50) were grafted into the framework of the human germline variable gene IGVH3-7 while retaining several mouse framework residues (D73, S76, M89, V93).

[0233] Humanized sp34 (hu-sp34) and chimeric sp34 (ch-sp34) were constructed as humanized full-length antibody formats using in-house developed expression vectors containing the constant regions of human IgG1 and kappa chains along with easily adaptable subcloning sites, respectively. Expression and preparation of humanized sp34 and chimeric sp34 antibodies were achieved by co-transfection of heavy chain and corresponding light chain constructs into 293G cells (in-house developed) and purification using a Protein A column. The purified antibodies were concentrated to 0.5–5 mg / mL in PBS and stored in aliquots in a -80°C freezer for the following assays.

[0234] For affinity measurements, antibodies were captured on anti-human Fc surfaces and used in affinity assays based on surface plasmon resonance (SPR) technology. The binding activity of humanized sp34 to native CD3 in viable cells was evaluated using HuT78 cells in a FACS-based assay. Viable HuT78 cells were seeded in 96-well plates and incubated with a series of dilutions of chimeric or humanized sp34. Mouse anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The EC50 values ​​for dose-dependent binding to human native CD3 were determined by fitting dose-response data to a 4-parameter logistic model using GraphPad Prism. Humanized sp34 BG53P (SEQ ID NOs. 48-53 and 58-61) showed comparable binding affinity to ch-sp34 in both SPR and FACS assays (Table 15 and Figure 4A). [Table 15]

[0235] Based on the BG53P template of humanized sp34, the inventors performed several single mutations that converted mouse residues retained within the framework region to residues containing four mouse residues retained in the VH region (D73, S76, M89, V93) and fifteen mouse residues retained in the VL region (Q1, A2, V4, V36, E38, L43, F44, T45, G46, G49, L66, D69, A71, I85, and F87) of the corresponding human germline. All humanization mutations were performed using primers containing mutations at specific locations and site-directed mutagenesis kits (catalog no. FM111-02, TransGen, Beijing, China). The desired mutations were validated by sequence analysis. These hu-sp34 variant antibodies were examined using the previously described binding assays. Compared to hu-sp34-1A-1f, mutations in VK V36Y, G46L, and G49Y (Kabat numbering) significantly impaired the binding affinity of the humanized variant, while the remaining types of hu-sp34 humanized variants exhibited comparable binding activity to hu-sp34-1A-1f. VH D73N significantly reduced expression levels (data not shown).

[0236] In summary, well-engineered humanized monoclonal antibody types, namely BG56P (SEQ ID NOs. 70-77 and 72-86), were obtained from the above mutation process and were characterized in detail (Table 16 and Figure 4B). [Table 16]

[0237] Example 9. ScFv operation of humanized sp34 To generate a plug-and-play bispecific format and avoid mispairing of light and heavy chains, the inventors reformatted the BG56P antibody to a single-stranded fragment variable (scFv) format using a 3xG4S linker between VH and VK. The reformatted scFv was fused to the N-terminus of the Fc region of human IgG1 to form the scFv-Fc format using an in-house developed expression vector containing an easily adaptable subcloning site. Expression and preparation of scFv-Fc from parental and remanufactured hu-sp34 were achieved by transfection of scFv-Fc constructs into 293G cells (in-house developed) and purification using a protein A column. The purified scFv-Fc format antibody was concentrated to 0.5–5 mg / mL in PBS and stored in aliquots in a -80°C freezer for the following assays. scFv-modified BG56P (referred to as BG561P, sequence numbers 48-53 and 62-65) showed binding affinity equivalent to that of the antibody form of BG56P in SPR and FACS (Table 17 and Figure 5). [Table 17]

[0238] Based on BG561P, the inventors made several mutations in the framework and CDR to remove potential PTM sites and improve thermal and colloidal stability for therapeutic use in humans. The L4V mutation in VL (the resulting humanized scFv designated as BG562P, SEQ ID NOs. 48-53 and 69-70) showed a 5-degree improvement in the aggregation temperature (Tagg). The combination of L4V in VL and A49G and D65G in VH (the resulting humanized scFv designated as BG563P) (SEQ ID NOs. 48, 71, 50, 51-53, 73, and 74) showed improved thermal and colloidal stability compared to BG561P and a slight improvement in binding affinity to human CD3 in FACS assays. Potential PTM sites include the potential deamidation site N30 (NT) in the binding region between FR1 and HCDR1 (Kabat's definition of CDR), and N100 (NS) in HCDR3. Each of these Ns was mutated to S to eliminate potential deamidation sites. All mutations were performed using primers containing mutations at specific locations and a site-directed mutagenesis kit (catalog number FM111-02, TransGen, Beijing, China). In summary, well-engineered humanized scFv types, namely BG564P (SEQ ID NOs. 48, 71, 75, 51-53, 77, and 78), were obtained from the above mutation process and were characterized in detail. These results showed that humanized scFv BG564P retained its binding affinity to CD3 (Tables 18-20 and Figure 6) and exhibited improved biophysical stability compared to humanized scFv BG561P (Table 20). [Table 18] [Table 19] [Table 20]

[0239] The melting temperature (Tm) was measured using a high-throughput MicroCal® VP-Capillary DSC (Malvern Instruments, Northampton, MA). Thermograms of each protein (350 μL at 0.5 mg / mL) were acquired at 20°C to 100°C using a scanning rate of 90°C / hour. Thermograms of the buffer solution alone were subtracted from each protein sample. The results show the midpoint of the transition temperature (Tm) and the calorific enthalpy (ΔH) of the sample. This suggests that the Tm of BG564P was improved compared to BG561P (Table 20).

[0240] The aggregation temperature Tag (°C) represents the colloidal stability of the sample and was obtained by monitoring the onset of aggregation using SLS266 with UNCLE® (Unchained lab, Pleasanton, CA). The sample was loaded into the Uni and the temperature was increased from 15°C to 95°C. The back-reflected optics could not detect near-ultraviolet light scattering by protein aggregates, and therefore only unscattered light reached the detector. Thus, the reduction in back-reflected light was a direct measure of aggregation in the sample, suggesting that the Tag of BG564P was improved compared to BG561P (Table 20).

[0241] Example 10. Production of CLDN6×CD3 BsAb BG143P Agonist anti-CD3 antibodies have shown toxicity in clinical settings, which may indicate that systemic FcγR crosslinking is not ideal for CD3 activation. The objective is to achieve potent CD3 stimulation specifically at tumor sites without systemic CD3 activation for a wide range of cancers. To overcome the dependence on FcγR crosslinking, the inventors have created CLDN6×CD3 BsAb BG143P, which has the following features as shown in Figure 7. This specific construct BG143P contains a module ratio 1:1 IgG fusion-like multispecific antibody format, well-engineered Fab fragments BG87P-34 that bind to CLDN6 and scFv of BG564P that binds to CD3 fusion at the N-terminus of CH2, and an Fc-null form of huIgG1 that lacks FcγR binding but retains FcRn binding. Knobs and holes (KIH) were also introduced to Fc to increase heterodimerization. Sequence information for BG143P is described in SEQ ID NOs. 79-84.

[0242] Example 11. Target binding activity of CLDN6×CD3 BsAb BG143P The binding reaction rate of CLDN6×CD3 BsAb BG143P was measured using SPR. The binding rate constant (k) of the antibody to recombinant CDεγ was measured using SPR. a ) and dissociation rate constant (k d ) is measured, and then the affinity constant (K D The following was determined. As shown in Table 21, these results indicate that CLDN6×CD3 BsAb has a strong binding affinity to human CDεγ. [Table 21]

[0243] FACS results further confirmed the binding activity of BG143P to CD3 and CLDN6. BsAb showed potent dose-response binding activity to CD3-expressing Jurkat, and EC 50 The concentration was 6.98 nM. (Figure 8A) Similarly, BG143P showed dose-response potent binding activity to CLDN6-expressing PA-1, and EC 50The value was 81.26 nM (Figure 8B).

[0244] Example 12. In vitro functional activity of CLDN6 × CD3 antibody [On-target T cell redirection, cytotoxicity, and cytokine release] The T-cell redirection cytotoxicity of BG143P against PA-1 (high CLDN6-expressing cancer cell line), Hutu80 (medium CLDN6-expressing cancer cell line), AGS (low and heterogeneous CLDN6-expressing cancer cell line), and NCI-H1299 (CLDN6-negative cancer cell line) was evaluated using human PBMCs as effector cells. To measure cytotoxicity, target cancer cell lines were modified to express nanoluciferase. Approximately 10,000 target cells and 25,000 human PBMCs (E / T=2.5) were seeded in each well of a 96-well U-bottom plate and incubated with various antibody concentrations at 37°C and 5% CO2 for 48 hours. The supernatant was collected for cytokine detection. Target cell killing was measured using the Nano-Glo detection kit (Promega). The cytotoxic activity (%) of the antibody was calculated using the following formula: Cytotoxic activity (%) = (AB) / (AC) * 100%. "A" represents the average luminescence signal of wells containing only untreated target cells, "B" represents the average luminescence signal of wells containing antibodies and PBMCs, and "C" represents the average luminescence signal of wells containing target cells completely lysed with Triton-X100. IFN-γ and IL-2 were detected from the supernatant using an HTRF kit (Cisbio).

[0245] As shown in Figure 9, BG143P demonstrated dose-dependent potent T cell redirection-killing and cytokine release-inducing efficacy at the pM EC50 level.

[0246] Functional specificity for human CLDN6 and CLDN9 The amino acid sequences of human CLDN6 and CLDN9 are highly conserved, differing only in three amino acids in the extracellular domain. Since CLDN9 is widely expressed in normal human tissues, the binding specificity between CLDN6 and CLDN9 is important and will be investigated by FACS analysis.

[0247] Human CLDN6 and CLDN9 expression vectors were established by inserting the synthesized cDNA coding sequences into mammalian expression vectors. Stable NCI-H1299 cells expressing human CLDN6 and CLDN9 were generated by transfection with the corresponding plasmids. Cells were then placed in FACS buffer (2% FBS, 1×PBS) at a rate of 1×10⁶ cells. 6 The cells were suspended at their concentration, and this cell suspension was dispensed into a U-bottom 96-well plate (100 μL / well). The antibody was added to the cells at a final peak concentration of 100 nM and 11 2-fold dilutions, then mixed with the cells and incubated at 4°C for 1 hour. After centrifugation, the reaction solution was removed, and the cells were washed twice with 200 μL / well of FACS buffer. Next, APC-anti-human Fcγ was diluted 500-fold with FACS buffer and added to the cells as a secondary antibody. The cells were incubated at 4°C for 30 minutes, then washed twice as described above, and suspended in 100 μL of FACS buffer. The cell suspension was subjected to flow cytometry.

[0248] The killing of NCI-H1299-CLDN6 / CLDN9 cells by BG143P was measured using human PBMCs by Nano-Glo assay. Approximately 10,000 target cells and 25,000 human PBMCs (E / T=2.5) were seeded in each well of a 96-well U-bottom plate and incubated with varying concentrations of antibody at 37°C and 5% CO2 for 48 hours. The supernatant was collected for cytokine detection. Killing of target cells was measured using the Nano-Glo detection kit (Promega). The cytotoxic activity (%) of the antibody was calculated using the following formula: Cytotoxic activity (%) = (AB) / (AC) * 100%. "A" represents the mean luminescence signal of the well containing only untreated target cells, "B" represents the mean luminescence signal of the well containing antibody and PBMCs, and "C" represents the mean luminescence signal of the well containing target cells completely lysed with Triton-X100. IFN-γ was detected using an HTRF kit (Cisbio).

[0249] As shown in Figure 10, BG143P was an antibody that exhibited specific binding (Figure 10A), cell killing (e.g., lysis) (Figure 10B), and IFN-γ inducing activity (Figure 10B) against human CLDN6, but not against human CLDN9.

[0250] Example 13. In vivo efficacy of CLDN6×CD3 BsAb BG143P in an OV90 xenograft model. The in vivo antitumor effect of CLDN6×CD3 BsAb BG143P was evaluated in a xenograft model of PBMC humanized mice. Human ovarian cancer cell line OV-90 (ATCC) expressing human CLDN6 was used in NCG(NOD / ShiLtJGpt-Prkdc em26Cd52 Il2rg em26Cd22 Human PBMCs were subcutaneously transplanted into mice (Gpt), and the following day, these mice were intravenously injected. The tumor volume was approximately 200 mm². 3 When tumor-bearing mice reached a certain stage, they were randomized to a treatment group and administered either an antibody or a control medium (PBS). The antibody / medium was administered once weekly. The length (L) and width (W) of the tumor mass, as well as body weight, were measured three times a week for each mouse. Tumor volume (TV) was calculated as TV = (L × W). 2The calculation was performed using ) / 2. Figure 11A shows the in vivo antitumor effect of BG143P. It showed a strong effect at 0.03 mg / kg and 0.1 mg / kg, with TGI% (tumor growth inhibition ratio, %) of 115.43% and 125.92%.

[0251] hPBMC rearrangement in mice was confirmed at weeks 2, 3, and 4 after PBMC injection. Among the viable cells in peripheral blood, hCD45+ cells increased from 20% at week 2 to 60% at week 4. Figure 11B shows the rearrangement of hPBMCs.

[0252] Example 14. In vivo efficacy of CLDN6×CD3 BsAb BG143P in a B16F10- / hCLDN6 syngeneic model. Another type of efficacy model was performed to evaluate the in vivo efficacy of CLDN6×CD3 BsAb BG143P. A human CLDN6 expression plasmid was constructed and stably transfected into the B16F10 cell line. The resulting B16F10 / human CLDN6 cell line was able to proliferate in human CD3EDG transgenic mice, and hCLDN6 expression was maintained even after tumor formation. To establish this model, B16F10 / human CLDN6 cells were subcutaneously transplanted into hCD3EDG transgenic mice, and the mouse CD3 gene was replaced with a human counterpart. The tumor volume was approximately 100 mm². 3 Mice were randomized after reaching a certain stage. The test substance or PBS was injected intraperitoneally into the mice weekly. The length (L) and width (W) of the tumor mass and body weight of each mouse were measured three times a week. Tumor volume (TV) was calculated as TV = (L × W²) / 2. BG143P showed a strong effect at 0.1 mg / kg, with a TGI% of 93.54%, as shown in Figure 12A. No significant weight loss was observed in this study, as shown in Figure 12B.

[0253] literature 1.Krause,G.,et al.,Structure and function of claudins.Biochimica et Biophysica Acta(BBA)-Biomembranes,2008.1778(3):p.631-645. 2.Gunzel,D.and A.S.Yu,Claudins and the modulation of tight junction permeability.Physiol Rev,2013.93(2):p.525-69. 3.Tsukita,S.,H.Tanaka,and A.Tamura,The Claudins:From Tight Junctions to Biological Systems.Trends in Biochemical Sciences,2019.44(2):p.141-152. 4.Matter,K.and M.S.Balda,Signalling to and from tight junctions.Nat Rev Mol Cell Biol,2003.4(3):p.225-36. 5.Singh,A.B.,S.B.Uppada,and P.Dhawan,Claudin proteins,outside-in signaling,and carcinogenesis.Pflugers Arch,2017.469(1):p.69-75. 6.Turksen,K.and T.-C.Troy,Claudin-6:A novel tight junction molecule is developmentally regulated in mouse embryonic epithelium.Developmental Dynamics,2001.222(2):p.292-300. 7.Du,H.,et al.,Claudin 6:Therapeutic prospects for tumours,and mechanisms of expression and regulation(Review).Mol Med Rep,2021.24(3). 8.Ben-David,U.,N.Nudel,and N.Benvenisty,Immunologic and chemical targeting of the tight-junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells.Nat Commun,2013.4:p.1992. 9.Reinhard,K.,et al.,An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors.Science,2020.367(6476):p.446-453. 10.Abuazza,G.,et al.,Claudins 6,9,and 13 are developmentally expressed renal tight junction proteins.Am J Physiol Renal Physiol,2006.291(6):p.F1132-41. 11.Hashizume,A.,et al.,Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland.Dev Dyn,2004.231(2):p.425-31. 12.Kohmoto,T.,et al.,Claudin-6 is a single prognostic marker and functions as a tumor-promoting gene in a subgroup of intestinal type gastric cancer.Gastric Cancer,2020.23(3):p.403-417. 13.Kojima,M.,et al.,Prognostic Significance of Aberrant Claudin-6 Expression in Endometrial Cancer.Cancers(Basel),2020.12(10). 14.Micke,P.,et al.,Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer.Int J Cancer,2014.135(9):p.2206-14. 15.Sullivan,L.M.,et al.,Claudin-6 is a nonspecific marker for malignant rhabdoid and other pediatric tumors.Am J Surg Pathol,2012.36(1):p.73-80. 16.Ushiku,T.,et al.,Distinct expression pattern of claudin-6,a primitive phenotypic tight junction molecule,in germ cell tumours and visceral carcinomas.Histopathology,2012.61(6):p.1043-56. 17.Kamakura,D.,R.Asano,and M.Yasunaga,T Cell Bispecific Antibodies:An Antibody-Based Delivery System for Inducing Antitumor Immunity.Pharmaceuticals(Basel),2021.14(11). 18.Middelburg,J.,et al.,Overcoming Challenges for CD3-Bispecific Antibody Therapy in Solid Tumors.Cancers(Basel),2021.13(2). 19.Singh,A.,S.Dees,and I.S.Grewal,Overcoming the challenges associated with CD3+ T-cell redirection in cancer.Br J Cancer,2021.124(6):p.1037-1048. 20.Baeuerle,P.A.and H.Wesche,T-cell-engaging antibodies for the treatment of solid tumors:challenges and opportunities.Current Opinion in Oncology,2022.34(5). 21.Stadler,C.R.,et al.,Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6.Oncoimmunology,2016.5(3):p.e1091555. 22.Stadler,C.R.,et al.,Elimination of large tumors in mice by mRNA-encoded bispecific antibodies.Nat Med,2017.23(7):p.815-817.

Claims

1. An antibody or its antigen-binding fragment, comprising an antigen-binding domain that specifically binds to human claudin 6 (CLDN6).

2. The antibody or antigen-binding fragment according to claim 1, wherein the antigen-binding domain does not bind to other members of the claudin (CLDN) protein family.

3. The antibody or antigen-binding fragment according to claim 2, wherein the antigen-binding domain does not bind to human claudin 9 (CLDN9).

4. The antibody or antigen-binding fragment according to claim 3, wherein the antigen-binding domain has higher selectivity for human CLDN6 than for human CLDN9.

5. The antigen-binding domain that specifically binds to the aforementioned human CLDN6 is (a) (i) Heavy chain complementarity determination region (HCDR) 1 having the amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 39, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 3 Heavy chain variable region including, (iv) Light chain complementarity determining region (LCDR) 1 having the amino acid sequence of SEQ ID NO: 40, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 5, (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6 Light chain variable region including, (b) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 2, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 3 Heavy chain variable region including, (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 4, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 5, (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6 Light chain variable region including, (c) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 23, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 3 Heavy chain variable region including, (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 4, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 5, (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6 Light chain variable region including, or (d) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 45, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 3 Heavy chain variable region including, (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 40, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 5, (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6 Light chain variable region including An antibody or antigen-binding fragment according to any one of the prior claims, comprising:

6. The antigen-binding domain, (a) A heavy chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 43, and a light chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 44, (b) A heavy chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to Sequence ID No. 7, and a light chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to Sequence ID No. 8, (c) A heavy chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 24, and a light chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 12, (d) A heavy chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 41, and a light chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 42, (e) A heavy chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 46, and a light chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence to SEQ ID NO: 47, or (f) Heavy chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 46, and light chain variable region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequences to SEQ ID NO: 42 An antibody or antigen-binding fragment according to any one of the prior claims, comprising:

7. An antibody or antigen-binding fragment according to any one of the prior claims, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids of sequence number 7, eight, twelve

8. The antigen-binding domain, (a) A heavy chain variable region having an amino acid sequence including SEQ ID NO: 43, and a light chain variable region having an amino acid sequence including SEQ ID NO: 44, (b) A heavy chain variable region having an amino acid sequence including SEQ ID NO: 7, and a light chain variable region having an amino acid sequence including SEQ ID NO: 8, (c) A heavy chain variable region having an amino acid sequence including SEQ ID NO: 24, and a light chain variable region having an amino acid sequence including SEQ ID NO: 12, (d) A heavy chain variable region having an amino acid sequence including SEQ ID NO: 41, and a light chain variable region having an amino acid sequence including SEQ ID NO: 42, (e) A heavy chain variable region having an amino acid sequence including SEQ ID NO: 46, and a light chain variable region having an amino acid sequence including SEQ ID NO: 47, or (f) Heavy chain variable region having an amino acid sequence including SEQ ID NO: 46, and light chain variable region having an amino acid sequence including SEQ ID NO: 42 An antibody or antigen-binding fragment according to any one of the prior claims, comprising:

9. An antibody or antigen-binding fragment according to any one of the prior claims, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

10. The antibody or antigen-binding fragment according to any one of the prior claims, wherein the antibody is a multispecific antibody.

11. The antibody or antigen-binding fragment according to any one of the prior claims, wherein the antibody is a bispecific antibody.

12. The antibody or antigen-binding fragment thereof has antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC), as described in any one of the prior claims.

13. The antibody or antigen-binding fragment according to any one of the prior claims, wherein the antibody or antigen-binding fragment has reduced glycosylation, is not glycosylated, or is low-fucosylated.

14. The antibody or antigen-binding fragment according to any one of the prior claims, comprising an increased bisected GlcNac structure.

15. The antibody or antigen-binding fragment according to any one of the prior claims, wherein the Fc domain is IgG1.

16. The antibody or antigen-binding fragment according to any one of the prior claims, wherein the Fc domain is IgG1 with reduced effector function.

17. The antibody or antigen-binding fragment according to any one of the prior claims, wherein the Fc domain is IgG4.

18. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 17, further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition.

19. The pharmaceutical composition according to claim 18, further comprising histidine / histidine HCl, trehalose dihydrate, and / or polysorbate 20.

20. A method for treating cancer, comprising administering an effective amount of an antibody or antigen-binding fragment according to claims 1 to 17 to a patient who requires treatment for cancer.

21. The method according to claim 20, wherein the cancer is a solid tumor.

22. The method according to claim 20, wherein the cancer is selected from gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor, and / or glioma.

23. The method according to claim 22, wherein the antibody or antigen-binding fragment is administered in combination with one or more further therapeutic agents.

24. The method according to claim 23, wherein the one or more therapeutic agents are selected from paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacitidine.

25. The method according to claim 24, wherein the one or more therapeutic agents are paclitaxel, lenalidomide, or 5-azacitidine.

26. The method according to claim 23, wherein the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

27. The method according to claim 25, wherein the anti-PD1 antibody is tislerizumab.

28. An isolated nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 1 to 17.

29. A vector comprising the nucleic acid described in claim 28.

30. A host cell comprising the nucleic acid described in claim 28 or the vector described in claim 29.

31. A process for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 30, and recovering the antibody or the antigen-binding fragment from the culture.

32. An antibody or antigen-binding fragment according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18 or 19, for use in pharmaceuticals or therapeutics.

33. An antibody or antigen-binding fragment according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18 or 19, for use in a method of treating cancer.

34. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 17 in the manufacture of a drug for the treatment of cancer.

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