CLDN6 binding molecules and uses thereof

Abstract

The invention provides a separated CLDN6 binding molecule and a preparation method of the separated CLDN6 binding molecule. In particular to a separated CLDN6 single domain antibody or an antigen binding fragment thereof, nucleic acid for coding the CLDN6 single domain antibody or the antigen binding fragment thereof, and an expression vector or host cell containing the nucleic acid. And an antibody drug conjugate composition and a drug or kit comprising the antibody drug conjugate composition of the CLDN6 single domain antibody or the antigen binding fragment thereof. The invention also relates to application of the binding molecule or the single domain antibody or the antigen binding fragment thereof or the antibody drug conjugate composition in treatment of CLDN6 related diseases.

Description

Technical Field

[0001] This application belongs to the field of biotechnology. In general, the present invention relates to CLDN6-binding molecules. More specifically, the present application relates to single-domain antibodies or antigen-binding fragments thereof or antibody-drug conjugate (ADC) compositions that specifically bind to CLDN6, methods for preparing the same, and uses thereof. Background Art

[0002] CLDN6 (also known as Claudin-6, Skullin), a member of the claudin family (abbreviated as the CLDN family, which includes 27 CLDN members), is an important molecule in tight junctions between cells. CLDN6 is located on chromosome 16p3.3 and has a molecular weight of approximately 20-40 kDa. Claudin members share a similar structure, with each molecule having four transmembrane domains, two extracellular loops, and two intracellular tails (Lal-Nag, M., Battis, M., Santin, A.D., & Morin, P.J. (2012). Claudin-6: a novel receptor for CPE-mediated cytotoxicity in inovarian cancer. Oncogenesis). CLDN6 has a C-terminal PDZ binding site, which enables tight junction proteins to interact with certain proteins in the cell, playing an important role in cell connection and epithelial tissue permeability (Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions. Oncology. 2017).

[0003] CLDN6, as a potential tumor-specific target, is almost not expressed in normal tissues (US9487584) but is expressed in a variety of solid tumors, including ovarian cancer (24-54%), gastric cancer (9.7-51.6%), germ cell cancer (100%), non-small cell lung cancer (NSCLC) (6.5-10.9%), liver cancer (79.2-80.9%), and myxofibrosarcoma (63.9%) (Identification of Claudin-6 as a Molecular Biomarker in Pan-Cancer Through Multiple Omics Integrative Analysis. Front. Cell Dev. Biol. 2021; Qu, Huinan et al. “CLDN6: From Traditional Barrier Function to Emerging Roles in Cancers.” International journal of molecular sciences vol. 22, 24 13416. 14 Dec. 2021). At the same time, in ovarian cancer, endometrial cancer, non-small cell lung cancer, cervical cancer, gastric cancer, and liver cancer, high expression of CLDN6 is associated with lower survival rate and prognosis (Gao, Peipei et al. "Association of CLDN6 and CLDN10 With ImmuneMicroenvironment in Ovarian Cancer: A Study of the Claudin Family." Frontiers in genetics vol. 12 595436. 23Jun. 2021; Targeting tumor lineage plasticity in hepatocellular carcinoma using an anti-CLDN6 antibody-drug conjugate. Science Translational Medicine. 03Feb 2021; Yu, S., Zhang, Y., Li, Q. et al. CLDN6 promotes tumor progression through the YAP1-snail1 axis in gastric cancer. Cell Death Dis 10, 949 (2019)).

[0004] In addition, CLDN6 is associated with drug resistance in various malignant tumors and can participate in tumor cell invasion and metastasis through multiple signaling pathways such as TGF-β / SMAD, p38-MAPK, JAKs-STATs, ASK1-p38 / c-Jun (Ito, Yui et al. “Aberrant expression of claudin-6 contributes to malignant potentials and drug resistance of cervical adenocarcinoma.” Cancer science vol. 113, 4 (2022)). CLDN6 is a cancer stem cell marker (Abstract 1907: Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features. AACR Annual Meeting 2018 / US20190077876A1). In liver cancer, sorafenib treatment leads to the directed differentiation of CLDN6-positive cancer stem cells, thereby mediating sorafenib resistance (Kong, Fan-En et al. “Targeting tumor lineage plasticity in hepatocellular carcinoma using an anti-CLDN6 antibody-drug conjugate.” Sciencetranslational medicine vol.13,579(2021)).

[0005] Sequence alignment revealed a high degree of sequence conservation between CLDN6 and other CLDN family proteins, with a sequence similarity of 25-70%. It also exhibits high sequence similarity with CLDN9, CLDN3, and CLDN4, differing from CLDN9 by only one amino acid in the first extracellular region and two amino acids in the second (Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions. Oncology. 2017). CLDN9 is lowly expressed in normal tissues but highly expressed in ovarian and cervical cancers (Bispecific claudin-6 x CD3 antibodies in a 2+1 format demonstrate selectivity and activity on human ovarian cancer cells. 2021. AACR). Therefore, the development of antibody drugs that specifically target CLDN6 or simultaneously target both CLDN6 and CLDN9 could potentially extend its reach to a wider patient population, potentially offering enhanced synergistic efficacy in indications such as ovarian cancer.

[0006] Nanobodies have smaller relative molecular weight and better tissue penetration, and can potentially be used in CAR-T, bispecific antibodies and radionuclide therapy ( I, Muyldermans S. The Therapeutic Potential of Nanobodies. BioDrugs. 2020; 34(1):11-26.).

[0007] There is an urgent need in this field for nanobodies that can strongly bind to CLDN6 and weakly bind to (or not bind to) CLDN9 and not bind to CLDN3 and CLDN4, or strongly bind to CLDN6 and CLDN9 and not bind to CLDN3 and CLDN4, so as to achieve better antibody therapeutic effects. Summary of the Invention

[0008] The purpose of the present invention is to provide a new CLDN6 binding molecule, specifically, to provide a new single-domain antibody that specifically recognizes CLDN6. The single-domain antibody that specifically recognizes CLDN6 has lower toxic side effects and better clinical efficacy, and can more effectively treat cancer.

[0009] In general, the present invention provides a binding molecule that specifically binds to CLDN6, specifically, a single-domain antibody that specifically binds to CLDN6, also known as a CLDN6 single-domain antibody. The term "CLDN6 single-domain antibody" is also referred to hereinafter as a CLDN6 single-domain antibody, a CLDN6 antibody in the form of a nanobody, a CLDN6 nanobody, or a CLDN6 VHH antibody, and the above terms are used interchangeably. The present application also provides methods for constructing and screening such CLDN6-binding molecules or single-domain antibodies, nucleic acid molecules encoding such CLDN6-binding molecules or single-domain antibodies, vectors and host cells for expressing such CLDN6-binding molecules or single-domain antibodies, and compositions or kits comprising such CLDN6-binding molecules or single-domain antibodies.

[0010] Specifically, the present invention provides CLDN6 antibodies in the form of nanobodies and their humanized or affinity-matured derivatives, which, while maintaining advantages such as high affinity and low molecular weight, undergo immunogenicity modification, improve drugability, and possess great therapeutic advantages.

[0011] In some aspects, the present invention provides an isolated CLDN6 binding molecule that is capable of specifically binding to CLDN6 and comprises a heavy chain variable region comprising the following CDR1, CDR2, and CDR3:

[0012] (a) comprising SEQ ID NO: 1-3, respectively, or

[0013] (b) comprising SEQ ID NO: 1 and 4-5, respectively, or

[0014] (c) comprising SEQ ID NO: 1 and 6-7, respectively, or

[0015] (d) comprising SEQ ID NOs: 8-9 and 3, respectively, or

[0016] (e) comprising SEQ ID NOs: 10-11 and 3, respectively, or

[0017] (f) comprising SEQ ID NO: 12 and 2-3, respectively, or

[0018] (g) comprising SEQ ID NOs: 13-14 and 3, respectively, or

[0019] (h) comprising SEQ ID NO: 15-17, respectively, or

[0020] (i) comprising SEQ ID NOs: 1 and 18-19, respectively, or

[0021] (j) comprising SEQ ID NOs: 1 and 20-21, respectively, or

[0022] (k) comprise SEQ ID NOs: 1 and 22-23, respectively.

[0023] In some embodiments, the CLDN6 binding molecule comprises the following heavy chain variable region CDR1, CDR2, and CDR3:

[0024] (a) as shown in SEQ ID NO: 1-3, respectively, or

[0025] (b) as shown in SEQ ID NO: 1 and 4-5, respectively, or

[0026] (c) as shown in SEQ ID NO: 1 and 6-7, respectively, or

[0027] (d) as shown in SEQ ID NO: 8-9 and 3, respectively, or

[0028] (e) as shown in SEQ ID NO: 10-11 and 3, respectively, or

[0029] (f) as shown in SEQ ID NO: 12 and 2-3, respectively, or

[0030] (g) as shown in SEQ ID NO: 13-14 and 3, respectively, or

[0031] (h) as shown in SEQ ID NO: 15-17, respectively, or

[0032] (i) as shown in SEQ ID NO: 1 and 18-19, respectively, or

[0033] (j) as shown in SEQ ID NO: 1 and 20-21, respectively, or

[0034] (k) are shown in SEQ ID NOs: 1 and 22-23, respectively.

[0035] In some embodiments, the heavy chain variable region (VH) of the CLDN6 binding molecule comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-44.

[0036] In some embodiments, the heavy chain variable region of the CLDN6 binding molecule consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-44.

[0037] In some embodiments, the heavy chain variable region (VH) of the CLDN6 binding molecule comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

[0038] In some embodiments, the heavy chain variable region (VH) of the CLDN6-binding molecule comprises an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

[0039] In some preferred embodiments, the number of additions, deletions and / or substitutions (eg, conservative substitutions) of the one or more amino acids is no more than five, preferably no more than three.

[0040] In some embodiments, the CLDN6 binding molecule is an antibody, such as, but not limited to, a camelid antibody, a humanized antibody, or an affinity matured antibody.

[0041] In some embodiments, the CLDN6 binding molecule is fused to another molecule, such as the Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen-binding fragment of an antibody, an antibody-drug conjugate, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, or a fluorescent protein.

[0042] In some preferred embodiments, the CLDN6 binding molecule is fused to the Fc domain of human IgG (such as human IgG1 or human IgG4).

[0043] In some aspects, the CLDN6 binding molecule is a single domain antibody comprising a heavy chain variable region as defined above. Thus, the present invention also provides an isolated CLDN6 single domain antibody in the form of a nanobody.

[0044] In some embodiments, the CLDN6 single domain antibody or antigen-binding fragment thereof specifically binds to CLDN6.

[0045] In some embodiments, the CLDN6 single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises the following CDR1, CDR2, and CDR3:

[0046] (a) comprising SEQ ID NO: 1-3, respectively, or

[0047] (b) comprising SEQ ID NO: 1 and 4-5, respectively, or

[0048] (c) comprising SEQ ID NO: 1 and 6-7, respectively, or

[0049] (d) comprising SEQ ID NOs: 8-9 and 3, respectively, or

[0050] (e) comprising SEQ ID NOs: 10-11 and 3, respectively, or

[0051] (f) comprising SEQ ID NO: 12 and 2-3, respectively, or

[0052] (g) comprising SEQ ID NOs: 13-14 and 3, respectively, or

[0053] (h) comprising SEQ ID NO: 15-17, respectively, or

[0054] (i) comprising SEQ ID NOs: 1 and 18-19, respectively, or

[0055] (j) comprising SEQ ID NOs: 1 and 20-21, respectively, or

[0056] (k) comprise SEQ ID NOs: 1 and 22-23, respectively.

[0057] In some embodiments, the CLDN6 single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises the following CDR1, CDR2, and CDR3:

[0058] (a) as shown in SEQ ID NO: 1-3, respectively, or

[0059] (b) as shown in SEQ ID NO: 1 and 4-5, respectively, or

[0060] (c) as shown in SEQ ID NO: 1 and 6-7, respectively, or

[0061] (d) as shown in SEQ ID NO: 8-9 and 3, respectively, or

[0062] (e) as shown in SEQ ID NO: 10-11 and 3, respectively, or

[0063] (f) as shown in SEQ ID NO: 12 and 2-3, respectively, or

[0064] (g) as shown in SEQ ID NO: 13-14 and 3, respectively, or

[0065] (h) as shown in SEQ ID NO: 15-17, respectively, or

[0066] (i) as shown in SEQ ID NO: 1 and 18-19, respectively, or

[0067] (j) as shown in SEQ ID NO: 1 and 20-21, respectively, or

[0068] (k) are shown in SEQ ID NOs: 1 and 22-23, respectively.

[0069] In some embodiments, the CLDN6 single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), which comprises an amino acid sequence selected from any one of SEQ ID NOs: 24-44.

[0070] In some embodiments, the CLDN6 single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), which consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-44.

[0071] In some embodiments, the heavy chain variable region (VH) of the CLDN6 single domain antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

[0072] In some embodiments, the heavy chain variable region (VH) of the CLDN6 single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

[0073] In some preferred embodiments, the number of additions, deletions and / or substitutions (eg, conservative substitutions) of the one or more amino acids is no more than five, preferably no more than three.

[0074] In some embodiments, the CLDN6 single-domain antibody is a camelid antibody, a humanized antibody, or an affinity-matured antibody.

[0075] In some embodiments, the CLDN6 single-domain antibody or antigen-binding fragment thereof is fused to another molecule, such as the Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen-binding fragment of an antibody, a drug molecule, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, or a fluorescent protein.

[0076] In some preferred embodiments, the antibody or antigen-binding fragment thereof is fused to the Fc domain of human IgG (such as human IgG1 or human IgG4).

[0077] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CLDN6 binding molecule as disclosed herein or comprising a nucleotide sequence encoding a CLDN6 single domain antibody or antigen-binding fragment thereof as disclosed herein.

[0078] In some aspects, the present invention relates to an expression vector comprising a nucleotide sequence encoding a CLDN6 binding molecule as disclosed herein or an expression vector comprising a nucleic acid molecule encoding a CLDN6 single domain antibody or antigen-binding fragment thereof as disclosed herein.

[0079] In some aspects, the present invention relates to a host cell comprising a nucleic acid molecule or expression vector as disclosed herein.

[0080] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0081] In other aspects, the present invention relates to an antibody drug conjugate (ADC) composition comprising a conjugate of a CLDN6 binding molecule disclosed herein or a CLDN6 single domain antibody or antigen-binding fragment thereof disclosed herein and a drug molecule.

[0082] In some embodiments, the drug molecule can be a cytotoxic agent, such as a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, or an antimicrobial agent. In preferred embodiments, the drug molecule can be selected from, but not limited to, a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder. In more preferred embodiments, the drug molecule can be selected from MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0083] In some embodiments, the CLDN6-binding molecules or CLDN6 single-domain antibodies or antigen-binding fragments thereof described herein can be combined with drug molecules and appropriate drug molecules according to antibody-drug conjugation methods commonly used in the art to form an antibody-drug conjugate (ADC) mixture. Specifically, disulfide bonds in the antibody are reduced to free sulfhydryl groups using a reducing agent (e.g., TCEP). Drug molecules can be coupled to the antibody by directly forming disulfide bonds with these free sulfhydryl groups or by forming disulfide bonds with these free sulfhydryl groups via a linker, thereby generating an antibody-drug conjugate (ADC). Furthermore, since antibodies comprising heavy and light chains typically contain four interchain disulfide bonds, theoretically, one antibody molecule can be coupled to 0, 2, 4, 6, or 8 drug molecules. The resulting antibody-drug conjugate (ADC) is typically a mixture of conjugate molecules having these different coupling numbers (DARs) (also referred to herein as an "antibody-drug conjugate (ADC) composition"). Regarding the linker used in the preparation of antibody-drug conjugates, those skilled in the art can make an appropriate selection based on the drug molecule to be conjugated. Generally, the drug can be coupled to the linker via an amine coupling method to form a drug-linker complex, which is then coupled to the reduced antibody molecule. The linker used generally contains a reactive group capable of coupling to the antibody, such as maleimide, organobromide, iodide, and the like.

[0084] In some aspects, the present invention relates to a pharmaceutical composition comprising a CLDN6 binding molecule as disclosed herein or a CLDN6 single domain antibody or antigen-binding fragment thereof as disclosed herein, and a pharmaceutically acceptable carrier; or comprising an antibody drug conjugate (ADC) composition as disclosed herein, and a pharmaceutically acceptable carrier.

[0085] In some aspects, the present invention relates to a method for preparing a CLDN6 binding molecule, comprising expressing the CLDN6 binding molecule in a host cell and isolating the CLDN6 binding molecule from the host cell.

[0086] In some aspects, the present invention relates to a method for preparing a CLDN6 single domain antibody or an antigen-binding fragment thereof, comprising expressing the CLDN6 single domain antibody or the antigen-binding fragment thereof in a host cell and isolating the antibody or antigen-binding fragment from the host cell.

[0087] In some embodiments, the subject is a human or mammal suffering from a disease associated with CLDN6. Specifically, the subject may suffer from, but is not limited to, the following diseases: ovarian cancer, endometrial cancer, gastric cancer, germ cell cancer, non-small cell lung cancer (NSCLC), liver cancer, myxofibrosarcoma, cervical cancer, etc.

[0088] In some aspects, the present invention relates to a method for treating or preventing a disease associated with CLDN6, comprising administering to a patient suffering from the disease associated with CLDN6 or a subject predisposed to the disease associated with CLDN6 an effective amount of a CLDN6 binding molecule as disclosed herein, or administering to a patient suffering from the disease associated with CLDN6, or administering to ... subject predisposed to the disease associated with CLDN6, or administering to a subject predisposed to the disease associated with CLDN6 an effective amount of a pharmaceutical composition comprising a CLDN6 binding molecule, a CLDN6 single domain antibody or an antigen-binding fragment thereof, or an antibody drug conjugate (ADC) composition as disclosed herein.

[0089] In some aspects, the present invention relates to methods of treating any disease or condition that can be improved, alleviated, inhibited, or prevented by eliminating, inhibiting, or reducing CLDN6 activity.

[0090] In other aspects, the methods of the present invention also relate to methods for treating or preventing tumors by combination therapy, comprising administering to a subject an effective amount of a CLDN6 binding molecule, a CLDN6 single domain antibody or an antigen-binding fragment thereof as described herein and one or more other drugs.

[0091] In some embodiments, the methods disclosed herein further comprise co-administering an effective amount of a second drug to the subject, wherein the CLDN6 binding molecule or CLDN6 single-domain antibody or antigen-binding fragment thereof disclosed herein is the first drug. In one embodiment, the second drug is a chemotherapeutic agent, a radiotherapeutic agent, or a biomacromolecule drug used to treat the relevant disease. In one embodiment, the biomacromolecule drug is, for example, various monoclonal antibody drugs that attack tumor cells through T cell recognition, such as rituximab, cetuximab, and trastuzumab. As used herein, the expression "second drug" does not mean that it refers to the only drug other than the first drug. Therefore, the second drug does not have to be a single drug, but may constitute or contain more than one such drug.

[0092] In some embodiments, the subject or individual is a mammal, eg, a human and a non-human primate, eg, a mouse or rat, a monkey, etc., preferably a human.

[0093] In some aspects, the present invention relates to the use of a CLDN6 binding molecule as disclosed herein in the preparation of a medicament for treating or preventing a disease associated with CLDN6.

[0094] In some aspects, the present invention relates to the use of a CLDN6 single domain antibody or an antigen-binding fragment thereof as disclosed herein in the preparation of a medicament for treating or preventing a disease associated with CLDN6.

[0095] In some aspects, the present invention relates to the use of an antibody drug conjugate (ADC) composition as disclosed herein in the preparation of a medicament for treating or preventing a disease associated with CLDN6.

[0096] In some embodiments, the disease associated with CLDN6 is selected from, but not limited to, ovarian cancer, endometrial cancer, gastric cancer, germ cell cancer, NSCLC, liver cancer, myxofibrosarcoma, non-small cell lung cancer, cervical cancer, etc.

[0097] In some aspects, the present invention relates to kits or devices and related methods for using the CLDN6 binding molecules, CLDN6 single domain antibodies or antigen-binding fragments thereof, or antibody drug conjugate (ADC) compositions as disclosed herein, as well as pharmaceutical compositions as disclosed herein, which can be used to treat diseases associated with CLDN6, such as cancer. To this end, the present invention preferably provides an article of manufacture that can be used to treat such conditions, comprising a container containing the CLDN6 binding molecules, CLDN6 single domain antibodies or antigen-binding fragments thereof, or antibody drug conjugate (ADC) compositions as disclosed herein, and instructions for using the CLDN6 binding molecules, CLDN6 single domain antibodies or antigen-binding fragments thereof, or antibody drug conjugate (ADC) compositions as disclosed herein to treat, ameliorate, or prevent a disease associated with CLDN6, or its progression or recurrence.

[0098] The present invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof is applicable to any and all CLDN6 binding molecules, CLDN6 single domain antibodies or antigen-binding fragments thereof, methods and uses of the invention described herein.

[0099] In summary, the present invention relates to the following embodiments:

[0100] 1. An isolated CLDN6 binding molecule comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

[0101] (a) comprising SEQ ID NO: 1-3, respectively, or

[0102] (b) comprising SEQ ID NO: 1 and 4-5, respectively, or

[0103] (c) comprising SEQ ID NO: 1 and 6-7, respectively, or

[0104] (d) comprising SEQ ID NOs: 8-9 and 3, respectively, or

[0105] (e) comprising SEQ ID NOs: 10-11 and 3, respectively, or

[0106] (f) comprising SEQ ID NO: 12 and 2-3, respectively, or

[0107] (g) comprising SEQ ID NOs: 13-14 and 3, respectively, or

[0108] (h) comprising SEQ ID NO: 15-17, respectively, or

[0109] (i) comprising SEQ ID NOs: 1 and 18-19, respectively, or

[0110] (j) comprising SEQ ID NOs: 1 and 20-21, respectively, or

[0111] (k) comprise SEQ ID NOs: 1 and 22-23, respectively.

[0112] 2. The CLDN6 binding molecule according to embodiment 1, wherein the CLDN6 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

[0113] (a) as shown in SEQ ID NO: 1-3, respectively, or

[0114] (b) as shown in SEQ ID NO: 1 and 4-5, respectively, or

[0115] (c) as shown in SEQ ID NO: 1 and 6-7, respectively, or

[0116] (d) as shown in SEQ ID NO: 8-9 and 3, respectively, or

[0117] (e) as shown in SEQ ID NO: 10-11 and 3, respectively, or

[0118] (f) as shown in SEQ ID NO: 12 and 2-3, respectively, or

[0119] (g) as shown in SEQ ID NO: 13-14 and 3, respectively, or

[0120] (h) as shown in SEQ ID NO: 15-17, respectively, or

[0121] (i) as shown in SEQ ID NO: 1 and 18-19, respectively, or

[0122] (j) as shown in SEQ ID NO: 1 and 20-21, respectively, or

[0123] (k) are shown in SEQ ID NOs: 1 and 22-23, respectively.

[0124] 3. The CLDN6 binding molecule according to embodiment 1, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-44, or

[0125] The heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6, or

[0126] The heavy chain variable region comprises an amino acid sequence having one or more amino acid additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

[0127] 4. The CLDN6 binding molecule according to embodiment 3, wherein the number of additions, deletions and / or substitutions (e.g., conservative substitutions) of one or more amino acids is no more than five, preferably no more than three.

[0128] 5. The CLDN6 binding molecule according to embodiment 1, wherein the CLDN6 binding molecule is a camelid antibody, a humanized antibody or an affinity matured antibody.

[0129] 6. The CLDN6 binding molecule according to any one of embodiments 1 to 5, wherein the CLDN6 binding molecule is fused to another molecule, wherein the other molecule is selected from the Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen-binding fragment of an antibody, a drug molecule, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, or a fluorescent protein.

[0130] 7. The CLDN6 binding molecule according to embodiment 6, wherein the CLDN6 binding molecule is fused to the Fc domain of human IgG (such as human IgG1 or human IgG4).

[0131] 8. An isolated single-domain antibody or antigen-binding fragment thereof that specifically binds to CLDN6, wherein the isolated single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises the following CDR1, CDR2 and CDR3:

[0132] (a) comprising SEQ ID NO: 1-3, respectively, or

[0133] (b) comprising SEQ ID NO: 1 and 4-5, respectively, or

[0134] (c) comprising SEQ ID NO: 1 and 6-7, respectively, or

[0135] (d) comprising SEQ ID NOs: 8-9 and 3, respectively, or

[0136] (e) comprising SEQ ID NOs: 10-11 and 3, respectively, or

[0137] (f) comprising SEQ ID NO: 12 and 2-3, respectively, or

[0138] (g) comprising SEQ ID NOs: 13-14 and 3, respectively, or

[0139] (h) comprising SEQ ID NO: 15-17, respectively, or

[0140] (i) comprising SEQ ID NOs: 1 and 18-19, respectively, or

[0141] (j) comprising SEQ ID NOs: 1 and 20-21, respectively, or

[0142] (k) comprise SEQ ID NOs: 1 and 22-23, respectively.

[0143] 9. The isolated single domain antibody or antigen binding fragment thereof according to embodiment 8, wherein the heavy chain variable region comprises the following CDR1, CDR2 and CDR3:

[0144] (a) as shown in SEQ ID NO: 1-3, respectively, or

[0145] (b) as shown in SEQ ID NO: 1 and 4-5, respectively, or

[0146] (c) as shown in SEQ ID NO: 1 and 6-7, respectively, or

[0147] (d) as shown in SEQ ID NO: 8-9 and 3, respectively, or

[0148] (e) as shown in SEQ ID NO: 10-11 and 3, respectively, or

[0149] (f) as shown in SEQ ID NO: 12 and 2-3, respectively, or

[0150] (g) as shown in SEQ ID NO: 13-14 and 3, respectively, or

[0151] (h) as shown in SEQ ID NO: 15-17, respectively, or

[0152] (i) as shown in SEQ ID NO: 1 and 18-19, respectively, or

[0153] (j) as shown in SEQ ID NO: 1 and 20-21, respectively, or

[0154] (k) are shown in SEQ ID NOs: 1 and 22-23, respectively.

[0155] 10. The isolated single domain antibody or antigen binding fragment thereof according to embodiment 8, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-44, or

[0156] The heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6, or

[0157] The heavy chain variable region comprises an amino acid sequence having one or more amino acid additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

[0158] 11. The isolated single domain antibody or antigen binding fragment thereof according to embodiment 8, wherein the isolated antibody is a camelid antibody, a humanized antibody or an affinity matured antibody.

[0159] 12. An isolated single-domain antibody or antigen-binding fragment thereof according to any one of embodiments 8-11, wherein the antibody or antigen-binding fragment thereof is fused to another molecule, wherein the other molecule is selected from the Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen-binding fragment of an antibody, a drug molecule, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, or a fluorescent protein.

[0160] 13. The isolated single-domain antibody or antigen-binding fragment thereof according to embodiment 12, wherein the antibody or antigen-binding fragment thereof is fused to the Fc domain of human IgG (such as human IgG1 or human IgG4).

[0161] 14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated CLDN6 binding molecule of any one of embodiments 1-7 or the single domain antibody or antigen-binding fragment thereof of any one of embodiments 8-13.

[0162] 15. A vector comprising the nucleic acid molecule of embodiment 14.

[0163] 16. A host cell comprising the nucleic acid molecule of embodiment 14 or the vector of embodiment 15.

[0164] 17. An antibody drug conjugate (ADC) composition comprising a conjugate of the isolated CLDN6 binding molecule of any one of embodiments 1-7 or the single domain antibody or antigen-binding fragment thereof of any one of embodiments 8-13 and a drug molecule.

[0165] 18. The antibody drug conjugate (ADC) composition according to embodiment 17, wherein the drug molecule is a cytotoxic agent, such as a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent or an antimicrobial agent, preferably selected from a microtubule inhibitor, a topoisomerase inhibitor or a DNA binding agent, more preferably selected from MMAE (monomethylauristatin E), MMAD (monomethylauristatin D) or MMAF (monomethylauristatin F).

[0166] 19. A pharmaceutical composition comprising at least one CLDN6 binding molecule according to any one of Embodiments 1-7 or the single domain antibody or antigen-binding fragment thereof according to any one of Embodiments 8-13, and a pharmaceutically acceptable carrier; or comprising the antibody drug conjugate composition according to Embodiment 17 or 18 and a pharmaceutically acceptable carrier.

[0167] 20. A method for preparing the CLDN6 binding molecule of any one of embodiments 1-7 or the single domain antibody or antigen-binding fragment thereof of any one of embodiments 8-13, comprising the following steps:

[0168] - expressing the CLDN6 binding molecule of any one of embodiments 1 to 7 or the single domain antibody or antigen-binding fragment thereof of any one of embodiments 8 to 13 in the host cell of embodiment 16; and

[0169] - isolating the CLDN6 binding molecule or single domain antibody or antigen-binding fragment thereof from the host cell.

[0170] 21. Use of the CLDN6 binding molecule of any one of embodiments 1-7, or the single domain antibody or antigen-binding fragment thereof of any one of embodiments 8-13, or the antibody drug conjugate (ADC) composition of embodiment 17 or 18, or the pharmaceutical composition of embodiment 19 in the preparation of a medicament for preventing or treating a disease associated with CLDN6 in a subject.

[0171] 22. The use according to embodiment 21, wherein the subject is a human or a non-human primate, preferably a human.

[0172] 23. The use according to embodiment 21, wherein the disease associated with CLDN6 is selected from ovarian cancer, endometrial cancer, gastric cancer, germ cell cancer, non-small cell lung cancer (NSCLC), liver cancer, myxofibrosarcoma or cervical cancer.

[0173] 24. A kit for preventing or treating a disease associated with CLDN6 in a subject, comprising a container comprising at least one CLDN6 binding molecule according to any one of embodiments 1-7 or a single domain antibody or antigen-binding fragment thereof according to any one of embodiments 8-13, an antibody drug conjugate (ADC) composition according to embodiment 17 or 18, or a pharmaceutical composition according to embodiment 19, and instructions for use.

[0174] 25. A method for preventing or treating a disease associated with CLDN6 in a subject, the method comprising administering to the subject a therapeutically effective amount of the CLDN6 binding molecule of any one of embodiments 1-7, or the single domain antibody or antigen-binding fragment thereof of any one of embodiments 8-13, the antibody drug conjugate (ADC) composition of embodiment 17 or 18, or the pharmaceutical composition of embodiment 19. BRIEF DESCRIPTION OF THE DRAWINGS

[0175] Figure 1A 、 Figure 1B and Figure 1C The expression detection results of cells overexpressing human CLDN6 are shown, wherein Figure 1A For huCLDN-HEK293, Figure 1B For huCLDN6-CHO, Figure 1C PA-1 tumor cells.

[0176] Figure 2 The expression detection results of huCLDN9-HEK293, huCLDN3-HEK293, huCLDN4-HEK293 and huCLDN6-OV90 cells are shown.

[0177] Figure 3 Shown are FACS binding results for huCLDN6-CHO levels of candidate antibodies A11 and A149.

[0178] Figure 4 Shown are FACS binding results of huCLDN9-HEK293 levels for candidate antibodies A11 and A149.

[0179] Figure 5A 、 Figure 5B and Figure 5CFACS binding results for huCLDN3-HEK293, huCLDN4-HEK293, and HEK293 levels are shown for candidate antibodies A11 and A149, respectively.

[0180] Figure 6A and Figure 6B The FACS binding results of huCLDN6-CHO level of humanized antibody are shown, wherein Figure 6A FACS binding results showing huCLDN6-CHO levels of A11-VHH1-Fc, A11-VHH2-Fc, A11-VHH3-Fc, A11-VHH4-Fc, and A11-VHH5-Fc, Figure 6B FACS binding results of huCLDN6-CHO levels of A149-VHH2-Fc, A149-VHH3-Fc, A149-VHH4-Fc, A149-VHH5-Fc, and A149-VHH6-Fc are shown.

[0181] Figure 7A and Figure 7B The FACS binding results of huCLDN6-CHO levels of affinity matured antibodies are shown. Figure 7A FACS binding results showing huCLDN6-CHO levels for A11-11, A11-32, A11-48, A11-68, and A11-71, Figure 7B FACS binding results of huCLDN6-CHO levels of A149-32, A149-38, A149-53, and A149-80 are shown.

[0182] Figure 8A and Figure 8B The FACS binding results of huCLDN9-HEK293 level of affinity matured antibodies are shown, wherein Figure 8A FACS binding results showing huCLDN9-HEK293 levels of A11-11, A11-32, A11-48, A11-68, and A11-71, Figure 8B FACS binding results of huCLDN9-HEK293 levels are shown for A149-32, A149-38, A149-53, and A149-80.

[0183] Figure 9A 、 Figure 9B 、 Figure 9C and Figure 9D The FACS binding results of the affinity matured antibodies at the huCLDN3-HEK293 level are shown, wherein Figure 9AFACS binding results of huCLDN3-HEK293 levels of A11-11, A11-48, and A11-71 are shown. Figure 9B FACS binding results of huCLDN3-HEK293 levels of A11-32 and A11-68 are shown. Figure 9C FACS binding results showing huCLDN3-HEK293 levels of A149-32 and A149-38, Figure 9D FACS binding results of huCLDN3-HEK293 levels of A149-53 and A149-80 are shown.

[0184] Figure 10A 、 Figure 10B 、 Figure 10C and Figure 10D The FACS binding results of huCLDN4-HEK293 levels of affinity matured antibodies are shown. Figure 10A FACS binding results of huCLDN4-HEK293 levels of A11-11, A11-48, and A11-71 are shown. Figure 10B FACS binding results of huCLDN4-HEK293 levels of A11-32 and A11-68 are shown. Figure 10C FACS binding results showing huCLDN4-HEK293 levels of A149-32 and A149-38, Figure 10D FACS binding results of huCLDN4-HEK293 levels of A149-53 and A149-80 are shown.

[0185] Figure 11A and Figure 11B The FACS binding results of HEK293 level of affinity matured antibodies are shown. Figure 11A FACS binding results of HEK293 levels of A11-11, A11-48, A11-71, A11-32 and A11-68 are shown. Figure 11B FACS binding results for HEK293 levels of A149-32, A149-38, A149-53, and A149-80 are shown.

[0186] Figure 12A 、 Figure 12B 、 Figure 12C and Figure 12D The FACS binding results of the PA-1 level of the affinity matured antibody are shown. Figure 12A FACS binding results showing PA-1 levels of A11-11, A11-48, and A11-71, Figure 12B FACS binding results showing PA-1 levels of A11-32 and A11-68, Figure 12C FACS binding results showing PA-1 levels of A149-32 and A149-38, Figure 12D FACS binding results of PA-1 levels of A149-53 and A149-80 are shown.

[0187] Figure 13A and 13B The cross-species activity of candidate antibodies A11-11, A11-68, A149-32 and A149-38 is shown, wherein Figure 13A The binding activities of A11-11, A11-68, A149-32, and A149-38 at the cynoCLDN6-HEK293 level are shown; Figure 13B The binding activities of A11-11, A11-68, A149-32 and A149-38 at the musCLDN6-HEK293 level are shown.

[0188] Figure 14 Shown are the FACS competition results of huCLDN6-CHO levels of candidate antibodies A11-11, A11-68, A149-32, and A149-38 with DS9606a.

[0189] Figures 15A-15C The anti-tumor efficacy of the antibody drug conjugate (ADC) of the candidate antibodies A149-32, IMAB027 and DS9606a in the OV90 ovarian cancer mouse model is shown. Figure 15A The time-varying curve of tumor volume is shown; Figure 15B Tumor weights on day 30 are shown; Figure 15C Shown is the curve of mouse body weight changes over time.

[0190] Sequence Listing Overview

[0191] This application is accompanied by a sequence listing comprising a number of nucleotide and amino acid sequences. Tables A, B and C below provide an overview of the sequences included.

[0192] Table A. CDR sequences of the heavy chain variable regions of antibodies

[0193]

[0194]

[0195] Table B. VH sequences of antibodies

[0196]

[0197]

[0198] Table C. Other sequences

[0199]

[0200] DETAILED DESCRIPTION

[0201] It will be understood by those skilled in the art that the present invention is not limited to the specific methodology, embodiments and reagents described herein, as these are exemplary illustrations. It will also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0202] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0203] Furthermore, unless the context requires otherwise, terms in the singular shall include plural referents and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies.

[0204] definition

[0205] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0206] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0207] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.) and antibody fragments, so long as they exhibit the desired antigen-binding activity. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain circumstances, for example, antibodies naturally occurring in camelids may comprise only heavy chains.

[0208] The term "single-domain antibody," also known as a single-domain antibody (sdAb), refers to an antibody consisting of a single heavy chain variable region. Similar to IgG antibodies, they can selectively bind to specific antigens, but their molecular weight is much smaller than that of IgG antibodies.

[0209] As used herein, the term "antigen-binding portion" refers to a portion that specifically binds to a target antigen. The term includes antibodies and other natural molecules (e.g., receptors, ligands) or synthetic molecules (e.g., DARPins) that are capable of specifically binding to a target antigen. In a preferred embodiment, the antigen-binding portion of an antibody of the invention is an antibody fragment.

[0210] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules having a single amino acid composition, regardless of the method by which they are produced. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or a combination of these or other techniques known in the art.

[0211] As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or antigen-binding portion to an antigen epitope in an in vitro assay, preferably in biointerferometry (ForteBio) using purified wild-type antigen. In certain embodiments, an antibody or antigen-binding portion is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0212] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to the antigen. The variable region typically exhibits the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which allow the antibody to bind to a specific epitope. The two light and heavy chain variable regions typically comprise, in order from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0213] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that is capable of binding to the antigen to which the intact antibody binds.

[0214] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

[0215] As used herein, the term "EC 50”, also known as the “half-maximal effective concentration”, is the concentration of a drug, antibody, or toxicant that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM" or "μg / mL".

[0216] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0217] The term "conservative substitution" refers to the substitution of one amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid. Exemplary substitutions are shown in Table D below:

[0218] Amino acids can be grouped according to the properties of their common side chains:

[0219] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0220] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0221] (3) Acidic: Asp, Glu;

[0222] (4) Basic: His, Lys, Arg;

[0223] (5) Residues that affect chain orientation: Gly, Pro;

[0224] (6) Aromatic: Trp, Tyr, Phe.

[0225] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0226] Table D. Exemplary Substitutions

[0227] original residue Exemplary substitutions Conservative substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

[0228] A type of substitution variant includes one or more hypervariable region residues that replace the parent antibody (e.g., humanized antibody). Generally, one or more variants selected for further study have improvements (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity), and / or will have specific biological properties that the parent antibody substantially retains. An exemplary substitution variant is an affinity-matured antibody, which can be easily produced, for example, using phage-based affinity maturation techniques, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0229] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of identical amino acid residues in the candidate sequence and the reference polypeptide sequence, after the sequences are aligned (and, if necessary, introduced into spaces) to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain maximum alignment for the full length of the compared sequences. When referring to percentages of sequence identity in this application, if not otherwise specifically noted, these percentages are calculated relative to the full length of the longer sequence. Calculations relative to the full length of the longer sequence are applicable to both nucleic acid sequences and polypeptide sequences.

[0230] The terms "effective amount" and "therapeutically effective amount" refer to an amount or dosage of an antibody or antigen-binding fragment of the present invention that, after single or multiple doses, produces the desired effect in the treated subject, including improvement of the subject's condition (e.g., improvement of one or more symptoms) and / or delay in symptom progression. "Effective amount" and "therapeutically effective amount" may also refer to an amount sufficient to reduce CLDN6 signaling.

[0231] An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors, such as the species of the mammal; its size, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosing regimen selected; and the use of any concomitant therapy.

[0232] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0233] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0234] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, alpacas, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0235] The terms "antibody drug conjugate" or "ADC" or "antibody drug conjugate" are used interchangeably herein and refer to a conjugate formed by directly coupling a drug to an antibody or indirectly covalently coupling the drug via one or more suitable linkers. ADCs are typically in the form of "antibody-linker-drug conjugates." Antibody-drug conjugates combine the desirable properties of both antibodies and cytotoxic drugs, thereby enhancing their anti-tumor activity by targeting potent cytotoxic drugs to tumor cells expressing the antigen.

[0236] As used herein, the term "drug" refers to any cytotoxic molecule that has an anti-tumor effect and at least one substituent group or moiety that allows attachment to a linker structure. The drug can kill cancer cells and / or inhibit the growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or condition.

[0237] As used herein, the term "linker" refers to a reactive molecule containing at least two reactive groups, one of which can be covalently bonded to a drug molecule and the other can be covalently coupled to an antibody.

[0238] Example

[0239] The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0240] Those skilled in the art should understand that, unless otherwise noted, the reagents, plasmids, cells, etc. used in the following examples are all commercially available products.

[0241] Example 1 Preparation of raw materials

[0242] 1.1 Preparation of CLDN6 positive control antibody

[0243] The following two positive control antibodies were used: IMAB027 (heavy chain SEQ ID NO: 45, light chain SEQ ID NO: 46) obtained from US9487584, and DS9606a (heavy chain SEQ ID NO: 47, light chain SEQ ID NO: 48) obtained from CN111164208A. These sequences were converted into coding nucleotide sequences and synthesized by General Biotech Co., Ltd. Each target fragment was amplified by PCR and then constructed into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) via homologous recombination. Each recombinant protein expression vector was transformed into Escherichia coli SS320, cultured overnight at 37°C, and then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression.

[0244] The positive control antibody was expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) as follows: on the day of transfection, the cell density was confirmed to be 7 × 10 6 -1×10 7 viable cells / mL, and the cell viability was >98%. At this time, the cells were adjusted to a cell density of 6 × 10 6 Cells / mL. Dilute the target plasmid in 4°C pre-cooled OptiPRO™ SFM (ThermoFisher) (add 1 μg of plasmid to 1 mL of OptiPRO™ SFM). Simultaneously, dilute ExpiFectamine™ CHO in OptiPRO™ SFM. Mix equal volumes of the two and gently pipette to mix thoroughly to prepare the ExpiFectamine™ CHO / plasmid DNA mixture. Incubate at room temperature for 1-5 minutes. Slowly add the mixture to the prepared cell suspension while gently shaking. Finally, incubate in a cell culture shaker at 37°C, 8% CO2.

[0245] 18-22 hours after transfection, add ExpiCHOTMEnhancer and ExpiCHOTMFeed to the culture medium and continue culturing in a shaker at 32°C and 5% CO2. On the fifth day of transfection, add the same volume of ExpiCHOTMFeed and gently mix the cell suspension while slowly adding. 7-15 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 minutes. The resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl. Finally, the resulting protein was replaced with PBS buffer using an ultrafiltration concentration tube (Millipore, UFC901096). Finally, control antibodies (IMAB027 and DS9606a) were obtained. It is known that the DS9606a antibody can simultaneously target CLDN6 and CLDN9, and the IMAB027 antibody can target CLDN6.

[0246] 1.2 Construction of overexpression cell lines

[0247] Stable overexpression cells were obtained by lentiviral infection: huCLDN6-HEK293, huCLDN9-HEK293, huCLDN3-HEK293, huCLDN4-HEK293, huCLDN6-CHO, cynoCLDN6-HEK293, and musCLDN6-HEK293. The specific preparation method is as follows:

[0248] DNA fragments of human CLDN6 (huCLDN6, UniProtKB-P56747, SEQ ID NO: 49), human CLDN9 (huCLDN9, UniProtKB-O95484, SEQ ID NO: 50), human CLDN3 (huCLDN3, UniProtKB-O15551, SEQ ID NO: 51), human CLDN4 (huCLDN4, UniProtKB-O14493, SEQ ID NO: 52), murine CLDN6 (musCLDN6, UniProtKB-Q9Z262, SEQ ID NO: 53) and monkey CLDN6 (cynoCLDN6, UniProtKB-F7AWZ8, SEQ ID NO: 54) were synthesized by gene synthesis technology and cloned into the expression vector pLVX-puro (Clontech, Cat# 632164). The plasmid was introduced into Escherichia coli through transformation, and the correct plasmid clone was obtained by sequencing after picking a single E. coli clone. The plasmid was extracted and sequenced again for confirmation.

[0249] Recovery and culture of HEK293 ( CRL-1573 TM ), CHO (ATCC CCL-61) and OV90 (ovarian cancer cells, CRL-11732 TM ) cells, and were passaged 2-3 times. The day before transfection, the cells were plated at 3×10 5 The cells were inoculated into a cell culture dish at a density of 100 / mL. The cells were used the next day when the confluence reached about 70%. The cells were digested with trypsin containing 0.25% EDTA (Gibco, 25200-072) for 2 minutes and then collected. The cells were centrifuged at 100g for 5 minutes at room temperature, and the supernatant was discarded. After that, 1× DPBS (Shanghai Yuanpei Biological, B210) was added to resuspend the cells and counted. After taking enough CHO cells, the cells were centrifuged at 100g for 5 minutes and resuspended and counted (Shanghai Yuanpei Biological, L410KJ). 5×10 6 The cells were collected by centrifugation and washed with 250 μL Buffer R (Invitrogen, Neon TM Resuspend the cells in a buffer containing 5% dapoxetine (Kit, PK10096) and add 25 μg of the target plasmid. Mix gently with a pipette. Place the suspension in an electroporator (Invitrogen, Neon™ Transfection System, MP922947) for electroporation. The reaction conditions are 1100 V / 20 ms / 2 cycles. After electroporation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and without antibiotics. The cells were then seeded into 10 cm × 10 cm cell culture dishes and cultured for 48 h. The cells were then aliquoted into 96-well cell culture plates at an average density of 0.5 cells / well. Puromycin (Gibco, A111138-03) was added at a final concentration of 2 μg / mL as a screening pressure. The growth of cell line clones was observed for approximately 2 weeks. Single cell clones grown in the 96-well plates were picked and transferred to 24-well culture plates for further expansion.

[0250] Identification was performed by FACS: the huCLDN6 overexpressing cell line was identified by IMAB027 prepared in Example 1.1; the huCLDN3, huCLDN4, and huCLDN9 overexpressing cell lines were identified by anti-CLDN3 antibody (Miltenyi Biotec's, 130-110-834), anti-CLDN4 antibody (R&D, FAB4219A), and anti-CLDN9 antibody (origene, AM26751PU-N), respectively.

[0251] The tumor cell line PA-1 ( CRL-1572 TM ).

[0252] The results are as follows Figures 1A-1C and Figure 2 As shown, the results showed that a recombinant cell line stably overexpressing the above target antigen was obtained, and the cell line PA-1 (PA-1 is a germ cell cancer cell line that expresses CLDN6 and CLDN9) that endogenously expresses the above target antigen was verified.

[0253] Example 2 Alpaca immunization and immune library construction

[0254] In this example, alpacas were immunized using a cross-immunization approach of huCLDN6-OV90, huCLDN6-CHO, and huCLDN6-HEK293, and the antibody genes of peripheral blood B cells of alpacas immunized with CLDN6-overexpressing cell lines were cloned to construct a nanobody gene phage display library.

[0255] 2.1 Immunization regimen

[0256] In this example, two alpacas (Nanchang Dajia Technology Co., Ltd.) were immunized by intravenous injection. The cells used in the two alpacas were huCLDN6-OV90 / huCLDN6-CHO and huCLDN6-HEK293 / huCLDN6-OV90 as described above. The single immunization dose was 1 x 10 7 Alpaca cells were immunized once every two weeks for a total of five immunizations. After the third and fourth immunizations and the final immunization, blood was collected to test serum titers. Alpaca blood cells after the third, fourth, and final immunizations were used to construct a phage display library and further screening.

[0257] 2.2 Construction of camel-derived nanoantibody gene library

[0258] Take 15mL of Ficoll-Paque density gradient separation solution (GE, 17144003S) and slowly add it to a 50mL centrifuge tube. Tilt the centrifuge tube and slowly add 15mL of the collected immunized alpaca blood along the tube wall in batches so that the Ficoll-Paque density gradient separation solution and the alpaca blood maintain a clear separation interface. The 50mL centrifuge tube containing the blood and separation solution is centrifuged at 15°C for 20min, wherein the centrifuge is set to 400g, the acceleration is 3, and the deceleration is 0. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. There is a narrow band of white cloud layer mainly composed of PBMC at the junction of the upper and middle layers, i.e., the PBMC cell layer. Carefully remove the plasma mixture of the upper layer with a sterile Pasteur pipette, and then use a new sterile Pasteur pipette to absorb the PBMC to obtain separated PBMC. The separated PBMCs were first rinsed twice with PBS, then centrifuged at 1500 rpm at 4°C for 10 min, and finally resuspended with 1.5 mL of PBS and counted using a cell counter (CountStar, CountStar Altair).

[0259] Total RNA was extracted from the PBMC cells isolated by conventional methods. The total RNA extracted was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Based on the situation of VHH antibody germline (germline), degenerate primers were designed in the middle of the front end of the variable region of VHH antibody and the second constant region (CH2), and the VHH-CH2 fragment and the VH-CH1-CH2 fragment of the antibody were obtained after PCR amplification. By the length difference of the two fragments, the PCR product was identified with agarose gel electrophoresis, and the VHH-CH2 fragment was recovered. The VHH-CH2 fragment recovered was amplified by the method of secondary PCR using the forward and reverse primers of the amplification of VHH, using VHH-CH2 as a template to amplify the VHH antibody fragment (Sabir JS, El-Domyati FM et al., Construction of Camelids VHH repertoire in phage display-based library. CR Biol. 2014; 337(4): 244-249). Next, the PCR product and the phage display vector were digested, recovered, and ligated, and the ligated product was recovered using a recovery kit (Omega, D6492-02). Finally, the product was transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) using an electroporator (Bio-Rad, MicroPulser), and the transformed E. coli SS320 bacterial solution was spread on a 2-YT solid plate with ampicillin resistance (the solid plate was prepared by 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar, according to the mass volume g / mL).

[0260] 2.3 Calculation of Antibody Gene Reservoir Capacity

[0261] Inoculate the transformed E. coli SS320 culture with antibiotic-free 2YT medium at a 1:50 ratio. Incubate at 37°C, 220 rpm for 1.5-2 hours until the OD600 reaches 0.5-0.6, then remove to room temperature. Add 90 μL of the culture to a 96-well round-bottom dilution plate. Perform a 10-fold serial dilution of each sample, for a total of 12 dilutions. Using an 8-channel 10 μL pipette, pipette 2 μL of the diluted sample onto a 2YT plate containing 50 μg / mL carbenicillin and 50 μg / mL tetracycline (hereinafter referred to as C+ / T+2YT) containing 50 μg / mL carbenicillin and 50 μg / mL tetracycline, respectively. Incubate upright for 5 minutes, then invert and incubate at 37°C overnight. Observe colony growth the next day and calculate reservoir capacity. The reservoir capacity is calculated as follows: Starting with row A, label rows 1, 2, 3, 4, 5, 6, 7, 8, and so on, through row X. First, select the counting wells. Select the counting wells with 3-20 clones, get the row number X, and count the number of clones n in the corresponding well. The calculation formula is 5×100×10X×n. After calculation, the storage capacity of each milliliter of bacterial liquid is 7×10 8 cfu, i.e. 7 × 10 8 An antibody gene library containing 10 antibody genes.

[0262] 2.4 Preparation of Antibody Gene Phage Display Library

[0263] Based on the antibody gene library capacity, 50 OD (1 OD is 5×10 8cfu) of the Nanobody gene library was added to fresh 2YT liquid culture medium to an initial OD value of 0.1. The resulting mixture was cultured in a shaker at 37°C and 220 rpm until the logarithmic growth phase (OD600 = about 0.6), and then VSCM13 helper phage (Stratagene) was added at 50 times the number of bacteria (i.e., the multiplicity of infection (MOI) was about 50). The mixture was thoroughly mixed, allowed to stand for 30 minutes, and then cultured in a shaker at 220 rpm for 1 hour. Subsequently, the culture was centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the culture medium was replaced with a 2-YT medium containing 50 μg / mL of carbenicillin and 40 μg / mL of kanamycin (hereinafter also referred to as C+ / K+2-YT medium) with dual resistance, and cultured overnight at 30°C and 220 rpm. The next day, the bacterial suspension was centrifuged at 13,000 g for 10 minutes. The supernatant was collected and 20% PEG / NaCl (prepared from 20% PEG6000 (Shanghai Biotechnology) and 2.5 M NaCl) was added to a final PEG / NaCl concentration of 4%. The mixture was mixed and placed on ice for 1 hour. The suspension was then centrifuged at 13,000 g for 10 minutes. The precipitated phage was rinsed with PBS and stored for subsequent phage screening.

[0264] Example 3 Screening of Antibody Gene Phage Display Library

[0265] In this example, multiple cross-screening approaches were used for screening, including protein-protein, protein-cell, and cell-cell cross-screening approaches. CLDN6 cell positive screening and CLDN9 cell negative screening were also performed to screen the library to obtain multiple nanobody molecules that simultaneously bind to CLDN6 / CLDN9 or differentially bind to CLDN6 / CLDN9 at the cellular level.

[0266] 3.1 Cell Screening of Antibody Gene Phage Display Library

[0267] Antibodies against human CLDN6 were screened from phage display libraries using cell-based screening, using huCLDN6-CHO and huCLDN6-CHO cell lines for positive screening, or using huCLDN6-CHO cells for positive screening and huCLDN9-HEK293 and CHO cells for negative screening. The specific methods are as follows. Human huCLDN9-HEK293 cells were cultured in T25 culture flasks. When they reached approximately 90% confluence, which indicates optimal growth, the culture supernatant was removed and the cells were rinsed once with PBS (Shanghai Yuanpei Biotechnology, B310KJ). The cells were then fixed with 5 mL of 4% paraformaldehyde (Shanghai Biotechnology, E672002-0500) for 1 hour and rinsed twice with PBS. For huCLDN6-CHO and CHO cells, sufficient cells were grown in the logarithmic phase and used as antigen material for phage cell-based screening. During screening, phage corresponding to the alpaca library were first incubated with huCLDN9-HEK293 or CHO cell culture flasks at room temperature for 1 hour. The adsorbed phage supernatant was then aspirated and incubated with human huCLDN6-CHO culture flasks for 2 hours. After two PBS rinses, 3 mL of glycine-HCl (pH 2.0) was added and gently mixed for 10 minutes to elute phage that specifically bound the target membrane protein CLDN6. The eluted supernatant was then infected with logarithmic-phase SS320 bacteria (Lucigen, 60512-1), allowed to stand for 30 minutes, and then incubated at 220 rpm for 1 hour. VSCM13 helper phage was then added, allowed to stand for 30 minutes, and incubated at 220 rpm for another hour. The cells were then centrifuged and exchanged into C+ / K+2-YT medium. The resulting phage was then used for a second round of screening. Results showed that after three rounds of screening, significant sequence enrichment was observed after the third round.

[0268] 3.2 Screening of antibody gene phage display library by immunotube method

[0269] The principle of immunotube screening is to coat the huCLDN6-VLP protein on the surface of an immunotube with high adsorption capacity. Then, a panning process of incubating the phage-displayed antibody library with the antigen protein adsorbed on the surface of the immunotube, washing, and eluting the antigen protein is performed. After 2-4 rounds of panning, the specific monoclonal antibodies against the antigen are finally enriched. The specific implementation method is as follows:

[0270] For the first round of screening, 0.1 mL of 100 μg / mL antigen was added to the immunotubes and coated overnight at 4°C. The next day, the coating solution was discarded and the tubes were blocked with 5% milk in PBS for 2 hours. After rinsing twice with PBS, the tubes were added with the phage library displaying the huCLDN6-VLP nanobody. After incubation for 2 hours, the tubes were rinsed eight times with PBS and then twice with PBST to remove nonspecifically bound phage. Next, 0.8 mL of 0.05% EDTA trypsin digestion buffer was added to the immunotubes to elute phage that specifically bound the target antigen. The tubes were then infected with logarithmic-phase SS320 bacteria (Lucigen, 60512-1) and incubated at 37°C for 30 minutes. The tubes were then incubated at 220 rpm for 1 hour, followed by the addition of VSCM13 helper phage and incubation for 30 minutes. Continue incubating at 220 rpm for 1 hour, centrifuge, and transfer to C+ / K+2-YT medium. Continue incubating overnight at 30°C and 220 rpm. The next day, precipitate the phage for the next 2-4 rounds of screening. Antigen coating concentrations typically decrease for the second and third rounds of phage screening, to 10 μg / mL and 2 μg / mL, respectively. In addition, the PBS wash intensity is gradually increased, with 12 and 16 PBS washes, respectively.

[0271] ELISA was performed on the eluted phage pools from each round to evaluate the enrichment effect. A large number of single clones were selected for initial ELISA screening in rounds with good enrichment. After three rounds of screening, clones from the third round were selected for positive clone screening using ELISA. Two antibody VHH molecules with excellent binding activity were screened and named according to their corresponding clone numbers (A11-VHH and A149-VHH, respectively). The amino acid sequences of the CDR regions of the resulting antibody VHHs are shown in Table A. The CDR sequences were determined using the AbM CDR definition method.

[0272] Example 4 Construction, expression and purification of anti-CLDN6 candidate antibodies

[0273] 4.1 Plasmid construction

[0274] The C-terminal fusion of the A11-VHH and A149-VHH candidate nanoantibodies obtained in the screening was a human IgG1 Fc fragment (SEQ ID NO: 57). It was constructed into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured overnight at 37°C. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free antibody plasmid for eukaryotic expression. The two designed VHH-Fc chimeric antibodies were named A11 and A149 respectively. The amino acid sequences of the designed chimeric antibodies are shown in Table C (wherein, the chimeric antibody A11 is shown in SEQ ID NO: 55, and the chimeric antibody A149 is shown in SEQ ID NO: 56).

[0275] 4.2 Expression and Purification of Anti-CLDN6 Candidate Antibodies

[0276] The antibody expression and purification method is described in Example 1. The purified antibody protein was measured using a validated ultramicro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the A280 value was divided by the theoretical extinction coefficient of the antibody as the antibody concentration for subsequent studies.

[0277] Example 5 Detection of the Binding Ability of Candidate Antibodies to huCLDN6-CHO Based on FACS Method

[0278] The huCLDN6-CHO cells in the exponential growth phase were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in FACS buffer (PBS containing 1% BSA), counted, and the cell suspension density was adjusted to 2×10 6 / mL. Subsequently, huCLDN6-CHO cells were added to a 96-well round-bottom plate at 100 μL per well, and the supernatant was removed by centrifugation. Equi-nanomolar gradient dilutions of candidate antibodies and control antibody IMAB027 were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 minutes. The incubated cell mixture was washed three times and then PE-labeled anti-human-IgG-Fc flow cytometry antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 minutes. The incubated cell mixture was washed three times and then 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). The data were analyzed using PRISMTM (GraphPad Software, San Diego, CA), and the EC was calculated. 50 value.

[0279] FACS binding assay results were as follows Figure 3As shown, antibody molecules A11 and A149 have strong binding ability on huCLDN6-CHO cells, EC 50 were 2.644 nM and 3.311 nM, respectively, but weaker than the control antibodies IMAB027 and DS9606a, EC 50 1.692nM and 1.833nM respectively.

[0280] Example 6 Detection of the Binding Ability of Candidate Antibodies to huCLDN9-HEK293 Based on FACS Method

[0281] The huCLDN9-HEK293 cells in the exponential growth phase were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in FACS buffer (PBS containing 1% BSA), counted, and the cell suspension density was adjusted to 2×10 6 / mL. Subsequently, huCLDN9-HEK293 cells were added to a 96-well round-bottom plate at 100 μL per well, and the supernatant was removed by centrifugation. Equal nanomolar gradient dilutions of candidate antibodies and control antibody IMAB027 were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 minutes. The incubated cell mixture was washed 3 times and then PE-labeled anti-human-IgG-Fc flow cytometry antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 minutes. The incubated cell mixture was washed 3 times and then 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). The data were analyzed using PRISMTM (GraphPad Software, San Diego, CA), and the EC was calculated. 50 value.

[0282] FACS binding assay results were as follows Figure 4 As shown in the table, A11 has better binding ability to huCLDN9-HEK293 than IMAB027 and slightly weaker binding ability to DS9606a, while A149 has weaker binding ability to huCLDN9-HEK293 than IMAB027. This indicates that A11 can strongly bind to both human CLDN6 and human CLDN9, while A149 strongly binds to human CLDN6 and weakly binds to human CLDN9.

[0283] Example 7 Detection of the Binding Ability of Candidate Antibodies to huCLDN3-HEK293, huCLDN4-HEK293, and HEK293 Based on FACS Method

[0284] In this example, huCLDN3-HEK293, huCLDN4-HEK293, and HEK293 cells were used to detect non-specific binding of candidate antibodies.

[0285] HuCLDN3-HEK293, huCLDN4-HEK293, and HEK293 cells in the exponential growth phase were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in FACS buffer (PBS containing 1% BSA), counted, and the cell suspension density was adjusted to 2 × 10 6 / mL. Subsequently, the cells were added to a 96-well round-bottom plate at 100 μL per well and centrifuged to remove the supernatant. Equal nanomolar gradient dilutions of candidate antibodies and control antibodies IMAB027 and DS9606a were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 minutes. The incubated cell mixture was washed three times and then PE-labeled anti-human-IgG-Fc flow cytometry antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 minutes. The incubated cell mixture was washed three times and then 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometer (Beckman, CytoFLEX AOO-1-1102). The data were analyzed using PRISMTM (GraphPad Software, San Diego, CA), and the EC was calculated. 50 value.

[0286] FACS binding assay results were as follows Figures 5A-5C As shown, the antibody molecules A11, A149 and the control substances IMAB027 and DS9606a did not bind to huCLDN3-HEK293, huCLDN4-HEK293 and HEK293, indicating that the candidate antibodies A11 and A149 of the present invention have good specificity.

[0287] Example 8 Humanized transformation of candidate nanobodies

[0288] In this example, in order to reduce the immunogenicity that may be caused by camel-derived nanobodies A11 and A149, the framework region of the nanobody VHH was humanized and mutated, and the degree of humanization of the antibody sequence was improved by back mutation.

[0289] The antibody sequences of A11-VHH and A149-VHH were compared with the human antibody germline database to identify 1-3 germline sequences with high homology to the maternal version. Taking into account the drugability of the germline sequences, appropriate germline templates were selected for alignment and homology modeling was performed. Homology modeling was performed with reference to the nanobody models from the PDB database (http: / / www.rcsb.org / ). Combining the structural model and the presence of non-human sites, combined backmutation design was performed to avoid the introduction of potential post-translational modification sites. Antibody sequences with varying degrees of humanization were designed for A11-VHH and A149-VHH, respectively. The degree of humanization for each sequence is shown in Tables 1 and 2.

[0290] Table 1A149-VHH antibody humanization design and data results

[0291] Clone number Humanization ratio Number of non-human loci A149-VHH 0.918699 10 A149-VHH2 0.95122 6 A149-VHH3 0.95935 5 A149-VHH4 0.96748 4 A149-VHH5 0.98374 2 A149-VHH6 0.99187 1

[0292] Table 2A11-VHH antibody humanization design and data results

[0293] Clone number Humanization ratio Number of non-human loci A11-VHH 0.926829 9 A11-VHH1 0.95122 6 A11-VHH2 0.95935 5 A11-VHH3 0.95935 5 A11-VHH4 0.96748 4 A11-VHH5 0.98374 2

[0294] Example 9 Humanized Antibody Construction, Expression and Purification

[0295] Humanized antibodies fused to human IgG1 Fc at the C-terminus were prepared by the method of Reference Example 4. The prepared antibodies were named A11-VHH2-Fc, A11-VHH3-Fc, A11-VHH4-Fc, A11-VHH5-Fc, A11-VHH6-Fc, A149-VHH1-Fc, A149-VHH2-Fc, A149-VHH3-Fc, A149-VHH4-Fc and A149-VHH5-Fc, respectively.

[0296] Example 10 Detection of affinity activity of humanized antibodies based on FACS method

[0297] This example tested the binding ability of the humanized antibody to the human CLDN6-overexpressing cell line huCLDN6-CHO. The experimental method is shown in Example 5.

[0298] The experimental results are as follows Figures 6A-6B As shown, Figure 6A The binding ability of the humanized antibodies to huCLDN6-CHO was comparable to that of the parent antibody A11, but weaker than that of the positive control antibody DS9606a; Figure 6B The results showed that the binding ability of humanized antibodies to huCLDN6-CHO was better than that of the parent antibody A149, but weaker than that of the positive control antibody DS9606a.

[0299] Example 11 Humanized Nanobody Candidate Molecule Antibody Engineering

[0300] This example mainly describes antibody engineering of humanized Nanobody molecules A11-VHH5 and A149-VHH4 to obtain high-affinity antibody molecules that bind to both human CLDN6 and human CLDN9, or to obtain antibody molecules that differentially bind to human CLDN6 / human CLDN9.

[0301] 11.1 Antibody Engineering Library Design and Construction

[0302] The antibody engineering library is designed to perform mutation design on the antibody CDR region. The mutation methods include single-point saturation mutation and 2-3-point continuous mutation strategy. The mutations of different CDRs are combined to construct a mutation combination library.

[0303] The specific library construction method is as follows: first, primers containing point mutations are synthesized (synthesis company: Jinweizhi Biotechnology Co., Ltd.); second, the molecule to be modified is used as a PCR amplification template to amplify the sequence of the CDR containing the designed mutation. The fragments containing different mutations are combined by the bridge PCR method. The combined complete VHH antibody is inserted into the nanobody phage display vector by enzyme ligation. Electroporation, library capacity calculation and phage library preparation are performed. The operation process is detailed in the library construction section of Example 3.

[0304] 11.2 Screening of Antibody Engineering Libraries

[0305] The specific operation method of library screening is detailed in Example 3, which includes library screening, primary screening, affinity sorting and sequence analysis.

[0306] Example 12 Construction, expression, purification and physicochemical property testing of antibodies after antibody engineering

[0307] 12.1 Construction, Expression, and Purification of Antibodies After Antibody Engineering

[0308] The construction, expression and purification of the engineered antibodies are described in Example 4. Affinity matured engineered molecules A11-11, A11-32, A11-48, A11-68, A11-71, A149-32, A149-38, A149-53 and A149-80 were obtained.

[0309] 12.2 SDS-PAGE Identification of Antibodies After Antibody Engineering

[0310] Preparation of non-reducing solution: 1 μg of candidate antibody and quality control IPI (i.e., ipilimumab) was added to 5× SDS loading buffer and 40 mM iodoacetamide, heated in a 75°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.

[0311] Preparation of reducing solution: 2 μg of candidate antibody and quality control IPI were added to 5× SDS loading buffer and 5 mM DTT, heated in a dry bath at 100°C for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.

[0312] The supernatant was added to a 4-15% Bis-tris gradient gel (GenScript Biotech Co., Ltd.) for gel electrophoresis and visualized with Coomassie Brilliant Blue staining. The protein gel with the visualized protein bands was scanned using an EPSON V550 color scanner (destained with destaining buffer until the gel background was transparent). The purity of the reduced and non-reduced bands was calculated using ImageJ using peak area normalization. The results are shown in Table 3.

[0313] Table 3 Physicochemical properties of 14 candidate molecules for antibody engineering

[0314] name MW Isoelectric point Extinction coefficient Flash transfer (10mL) SDS-PAGE IMAB027 150 8.56 1.46 0.30 A11-11 78.58 8.44 1.74 5.04 >95.0 A11-32 78.56 8.44 1.74 7.04 >95.0 A11-48 78.72 8.61 1.74 4.30 >95.0 A11-68 78.5 7.95 1.74 3.40 >95.0 A11-71 78.44 8.44 1.74 1.44 >95.0 A149-32 78.8 8.23 1.74 3.24 >95.0 A149-38 79.04 8.44 1.77 0.4 98.6 A149-53 78.98 8.61 1.73 3.14 >95.0 A149-80 78.98 8.44 1.73 2.94 93.1 A11-VHH5-Fc 78.48 8.44 1.74 6.94 >95.0 A149-VHH4-Fc 78.92 8.44 1.73 2.2 >95.0

[0315] Example 13 Affinity Activity Assessment of Antibody Engineering Candidate Molecules

[0316] This example uses FACS to test the binding ability of engineered candidate antibodies to huCLDN6-CHO, huCLDN9-HEK293, huCLDN3-HEK293, huCLDN4-HEK293, PA-1 (human ovarian teratoma cells), and HEK293. For experimental methods, see Examples 5-7. IMAB027 was used as a positive control, and IgG1 and / or anti-Claudin-3 PE (1:500) or anti-Claudin-4 APC (1:500) were used as nonspecific controls.

[0317] The results of huCLDN6-CHO cell level binding assay are shown in Figures 7A-7B As shown, A11-11 and A11-68 have improved binding activity at the huCLDN6-CHO cell level relative to A11-VHH5-Fc, and their binding activity is better than that of IMAB027 ( Figure 7A ); A149-32, A149-38, A149-53, and A149-80 showed improved binding activity relative to A149-VHH4-Fc in huCLDN6-CHO cells, and the binding activity of A149-80 was superior to that of IMAB027 ( Figure 7B ).

[0318] The results of huCLDN9-HEK293 cell level binding assay are shown in Figures 8A-8B As shown, A11-11, A11-32 and A11-68 have improved binding activity at the huCLDN9-HEK293 cell level relative to A11-VHH5-Fc, while A11-48 and A11-71 have decreased binding activity at the huCLDN9-HEK293 cell level. The binding activity of the above molecules at the huCLDN9-HEK293 cell level is better than that of IMAB027 ( Figure 8A ); A149-53 and A149-80 have improved binding activity in huCLDN9-HEK293 cells relative to A149-VHH4-Fc, the binding activity of A149-38 in huCLDN9-HEK293 is comparable to that of IMAB027, and A149-32 does not substantially bind to huCLDN9-HEK293 ( Figure 8B ).

[0319] The results of the binding assays of huCLDN3-HEK293, huCLDN4-HEK293 and HEK293 cells are shown in Figure 2. Figures 9A-9D 、 Figures 10A-10D and Figures 11A-11B As shown, the molecules after antibody engineering all had weak binding to huCLDN3-HEK293 and huCLDN4-HEK293 ( Figures 9A-9D and Figures 10A-10D ); the engineered molecules did not bind to HEK293 ( Figures 11A-11B ).

[0320] The results of the binding assay at the PA-1 (human ovarian teratoma) cell level were as follows Figures 12A-12D As shown, A11-11, A11-48, A11-VHH5-Fc, A11-32, A11-68, A149-38, A149-53, and A149-80 bound to EC in PA-1 cells. 50 and upper platforms are superior to IMAB027.

[0321] Example 14: Evaluation of species cross-activity of candidate antibody engineering molecules

[0322] This example uses FACS to test the binding ability of engineered candidate antibodies to cynoCLDN6-HEK293 (monkey) and musCLDN6-HEK293 (mouse). For experimental methods, see Examples 5-7.

[0323] The results of the binding assay in cynoCLDN6-HEK293 cells were as follows: Figure 13AAs shown, A11-11, A11-68, A49-32, and A149-38 bind to EC in cynoCLDN6-HEK293 cells. 50 and upper platforms were superior to or equivalent to IMAB027.

[0324] The results of the binding assay in musCLDN6-HEK293 cells were as follows: Figure 13B As shown, A11-11, A11-68, A49-32, and A149-38 bind to EC in musCLDN6-HEK293 cells. 50 and upper platforms were superior to or equivalent to IMAB027.

[0325] Example 15 Evaluation of epitope competition activity of candidate antibody engineering molecules

[0326] This example uses FACS to test the competition of engineered candidate antibodies against DS9606a on huCLDN6-CHO cells. HuCLDN6-CHO cells in the exponential growth phase were collected, centrifuged at 300 g, and the supernatant removed. The cells were resuspended in FACS buffer (PBS containing 1% BSA), counted, and the cell suspension density was adjusted to 2×10 6 Cells were then plated at 100 μL per well in a 96-well round-bottom plate and centrifuged to remove the supernatant. 70 μL of serially diluted candidate antibodies A149-32, A149-38, A11-11, and A11-68 (300 nM in the first well), along with 70 μL of biotinylated DS9606a (DS9606a-Biotin) were added and incubated at 4°C for 30 minutes. The antibodies and biotinylated antibodies were mixed, resuspended, and incubated at 4°C for 30 minutes. 100 μL of this mixture was added to the corresponding wells, mixed, and incubated at 4°C for 60 minutes. The incubated cell mixture was washed twice, and then PE-labeled Streptavidin-PE (Invitrogen 12-4317-087) was added, resuspended, and incubated at 4°C for 30 minutes. The incubated cell mixture was washed three times and then resuspended in 200 μL of FACS buffer. The cells were analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). The data were analyzed using PRISM™ (GraphPad Software, San Diego, CA).

[0327] FACS binding assay results were as follows Figure 14As shown, the antibodies A149-32, A149-38, A11-11, A11-68, and the control DS9606a all inhibited the expression of DS9606a-Biotin in huCLDN6-CHO cells, indicating that the candidate antibodies of the present invention compete with DS9606a for the epitope, i.e., the antibodies A149-32, A149-38, A11-11, and A11-68 bind to the same epitope as DS9606a.

[0328] Example 16 Preparation of Antibody Drug Conjugate (ADC)

[0329] In this example, antibody A149-32 and control antibodies IMAB027 and DS9606a were conjugated to the linker-toxin MC-VC-PAB-MMAE (which has anticancer activity and is composed of MMAE (Monomethyl Auristatin E, a microtubule inhibitor) and Vc) at the thiol group of cysteine ​​to produce an antibody-drug conjugate (ADC). VHH-IgG1 / Fab-IgG antibodies have multiple pairs of cysteine ​​residues, which exist in the form of disulfide bonds. Interchain disulfide bonds are solvent-accessible and can be reduced by reducing agents to form thiol groups, which then become conjugation targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015; 17: 339-51).

[0330] The specific preparation method is as follows:

[0331] Antibody A149-32 and control antibodies IMAB027 and DS9606a were taken out of the -80°C freezer, thawed, and transferred to 15 mL 30KD ultrafiltration centrifuge tubes. Coupling buffer (per 1 L: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, diluted to 1000 g with purified water, pH 7.4) was added to 15 mL. The tubes were centrifuged at 4500 rpm for approximately 30 min, concentrated to 2-3 mL, and refilled with dialysate (per 1 L: histidine 0.73 g, histidine hydrochloride monohydrate 1.12 g, diluted to 1000 g with purified water, pH 6.0) to 15 mL. The dialyzed tubes were repeated 8-10 times to obtain the antibody stock solution, and the antibody concentration after dialysis was detected.

[0332] To the reduction reaction system, the antibody stock solution, 10 mM disulfide bond reducing agent TCEP stock solution (i.e., tris(2-carboxyethyl)phosphine hydrochloride stock solution, content per 1L: TECP 2.86 g, Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, diluted to 1000 g with purified water), 10 mM DTPA stock solution (i.e., diethylenetriaminepentaacetic acid stock solution, content per 1L: DTPA 3.90 g, NaOH 1.20 g, diluted to 1000 g with purified water), and coupling buffer were added sequentially to achieve an antibody concentration of 5 mg / mL, a DTPA concentration of 1 mM, and a molar ratio of TCEP to A149-32 or control antibody IMAB027 or DS9606a of 2. After thorough mixing, the mixture was placed in a 25°C constant temperature mixer at 400 rpm for 2 hours.

[0333] Weigh MC-VC-PAB-MMAE (Medchemexpress) and dissolve it in DMSO to prepare a 5 mM MC-VC-PAB-MMAE stock solution. After the reduction reaction is complete, add the MC-VC-PAB-MMAE stock solution to the reaction system in an ice-water bath. Mix thoroughly and place in a thermomixer at 25°C at 400 rpm for 1 hour for the coupling reaction.

[0334] After conjugation, the ADC sample was centrifuged and then filtered, then transferred to a 15 mL 30KD ultrafiltration centrifuge tube. The sample was then filled with dialysate to 15 mL, centrifuged at 4500 rpm for 20 min, concentrated to 2-3 mL, and refilled with dialysate to 15 mL. The dialysis was repeated 8-10 times. Table 4 shows the test results for the ADC conjugate affinity, where DAR represents the average number of drug molecules conjugated per antibody molecule in the resulting antibody-drug conjugate (ADC) mixture.

[0335] Table 4 Quality inspection information of antibody drug conjugates (ADCs)

[0336] ADC DAR(UV) DAR(HIC) IMAB027 3.6 4.3 DS9606a 3.3 3.9 A149-32 3.26 3.31

[0337] The test data in Table 4 show that the average number of drug molecules coupled to each antibody molecule in the generated antibody-drug conjugate (ADC) mixture was substantially the same when detected using ultraviolet (UV) and hydrophobic interaction chromatography (HIC). For the candidate antibody A149-32, the DARs measured using these two methods were 3.26 and 3.31, respectively, indicating that an average of approximately 3-4 drug molecules were coupled to each antibody molecule in the generated antibody-drug conjugate (ADC) mixture.

[0338] Example 17 In vivo tumor inhibition evaluation of candidate antibody ADC molecules in mice

[0339] 6-8 week old female nude mice (purchased from Shanghai Weitonglihua, strain: BALB / c Nude) were used. The experimental mice were housed in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 21-24°C and the humidity was 30-53%. 6 OV90 ovarian cancer cells were injected subcutaneously on the right side of the back of each mouse (day 0). On day 16, the mice were randomly divided into three groups (5 mice per group): PBS treatment group, A149-32-MMAE (DAR3.3) treatment group, IMAB027-MMAE (DAR4.3) treatment group, and DS9606a-MMAE (DAR3.9) treatment group. Each treatment group was set with equimolar mass of antibody. The antibody-conjugated ADC drug was injected via tail vein (ip) for a total of 3 doses, once a week.

[0340] The results are as follows Figures 15A to 15C As shown: All ADC-treated groups showed tumor growth inhibition relative to the PBS-treated group; tumor volume curves and tumor mass indicated that at equimolar concentrations, the tumor inhibitory effect of A149-32-MMAE (DAR3.3) was comparable to that of IMAB027-MMAE (DAR4.3) and DS9606a-MMAE (DAR3.9), and there was no significant change in mouse body weight.

[0341] It will be clear to those skilled in the art that many modifications and variations of the present invention may be made without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not intended to be limiting in any way. The true scope and spirit of the present invention are shown by the appended claims, and the description and examples are merely exemplary.

[0342] Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central features. Since the foregoing description of the present invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims for an indication of the scope and content of the present invention.

Claims

1. An isolated single domain antibody or antigen binding fragment thereof, which specifically binds to CLDN6, wherein the isolated single domain antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises the following CDR1, CDR2 and CDR3: (a) comprising SEQ ID NO: 1-3, respectively, or (b) comprising SEQ ID NO: 1 and 4-5, respectively, or (c) comprising SEQ ID NO: 1 and 6-7, respectively, or (d) comprising SEQ ID NO: 8-9 and 3, respectively, or (e) comprising SEQ ID NOs: 10-11 and 3, respectively, or (f) comprising SEQ ID NO: 12 and 2-3, respectively, or (g) comprising SEQ ID NOs: 13-14 and 3, respectively, or (h) respectively comprising SEQ ID NO: 15-17, or (i) comprising SEQ ID NO: 1 and 18-19, respectively, or (j) comprising SEQ ID NO: 1 and 20-21, respectively, or (k) comprise SEQ ID NOs: 1 and 22-23, respectively.

2. The isolated single domain antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the following CDR1, CDR2 and CDR3: (a) as shown in SEQ ID NO: 1-3, respectively, or (b) as shown in SEQ ID NO: 1 and 4-5, respectively, or (c) as shown in SEQ ID NO: 1 and 6-7, respectively, or (d) as shown in SEQ ID NO: 8-9 and 3, respectively, or (e) as shown in SEQ ID NO: 10-11 and 3, respectively, or (f) as shown in SEQ ID NO: 12 and 2-3, respectively, or (g) as shown in SEQ ID NO: 13-14 and 3, respectively, or (h) as shown in SEQ ID NO: 15-17, respectively, or (i) as shown in SEQ ID NO: 1 and 18-19, respectively, or (j) as shown in SEQ ID NO: 1 and 20-21, respectively, or (k) are shown in SEQ ID NOs: 1 and 22-23, respectively.

3. The isolated single domain antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-44, or The heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6, or The heavy chain variable region comprises an amino acid sequence having one or more amino acid additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 24-44 and retains the ability to specifically bind to CLDN6.

4. The isolated single domain antibody or antigen binding fragment thereof according to claim 1, wherein the isolated antibody is a camelid antibody, a humanized antibody or an affinity matured antibody.

5. An isolated single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen binding fragment thereof is fused to another molecule, wherein the other molecule is selected from the Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen binding fragment of an antibody, a drug molecule, an antibody-like molecule, an antigen binding fragment of an antibody-like molecule, or a fluorescent protein.

6. The isolated single domain antibody or antigen binding fragment thereof according to claim 5, wherein the antibody or antigen binding fragment thereof is fused to the Fc domain of human IgG (such as human IgG1 or human IgG4).

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. A vector comprising the nucleic acid molecule according to claim 7 .

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8.

10. An antibody drug conjugate (ADC) composition comprising a conjugate of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6 and a drug molecule.

11. The antibody drug conjugate (ADC) composition according to claim 10, wherein the drug molecule is a cytotoxic agent, such as a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent or an antimicrobial agent, preferably selected from a microtubule inhibitor, a topoisomerase inhibitor or a DNA binding agent, more preferably selected from MMAE, MMAD or MMAF.

12. A pharmaceutical composition comprising at least one single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier; or comprising the antibody drug conjugate (ADC) composition according to claim 10 or 11 and a pharmaceutically acceptable carrier.

13. A method for preparing the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, comprising the following steps: - expressing the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in a host cell according to claim 9; and - isolating said single domain antibody or antigen-binding fragment thereof from said host cell.

14. Use of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, the antibody drug conjugate (ADC) composition according to claim 10 or 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing or treating a disease associated with CLDN6 in a subject, wherein the subject is a human or a non-human primate, preferably a human.

15. The use according to claim 14, wherein the disease associated with CLDN6 is selected from ovarian cancer, endometrial cancer, gastric cancer, germ cell cancer, non-small cell lung cancer (NSCLC), liver cancer, myxofibrosarcoma or cervical cancer.

16. A kit for preventing or treating a disease associated with CLDN6 in a subject, comprising a container and instructions for use, wherein the container comprises at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the antibody-drug conjugate (ADC) composition according to claim 10 or 11, or the pharmaceutical composition according to claim 12.

Images