Antigen-binding protein for hcldn6 and use thereof

Abstract

Provided are an antigen-binding protein for hCLDN6 and the use thereof. Said antigen-binding protein comprises a heavy chain variable region. The heavy chain variable region has HCDR1 having an amino acid sequence of X1YTMS; HCDR2 having an amino acid sequence of TISSGGGX2TYYPDSVKG in which X2 = R, N, Q, D or E; and HCDR3 having an amino acid sequence of GDX4RYDX3FAY in which X4 = Y, N or Q, wherein (1) X1 = Y, M, Q, D or E, and X3 = G; or (2) X1 = S or D, and X3 = A. Provided are novel humanized antibodies targeting CLDN6, and different compositions based on these antibodies, which have better cell proliferation inhibitory activity and tumor inhibitory activity, thus providing novel means for cancer diagnosis and treatment.

Description

hCLDN6 antigen binding protein and its use Technical Field

[0001] The present invention relates to an hCLDN6 antigen-binding protein, a nucleic acid molecule encoding the antigen-binding protein, a vector, a cell, a drug molecule, and uses thereof. Background Art

[0002] The Claudin (CLDN) family consists of 27 members, all of which are multi-transmembrane proteins. Some of these members participate in the formation of tight junctions. Tight junctions are a key form of cell adhesion. CLDN proteins, working with other extracellular and peripheral membrane proteins, form barriers between adjacent polarized epithelial and endothelial cells, preventing the transport of macromolecules. CLDN proteins also bind to signaling proteins and cytoskeletal proteins, participating in cell signaling. Among the members involved in tight junctions, CLDN6 is unique in that it is only expressed in embryos and is almost absent in normal adult tissues. CLDN6, on the other hand, is a tumor antigen specifically expressed in various cancers, including ovarian, testicular, non-small cell lung, endometrial, liver, pancreatic, and choriocarcinoma. CLDN6 may also participate in stem cell differentiation and is associated with cancer cell proliferation, apoptosis, migration and metastasis, and drug resistance. Inhibition of CLDN6 has been shown to slow tumor growth in mouse models.

[0003] Other members of the CLDN6 family are expressed to some extent in normal tissues. Therefore, antibodies with high selectivity for CLDN6 can improve the safety of antibodies and their compositions. CLDN9 shares the highest similarity with CLDN6, differing in sequence at only three amino acid residues in the extracellular region (as shown in Figure 1 , the extracellular terminal loop regions of human CLDN6 and human CLDN9 are underlined and contain two extracellular terminal loop regions, EL1 and EL2. EL1 for human CLDN6 and CLDN9 is 29-81 AA, and EL2 is 138-160 AA). CLDN9 is expressed in tissues such as the pituitary gland, nasal mucosa, and inner ear, and mutations in CLDN9 are associated with hereditary deafness. Therefore, antibodies with high selectivity for CLDN6, recognizing CLDN6 but not binding or weakly binding to CLDN9 or other CLDN family members, can improve the safety of antibodies and their compositions.

[0004] Furthermore, mouse antibodies have high immunogenicity, and humanization can reduce this, making them more suitable for clinical applications. However, humanization is complex, and humanized antibodies often exhibit reduced affinity, expression, and / or stability. Currently, there is no method that guarantees that a specific method will yield high-affinity humanized antibodies after humanizing mouse antibodies.

[0005] Summary of the Invention

[0006] The main purpose of the present application is to provide an antigen-binding protein that can specifically bind to CLDN6 and has good antigen-binding activity.

[0007] [Corrected 26.07.2024 according to Rule 91] A first aspect of the present invention relates to an isolated antigen-binding protein capable of binding to hCLDN6, comprising a heavy chain variable region, wherein the heavy chain variable region has:

[0008] HCDR1 with the amino acid sequence X1YTMS,

[0009] HCDR2 of the amino acid sequence TISSGGGX2TYYPDSVKG, X2=R, N, Q, D or E,

[0010] HCDR3 with the amino acid sequence of GDX4RYDX3FAY, X4=Y, N or Q,

[0011] in,

[0012] (1) X1 = Y, M, Q, D or E, X3 = G, or (2) X1 = S or D, X3 = A.

[0013] In some embodiments of the first aspect of the present invention, the heavy chain variable region comprises a human heavy chain framework region, wherein the human heavy chain framework region has one or more back mutations relative to a murine heavy chain framework region. The back mutation site in the human heavy chain variable region includes R44, and position numbering is determined by the Kabat numbering scheme.

[0014] In some embodiments of the first aspect of the present invention, the heavy chain variable region is: HFR1 to HCDR3 have 92.59% or more identity, have the said back mutation site, and have the said X1, X2, X3 and / or X4 site sequence.

[0015] In some embodiments of the first aspect of the present invention, the heavy chain variable region has a sequence shown in any one of SEQ ID No. 22, 23, 24, 59, 60, 61, 62, 63, 66, 67, 133 to 153, or a sequence having 92.59% or greater identity with HFR1 to HCDR3 of the heavy chain variable region.

[0016] In some embodiments of the first aspect of the present invention, the heavy chain framework region is a human IgG heavy chain framework region.

[0017] In some embodiments of the first aspect of the invention, the heavy chain variable region comprises a heavy chain framework region of murine origin.

[0018] In some embodiments of the first aspect of the present invention, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NO. 29, 30 or 45.

[0019] In some embodiments of the first aspect of the present invention, the invention further comprises a light chain variable region having an amino acid sequence shown in SEQ ID NO.5.

[0020] In some embodiments of the first aspect of the present invention, the invention further comprises a light chain variable region, wherein the light chain variable region comprises:

[0021] LCDR1 of amino acid sequence RASENI DS X5LA, X5 = Y, Q or R,

[0022] LCDR2 of the amino acid sequence ASTLLVD,

[0023] LCDR3 with amino acid sequence QHYYSX6PYT, X6=I or E.

[0024] In some embodiments of the first aspect of the present invention, the light chain variable region has a human light chain framework region, and the light chain framework region has one or more back mutations relative to the mouse light chain framework region, and the sites of the back mutations in the light chain framework region include one or more of R66, Q70, and R85, and the position numbers are determined by the Kabat numbering scheme.

[0025] In some embodiments of the first aspect of the present invention, the sequence having 95.88% or greater identity with HFR1 to HCDR3 of the light chain variable region has the back mutation site, and has the X5 and / or X6 site.

[0026] In some embodiments of the first aspect of the present invention, the light chain variable region has a sequence shown in any one of SEQ ID No. 25, 28, 154, 155, and 156, or has a sequence with 95.88% or more identity with HFR1 to HCDR3 of the light chain variable region.

[0027] In some embodiments of the first aspect of the present invention, the light chain framework region is a human IgG light chain framework region.

[0028] In some embodiments of the first aspect of the present invention, the heavy chain constant region and light chain constant region are human.

[0029] In some embodiments of the first aspect of the present invention, the heavy chain constant region is of human origin.

[0030] In some embodiments of the first aspect of the present invention, the constant region contains mutations at one or more sites.

[0031] In some embodiments of the first aspect of the present invention, the heavy chain constant region contains a LALA mutation.

[0032] In some embodiments of the first aspect of the present invention, the heavy chain constant region is a human IgG1 heavy chain constant region and contains one or more mutations of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.

[0033] In some embodiments of the first aspect of the present invention, the amino acid sequence of the heavy chain constant region is shown as SEQ ID No.9.

[0034] In some embodiments of the first aspect of the present invention, the amino acid sequence of the light chain constant region is shown as SEQ ID No.10.

[0035] In some embodiments of the first aspect of the present invention, the CDR sequences are determined according to the Kabat definition scheme.

[0036] In some embodiments of the first aspect of the present invention, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0037] In some embodiments of the first aspect of the present invention, the antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0038] In some embodiments of the first aspect of the present invention, the antigen-binding fragment is selected from a Fab antibody, a single-chain antibody or a single-domain antibody.

[0039] In some embodiments of the first aspect of the present invention, the heavy chain has a sequence as shown in any one of SEQ ID No. 157, 159 to 167, or a sequence having 92.59% or greater identity with HFR1 to HCDR3 of the heavy chain.

[0040] In some embodiments of the first aspect of the present invention, the heavy chain has a sequence as shown in any one of SEQ ID No. 158 and 171, or a sequence having 92.59% or greater identity with HFR1 to HCDR3 of the heavy chain.

[0041] In some embodiments of the first aspect of the present invention, the light chain has a sequence as shown in any one of SEQ ID No. 169 and 170, or a sequence having 95.88% or greater identity with HFR1 to HCDR3 of the light chain.

[0042] In some embodiments of the first aspect of the present invention, the light chain has a sequence as shown in SEQ ID No. 168, or a sequence having 95.88% or greater identity with HFR1 to HCDR3 of the light chain.

[0043] In some embodiments of the first aspect of the present invention, the antigen binding protein is the antibody hAb-13: the heavy chain variable region sequence is shown in SEQ ID No. 18, and the light chain variable region sequence is shown in SEQ ID No. 28.

[0044] The second aspect of the present invention relates to a nucleic acid molecule, characterized in that it is used to encode any of the antigen binding proteins described above.

[0045] The third aspect of the present invention relates to a vector, characterized in that it comprises the nucleic acid molecule.

[0046] The fourth aspect of the present invention relates to a cell comprising the nucleic acid molecule, and / or the vector.

[0047] The fifth aspect of the present invention relates to a drug molecule, characterized by comprising any one of the above-mentioned antigen-binding proteins.

[0048] In some embodiments of the fifth aspect of the present invention, a therapeutic agent or a detectable marker is further included that is linked to the antigen binding protein.

[0049] In some embodiments of the fifth aspect of the present invention, the therapeutic agent is selected from a cytotoxic agent, a cytostatic agent, a radioisotope, an anti-angiogenic agent or a liposome.

[0050] In some embodiments of the fifth aspect of the present invention, the therapeutic agent is an immunomodulator.

[0051] In some embodiments of the fifth aspect of the present invention, the cytotoxic agent is derived from a plant, fungal or bacterial molecule or a derivative thereof.

[0052] In some embodiments of the fifth aspect of the present invention, the cytotoxic agent is selected from a peptide toxin, a protein toxin, or an alkylating toxin.

[0053] In some embodiments of the fifth aspect of the present invention, the cytotoxic agent is selected from one or more of maytansine alkaloids, auristatins, eribulin, taxanes, calicheamicin, cemadin, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, α-amanitin and its derivatives, trabectedin and its derivatives, and rubectedin and its derivatives.

[0054] In some embodiments of the fifth aspect of the present invention, the therapeutic agent or detectable marker is coupled to the antigen binding protein via a cleavable or non-cleavable linker.

[0055] In some embodiments of the fifth aspect of the present invention, the linker has one or more linkers.

[0056] In some embodiments of the fifth aspect of the present invention, the linker is selected from one or more of an oligopeptide linker, a hydrazine linker, a thiourea linker, a triggered self-degradation linker, a succinimidyl trans-4-(maleimidomethyl)cyclohexane-1-carboxylate linker, a maleimide linker, a disulfide linker, a thioether linker, and an olefin linker.

[0057] In some embodiments of the fifth aspect of the present invention, the structure is as shown in Formula I:

[0058] Ab is the antigen binding protein, L is a linker containing one or more linkers, D is the therapeutic agent or detectable marker, and n is an integer selected from 1-20.

[0059] In some embodiments of the fifth aspect of the present invention, n is selected from an integer of 2-8.

[0060] The sixth aspect of the present invention relates to a pharmaceutical composition, characterized in that it comprises any of the above-mentioned antigen-binding proteins, the above-mentioned nucleic acid molecules, the above-mentioned vectors, the above-mentioned cells, and / or any of the above-mentioned drug molecules, as well as pharmaceutically acceptable carriers and / or additives.

[0061] A seventh aspect of the present invention relates to the use of any of the above antigen-binding proteins, nucleic acid molecules, vectors, cells, and drug molecules in the preparation of a drug, wherein the drug is used to diagnose, prevent, and / or treat hCLDN6-related diseases and / or conditions.

[0062] In some embodiments of the seventh aspect of the present invention, the disease and / or condition comprises a tumor.

[0063] In some embodiments of the seventh aspect of the present invention, the tumor is ovarian cancer, testicular cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, choriocarcinoma or endometrial cancer.

[0064] The present invention has produced highly selective humanized anti-CLDN6 antibodies with comparable or higher affinity than their murine parental antibodies. Compositions based on these antibodies have also been developed, such as ADCs combining different linkers and toxins. These antibodies and compositions provide new approaches for the diagnosis and treatment of tumors. In particular, anti-CLDN6 ADCs exhibit excellent anti-tumor activity and have potential for tumor treatment. The present invention provides novel humanized antibodies targeting CLDN6, as well as various compositions based on these antibodies, that exhibit enhanced cell proliferation inhibition and tumor suppression activities, providing new approaches for the diagnosis and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 shows the sequence alignment of human CLDN6 (hCLDN6) and human CLDN9 (hCLDN9). The extracellular EL1 and EL2 sequences are underlined.

[0066] FIG2 shows the binding activity of anti-CLDN6 mouse / human chimeric mAb and CDR-implanted humanized antibody to human ovarian cancer cell OV90.

[0067] Figure 3 shows the sequence alignment between the heavy chain variable regions (A) and the light chain variable regions (B) of a chimeric antibody, a CDR-implanted humanized antibody, and a representative humanized antibody with back mutations introduced after CDR implantation.

[0068] FIG4 shows the endocytic activity of humanized antibodies into which back mutations were introduced after selected CDR grafting on JEG-3 cells.

[0069] FIG5 shows the binding activity of cAb-2 to 31 to VLP-hCLDN6 (AB) and the endocytosis activity on JEG-3 cells (C).

[0070] FIG6 shows the binding activity detection of hAb-26-49 with VLP-hCLDN6 (AC, E) or VLP-hCLDN9 (D, F).

[0071] FIG7 schematically shows a process for producing antibody-drug conjugates (ADCs) using antibody interchain disulfide bonds and / or cysteine ​​mutation sites.

[0072] FIG8 shows the in vitro cytotoxicity of selected ADCs against human ovarian cancer cells OV90.

[0073] FIG9 shows the in vitro cytotoxicity of selected ADCs against human choriocarcinoma cell JEG3.

[0074] Figure 10 shows the efficacy of ADC-2, DAR2 (A) and ADC-46, DAR4 (B) in a xenograft model in female BALB / C nude mice established with hCLDN6-positive OV90 cells. DETAILED DESCRIPTION

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event of a conflict, the descriptions and definitions herein shall prevail.

[0076] In view of the problems existing in the prior art, the present invention transforms and humanizes murine antibodies to obtain antigen-binding proteins with higher hCLDN6 binding activity. The antigen-binding proteins involved in the present invention have the following features in the heavy chain variable region:

[0077] HCDR1 with the amino acid sequence X1YTMS,

[0078] HCDR2 of the amino acid sequence TISSGGGX2TYYPDSVKG, X2=R, N, Q, D or E,

[0079] HCDR3 with the amino acid sequence of GDX4RYDX3FAY, X4=Y, N or Q,

[0080] in,

[0081] (1) X1 = Y, M, Q, D or E, X3 = G, or (2) X1 = S or D, X3 = A.

[0082] The heavy chain variable region of the present invention involves specific mutations at one or more sites such as S31 and G100 in the HCDR region shown in SEQ ID Nos. 2 to 4. The resulting antigen binding protein has improved binding activity to hCLDN6.

[0083] The types of antigen-binding proteins include, but are not limited to, full-length antibodies, antibody fragments, immunoconjugates, antibody analogs, antibody derivatives, and fusion proteins. Antibodies include, but are not limited to, chimeric antibodies and humanized antibodies. Antibody fragments include, but are not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, VHH, and the like.

[0084] Chimeric antibodies generally refer to antibodies that have the V region (variable region) of a mouse monoclonal antibody and the C region (constant region) of a human immunoglobulin.

[0085] Humanized antibodies generally refer to antibodies formed by introducing the CDR sequences of mouse antibodies into the framework sequences of human VH and VL domains, respectively.

[0086] Chimeric and humanized antibodies can be produced using known methods. For example, a V region gene containing three CDRs and four FRs is amplified in full length using primers that anneal at the 5' and 3' ends and have appropriate restriction enzyme recognition sequences attached. The DNA obtained as described above is assembled into an expression vector with DNA encoding a human antibody C region for in-frame fusion. This recombinant vector is introduced into a host to establish recombinant cells, which are then cultured to express the antibody-encoding DNA to produce the antibody. For details, see Example 2.

[0087] The antibody forms include but are not limited to IgG, IgM, IgA, IgE, IgD, etc., with IgG being preferred. Furthermore, the IgG includes IgG1, IgG2, IgG3, IgG4, etc. In the constant regions of chimeric and humanized antibodies, heavy chains may include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε, etc., and light chains may include Cκ, Cλ, etc. LALA mutations may be introduced into the Fc region of the antibody to reduce binding of the antibody to Fcγ receptors and complement. Mutations in the heavy chain constant region include but are not limited to one or more mutations of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.

[0088] In the present invention, CDR is used to represent the complementarity determining region (or hypervariable loop) of an antibody. The amino acid sequence in the CDR determines the chemical structure and characteristics of the antibody antigen-binding site. The CDR in the heavy chain variable region (VH) is represented by HCDR, and the three heavy chain CDR regions are named HCDR1, HCDR2, and HCDR3 respectively; the CDR in the light chain variable region (VL) is represented by LCDR, and the three light chain CDR regions are named LCDR1, LCDR2, and LCDR3 respectively. It should be noted that the CDR regions in antibodies are generally predicted and identified through experiments. Unless otherwise specified, the sequences of the CDRs in the present invention are defined by the Kabat definition scheme, and the relevant amino acid numbering is determined by the Kabat numbering scheme. It should be understood that when different definition schemes are used to define CDRs, the predicted CDR region sequences may only partially overlap, or be shortened or lengthened. In antibody preparation, the CDR and FR gene sequences defined in different definition schemes can be concatenated and amplified to full length by known methods to further prepare antibodies or antibody fragments.

[0089] The antibody fragment Fab refers to the VH region of the heavy chain variable region, the CH1 functional region of the first constant region of the heavy chain, and the entire light chain connected by disulfide bonds, which mainly plays the antigen binding function of the antibody.

[0090] scFv is a single-chain antibody. Specifically, it refers to an antibody in which the light chain variable region gene and the heavy chain variable region gene are connected at the DNA level with a suitable oligonucleotide chain (linker), so that they are expressed in the form of a single peptide chain in a suitable organism and folded into a new antibody consisting only of the heavy chain and light chain variable regions. There are no special restrictions on the linker. For example, any single-chain peptide containing about 3 to 25 residues can be used as a connector.

[0091] dsFv is a disulfide-stabilized antibody, specifically an antibody formed by mutating one amino acid residue in the heavy chain variable region (VH) and light chain variable region (VL) of the antibody to cysteine, and then linking VH and VL through interchain disulfide bonds.

[0092] VHH is a heavy chain single-domain antibody, specifically an antibody containing only the VH functional domain but not the VL functional domain.

[0093] Bispecific antibodies are antibodies that have the binding properties of two antigens.

[0094] Fusion protein refers to the product of fusing antibody molecule fragments with other proteins (such as antibody enzymes, immunotoxins, immune cytokines, immunoadhesins, etc.) using genetic engineering technology.

[0095] Methods for preparing the various antibody fragments described above are known. One method involves enzymatic treatment of antibodies to produce antibody fragments. Enzymes used to produce antibody fragments include, but are not limited to, papain, pepsin, or plasmin. Another method involves constructing a gene encoding the antibody fragment, introducing the gene into an expression vector, and expressing the gene in an appropriate host cell to produce the antibody fragment.

[0096] In the present invention, CH is used to represent the heavy chain constant region of an antibody, and CL is used to represent the light chain constant region of an antibody. The heavy chain constant region generally further includes the CH1, CH2, and CH3 domains. FR is used to represent the framework of an antibody variable region. The framework region of the heavy chain variable region is represented by HFR, and the four heavy chain framework regions are named HFR1, HFR2, HFR3, and HFR4 respectively; the framework region of the light chain variable region is represented by LFR, and the four light chain framework regions are named LFR1, LFR2, LFR3, and LFR4 respectively.

[0097] The antibody derivatives in the present invention refer to products obtained by substituting, mutating, modifying, replacing, deleting and / or adding one or more amino acid residues in the antibody while retaining the endogenous function.

[0098] In the present invention, the binding activity of the antigen-binding protein is detected using existing methods, such as ELISA and flow cytometry, and can be detected by referring to the methods of Example 23 and Control Example 7.

[0099] The present invention employs existing methods for testing the endocytic activity of the prepared antigen-binding proteins. For example, a test antigen-binding protein containing a detectable marker is incubated with cells expressing CLDN6, and the fluorescence intensity elicited by the protein upon entry into the cells is measured by flow cytometry to analyze endocytic activity. See Example 24 for details.

[0100] The framework regions of the heavy chain variable regions can be derived from murine or human framework regions. The human heavy chain framework regions have one or more back mutations relative to the murine heavy chain framework regions. Preferred back mutation sites include R44, with position numbering determined by the Kabat numbering scheme.

[0101] In the antigen-binding protein, other heavy chain variable region sequences having 92.59% or greater identity with HFR1 to HCDR3 of the above-mentioned heavy chain variable region, having the above-mentioned back mutation site, and having the above-mentioned X1, X2, X3 and / or X4 sites can also be selected.

[0102] Preferably, the above antigen-binding protein further comprises a light chain variable region, wherein the light chain variable region comprises:

[0103] LCDR1 of amino acid sequence RASENI DS X5LA, X5 = Y, Q or R,

[0104] LCDR2 of the amino acid sequence ASTLLVD,

[0105] LCDR3 with amino acid sequence QHYYSX6PYT, X6=I or E.

[0106] The light chain variable region framework regions can be derived from murine or human framework regions, and can be combined with the corresponding heavy chain variable regions to form various types of antigen-binding proteins. The human light chain framework regions can have one or more back mutations relative to the murine light chain framework regions. Preferred back mutation sites include one or more of R66, Q70, and R85, with position numbering determined by the Kabat numbering scheme.

[0107] In the antigen-binding protein, other light chain variable region sequences having sequences with 95.88% or greater identity with the above-mentioned light chain variable region HFR1 to HCDR3 can also be selected.

[0108] The vectors of the present invention are used for the replication, transcription and / or translation of DNA or RNA. By cultivating cells containing the vectors, desired expression products can be produced. The selected vectors are known, such as plasmids.

[0109] Furthermore, the present invention also relates to drug molecules containing the aforementioned antigen-binding proteins. The drug molecules may comprise any known drug form, including but not limited to proteins, peptides, small molecule drugs, antibody-drug conjugates, or combinations of various drug forms.

[0110] Antibody-drug conjugates are antigen-binding proteins (usually antibodies or antibody fragments) coupled to therapeutic agents via a linker. Their structure can be shown in Formula I:

[0111] Ab is the antigen binding protein, L is a linker containing one or more linkers, D is the therapeutic agent or detectable marker, and n is an integer selected from 1-20, and further, can be an integer from 2-8.

[0112] Wherein, the connection between the linker and the drug is usually produced by a known chemical synthesis method to form a linker-therapeutic agent structure that can be bound to the antibody. In the preparation process of the antibody-drug conjugate, the antibody is coupled to one or more linker-therapeutic agent structures. The coupling of the antibody and the linker-therapeutic agent structure is usually established for the side chain of the lysine or cysteine ​​residue in the antibody. Wherein, for the conjugation of lysine, the linker usually has N-hydroxysuccinimide, which can react with the ε-amino group of the lysine to form a stable amide bond to form a coupling of the antibody and the therapeutic agent. For the conjugation of cysteine, the linker usually has a functional group such as a maleimide functional group, allenamide (allenamide), etc. that can mediate the coupling reaction. The cysteine ​​is reduced to a disulfide bond by a known method to produce a reactive thiol, thereby conjugating with maleimide to form a coupling of the antibody and the therapeutic agent. As can be seen in the methods exemplified in Examples 92 to 163 of the present invention.

[0113] Adoptable linkers include non-cleavable and cleavable ones. The linker may have one or more linkers, including but not limited to oligopeptide linkers (including cleavable and / or non-cleavable oligopeptide linkers), hydrazine linkers, thiourea linkers, triggered self-degradation (self-immolative) linkers, succinimidyl trans-4-(maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linkers, maleimide linkers, disulfide linkers, thioether linkers and / or olefin linkers.

[0114] Adoptable therapeutic agent can be cytotoxic agent, cytostatic agent etc.Cytotoxic agent can be molecule or its derivative from plant, fungus or bacterium, including but not limited to peptide toxin, protein toxin, alkylating agent toxin or other type toxin.Drug toxic agent can be selected from maytansine alkaloids (for example, maytansinol (maytansinol) or DM1 maytansine), auristatins (for example, monomethyl auristatin E or monomethyl auristatin F), eribulin, taxane, calicheamicin, cemadodine, pyrrolobenzodiazepine, anthracycline, camptothecin derivatives, α-amanitin and its derivatives, trabectedin and its derivatives, rubectedin and its derivatives One or more.The cytotoxic substance used in the present invention can be one, or two or more cytotoxic substances can be combined for use.

[0115] The therapeutic agent that can be used can also be a metabolite (for example, an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, cytarabine, or an analog of purine or adenosine); an intercalating agent (for example, an anthracycline such as doxorubicin, nemorubicin, or preferably a derivative of PNU-159682), daunorubicin, epirubicin, idarubicin, mitomycin-C, actinomycin D, or mithramycin, or other intercalating agents such as pyrrolobenzodiazepines; DNA-reactive agents such as calicheamicin, tiancimycins and other enediynes; platinum derivatives (for example, cisplatin or carboplatin); alkylating agents.

[0014] The therapeutic agent may be an anti-inflammatory agent (e.g., mechlorethamine, melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide nitrosourea, or thiotepa); an RNA polymerase inhibitor such as α-amanitin; an anti-mitotic agent (e.g., a vinca alkaloid such as vincristine, or a taxane such as paclitaxel or docetaxel); a topoisomerase inhibitor (e.g., etoposide, teniposide, amsacrine, topotecan, exitecan); a cell cycle inhibitor (e.g., flavopyridol); or an anti-microtubule agent (e.g., epothilone, tubulysine, pre-tubulysine, a picrotoxin analog, or a calendula analog). The therapeutic agent may be a proteasome inhibitor or a topoisomerase inhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin. Therapeutic radioisotopes include iodine (131I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi) and rhodium (Rh). Antiangiogenic agents include linoamide, bevacizumab, angiostatin and razoxane.

[0116] The therapeutic agent that can be used can be an immunomodulator, such as Sting agonist, TLR7, TLR8 and / or TLR9 agonist, PD-1 inhibitor, etc.

[0117] Drug molecules containing detectable markers can be used for diagnosis. Such as contrast agents can be included. Contrast agents can be radioisotope labels such as iodine (131I or 125I), indium (111In), technetium (99Tc), phosphorus (32P), carbon (14C), tritium (3H), other radioisotopes (such as radioactive ions), or therapeutic radioisotopes such as one of the therapeutic radioisotopes listed above. In addition, contrast agents can include radiopaque materials, magnetic resonance imaging (MRI) agents, ultrasonic imaging agents and any other contrast agents suitable for detection by the device of imaging animal body. The detectable marker can also be a fluorescent marker, a biologically active enzyme marker, a luminescent marker or a chromophore marker.

[0118] The present invention also relates to a pharmaceutical composition made from the above-mentioned antigen-binding protein, nucleic acid molecule, vector, cell, and pharmaceutically acceptable carrier and / or additive.

[0119] The pharmaceutical composition can be formulated according to conventional methods and can also contain pharmaceutically acceptable carriers and / or additives. The additives include, but are not limited to, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. Carriers for the pharmaceutical composition include, but are not limited to, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc.

[0120] Multiple antibodies may be incorporated into the above-described pharmaceutical compositions as needed. For example, by creating a mixture of multiple anti-CLDN6 antibodies, cytotoxicity against CLDN6-expressing cells may be enhanced. Alternatively, in addition to anti-CLDN6 antibodies, the therapeutic effect may be enhanced by incorporating antibodies that recognize other tumor-associated antigens.

[0121] The pharmaceutical composition can be administered orally or parenterally. Parenteral administration includes, for example, injection, nasal administration, pulmonary administration, transdermal administration, and the like. Injection administration can further include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. In addition, the dosage can be specifically adjusted considering the patient's age and symptoms. As for the dosage, for example, the dosage can be 0.0001 mg to 1000 mg per kg of body weight per administration. For another example, each administration for each patient is 0.001 mg to 100,000 mg. It should be noted that the above dosage is only for illustration and is not intended to limit the range of the dosage.

[0122] The type of cancer treated and diagnosed by the present invention is not particularly limited, and is generally a cancer expressing CLDN6 protein, preferably testicular cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, choriocarcinoma, or non-small cell lung cancer.

[0123] The present invention is further described below with reference to examples.

[0124] Example 1 Cell lines

[0125] Human ovarian cancer cells PA-1 and OV90, human triple-negative breast cancer cell line MCF-7, and human choriocarcinoma cell line JEG-3 were purchased from ATCC. OV90 cells were grown in a 1:1 MCDB 105 medium: medium 199 (Thermo Scientific; Logan, UT) supplemented with 15% (v / v) heat-inactivated FBS (Thermo Scientific; Logan, UT).

[0126] PA-1, JEG-3, and MCF-7 cell lines were grown in MEM (Thermo Scientific; Logan, UT) supplemented with 10% (v / v) heat-inactivated FBS (Thermo Scientific; Logan, UT), 1% (v / v) nonessential amino acids, and 1 mM sodium pyruvate to support adherent culture.

[0127] Example 2 Expression and purification of antibodies

[0128] The variable region coding region of the antibody was prepared by whole gene synthesis using MHSSALLCCLVLLTGVRA as a leading signal peptide and assembled with the human IgH-γ1 and IgL-κ coding regions through NotI / XbaI restriction sites into the expression vector pcDNA3.4. This vector has a CMV promoter that can enhance the high-level expression of multiple genes in mammalian cells and contains an ampicillin resistance gene for selective marker.

[0129] CHO-K1 cells were selected as the host for antibody expression. Cells were passaged three times under appropriate conditions (120 rpm, 8% CO₂, 37°C). Cells were harvested and mixed with electroporation buffer. Plasmids were then added, mixed, and transferred to electroporation tubes. The pcDNA3.4 expression vector was transfected into CHO-K1 cells using an electroporator. After 24 hours of incubation at 37°C, 270 rpm, and 8% CO₂, feed / sodium butyrate / dual antibody were added and cultured for 3-7 days. The harvested supernatant was filtered (0.22 μm) to remove cells and used for subsequent purification.

[0130] The treated supernatant was purified using a Protein A affinity chromatography column, followed by elution with citrate buffer (pH 3.4). The eluate was then placed in a dialysis bag for buffer exchange, ultimately yielding the purified AT03-2A antibody. The concentration and quantity of the antibody were determined by measuring the absorbance at A280 nM using a NanoDrop instrument. Purity was assessed by SDS-PAGE and SEC-HPLC, revealing a purity of >95%.

[0131] Each culture medium was supplemented with 100 U / mL penicillin and 100 mg / mL streptomycin (Sigma Aldrich).

[0132] Control Example 1 Mouse / human chimeric antibody cAb-1

[0133] According to the method of Example 2, mouse / human chimeric antibody cAb-1 was prepared.

[0134] The sequences of the complementarity determining regions (CDRs) in the heavy chain variable region and light chain variable region of the above-mentioned antibody are as follows:

[0135] Under different definition schemes, the CDR region sequences are as follows:

[0136] Antibody DTDH4 (INFECTION AND IMMUNITY, June 2006, p. 3682–3683) was used as an isotype control. This antibody is an anti-human DT antibody.

[0137] Control Examples 2 to 6 Humanized Antibodies hAb-1 to 5

[0138] According to the method of Example 2, the CDRs of Control Example 1 were implanted into the humanized framework by CDR transplantation to obtain humanized antibodies hAb-1 to 5. The variable region sequences of hAb-1 to 5 are shown in the following table.

[0139] Control Example 7: Binding Activity Detection of hAb-1 to 5 and cAb1 to hCLDN6-Positive Human Ovarian Cancer Cell Line OV90

[0140] Flow cytometry: cells were stained using standard flow cytometry methods. Briefly, for the antibodies hAb-1 to 5 and cAb1 to be tested, 4×10 5 hCLDN6-positive human ovarian cancer cell line OV90 cells were stained with serial dilutions of the test antibody (starting at 100 nM and diluted fourfold to 0.006 nM) on ice for 1 hour. After washing twice with ice-cold PBS containing 1% (v / v) FBS, the cells were incubated on ice for 1 hour with FITC-conjugated goat anti-human IgG (Abcam) in 100 μL of a 1:200 dilution of 1% (v / v) FBS in PBS. Cell-bound staining was analyzed using a flow cytometer (Agilent).

[0141] As shown in FIG2 , the CLDN6 mouse / human chimeric monoclonal antibody cAb-1 has strong binding activity to OV90 cells. However, the CDR-implanted humanized antibodies hAb-1 to hAb-5 almost completely lose their binding activity to OV90 cells.

[0142] Examples 3-22 Humanized antibodies hAb-6-25 with back mutations introduced after CDR grafting

[0143] Select appropriate human germline sequences and introduce back mutations for humanization design. The heavy chain variable region VH and light chain variable region VL sequences with back mutations include:

[0144] FIG3A shows the sequence alignment between heavy chain variable regions VH, and FIG3B shows the sequence alignment between light chain variable regions VL.

[0145] The method for small-scale expression of humanized antibodies with back mutations is as follows: Remove LVTransm transfection reagent and antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Remove PBS and warm to room temperature. Add 2 mL of PBS to one well of a 6-well plate and add 2 μg of pcDNA3.4-IgG1 and 2 μg of pcDNA3.4-IgKc. Mix thoroughly by pipetting up and down. Then add 12 μL of LVTransm and immediately mix by pipetting up and down. Let stand at room temperature for 10 minutes. Add the DNA / LVTransm complex to 3 mL of 293F cells and gently shake to mix thoroughly. Continue culturing the cells at 37°C, 5% CO2. After 48 hours of continuous culture, collect the supernatant by centrifugation and use it for ELISA to detect antigen binding. Antibodies are listed in the table below.

[0146] Example 23 Detection of Binding Activity of cAb-1 and hAb-6-25 to VLP-hCLDN6

[0147] Dilute AT-003-ag1 and the control protein to 4 μg / ml in sterile CBS (0.035 mol / L NaHCO₃, 0.015 mol / L Na₂CO₃). Add 100 μL / well of this solution to a new 96-well plate and coat overnight at 4°C. Incubate the plate at room temperature for 10 minutes, remove the antigen coating solution, and wash once with PBS (pH 7.4). Add 300 μL / well of blocking buffer (4% skim milk powder in PBS) and block at 37°C for 2 hours. Remove the blocking buffer and wash once with PBS (pH 7.4). Add varying volumes of the aforementioned cAb-1 and hAb-6-25 (total volume 100 μL) and incubate at 37°C for 1 hour. Control wells contain PBS. Remove the liquid from the wells and wash three times with PBST (0.1% Tween-20 in PBST, pH 7.4); then wash three times with PBS. Add 100 μL of HRP-Protein A (1:10,000 dilution) and incubate at 37°C for 1 hour. Remove the liquid from the wells and wash three times with PBST (0.1% Tween-20 in PBST, pH 7.4); then wash three times with PBS. Add 100 μL / well of TMB colorimetric solution and incubate at room temperature in the dark for 10 minutes. Add 50 μL / well of stop solution (2 M HCl). Read the OD450 value of the wells using a microplate reader. The results are shown in the table below:

[0148] As shown in the table above, these humanized antibodies all maintained good VLP-hCLDN6 activity. Compared to hAb-1-5 (Control Examples 2-6), these humanized antibodies retained the murine heavy chain framework region R44 in the VH0 region and the light chain framework region R66 in the VL0 region (Kabat numbering system), suggesting that backmutations at these two sites may be crucial.

[0149] Example 24 Detection of endocytic activity in human choriocarcinoma cell JEG3 cells with humanized antibodies introducing reverse mutations

[0150] The humanized antibodies hAb-6-25 with back mutations were expressed and purified according to the method of Example 2. Humanized antibodies with a culture volume exceeding 2 ml were selected. The selection results are shown in the following table.

[0151] The endocytic activity of human choriocarcinoma cell JEG3 was detected based on the selected humanized antibodies. Human choriocarcinoma cell JEG3 cells were cultured in a constant temperature incubator (37°C, 5% CO2) overnight. The selected antibodies were labeled with pHrodo iFL Green human IgG reagent, and the antibody-pHrodo iFL Green human IgG conjugates were incubated with human ovarian cancer cell PA-1 and human choriocarcinoma cell JEG3 cells for 24 hours. The highest concentration of the antibody was set to 10μg / ml and a 4-fold serial dilution was performed. After incubation, the cells were washed with PBS and resuspended in culture medium. The fluorescence intensity excited by pHrodo iFL Green entering the cells was measured on a NovoCyte flow cytometer to analyze the endocytic activity of the antibody.

[0152] As shown in Figure 4, these humanized antibodies all retained good endocytic activity. Among them, hAb-13, whose variable region combination was VH2VL4, showed higher endocytic activity than the mouse / human chimeric antibody cAb-1.

[0153] Examples 25-54 Murine / human chimeric antibodies cAb-2-31 containing heavy chain CDR region mutations

[0154] Based on cAb-1, different sites in the heavy chain CDR region were selected for mutation design. The designed antibodies, along with their final concentrations and buffers, are shown in the table below.

[0155] Example 55 Detection of Binding Activity of cAb-2-31 and VLP-hCLDN6

[0156] ELISA: For ELISA, each well of a 96-well 3590 plate (Corning, NY) was coated with 100 μL of coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.5) containing 400 ng of VLP-CLDN6 at 4°C overnight. After blocking with 150 μL of PBS buffer containing 4% (w / v) skim milk powder at 37°C for 2 h, the supernatant was discarded and the wells were patted dry on a clean paper towel. Then, 100 μL of a 4-fold serial dilution of the test antibody (cAb-1, cAb-2, cAb-3, cAB-18) was added to each well at 37°C as the primary antibody. After incubation at 37°C for 1 hour, the wells were washed three times with 1% PBST, the supernatant was discarded, and the wells were drained on a clean paper towel. Then, 100 μL of PBS buffer containing 1.25 ng / mL horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc (Invitrogen, California, CA) and 4% (w / v) skim milk powder were added to each well and incubated at 37°C for 45 minutes. After washing three times with 1% PBST and draining on a clean paper towel, the color reaction was developed using TMB color development solution (Beyotime, Shanghai, CN) according to the manufacturer's instructions. The wells were incubated at room temperature in the dark for 15 minutes until the desired color developed, and then the cells were stopped with TMB color development solution. Absorbance was measured at 450 nm using a SpectraMax i3X microplate reader (Molecular Devices; Sunnyvale, CA). The results are shown in Figure 5. Some specific EC50 values ​​are shown in the table below.

[0157] As shown in Figure 5 and the table above, cAb2, 3, and 18 all maintained good binding activity to VLP-hCLDN6, among which cAb-3 with the S31D mutation site significantly improved its binding activity to VLP-hCLDN6.

[0158] Examples 56-62 Antibodies containing heavy chain CDR region mutations and introduced back mutations hAb-26-30, 33-34, 44-49

[0159] Mutations were introduced into S31 and G100 of the heavy chain, and different humanized frameworks containing back mutations were implanted to obtain different humanized antibodies containing single mutations, double mutations, or multiple mutations, as shown in the table below.

[0160] Control Examples 8 to 18: Antibodies containing other heavy chain CDR region mutations and introduced back mutations

[0161] Different mutations from those in Examples 56 to 62 were introduced into the CDR region of the heavy chain, and a humanized VH2VL4 framework containing back mutations was implanted. Alternatively, S31D / G100A mutations were introduced into the CDR region of the heavy chain, and additional humanizing mutations were introduced into the framework region of VH2VL4 to obtain different humanized antibodies containing single mutations, double mutations, or multiple mutations, as shown in the table below.

[0162] Example 63 Detection of Binding Activity of hAb-26-39, 44-49 with VLP-hCLDN6 or VLP-hCLDN9

[0163] The binding activities of cAb-1, hAb-26-39, 44-49 and VLP-hCLDN6 or VLP-hCLDN9 were detected and compared using the detection method of Example 28. The detection results are shown in Figure 6.

[0164] As shown in Figures 6A-C, 6E, and the table above, when CDRs containing single mutations S31Y, S31M, S31Q, S31D, S31E, or G100A were combined with the human VH2VL4 framework region, the binding activity of antibodies to VLP-hCLDN6 was improved to varying degrees compared to the mouse / human chimeric antibody. The affinity of the humanized antibody containing the S31D / G100A double mutation was also comparable to that of the mouse / human chimeric antibody. On the other hand, although CDRs containing G100L and G100I mutations combined with the original mouse framework region produced antibodies with comparable or better affinity and internalization, CDRs containing these mutations combined with the human VH2VL4 framework region nearly lost their binding activity to VLP-hCLDN6. This indicates that the G100 position is crucial in humanized antibodies based on the human VH2VL4 framework region, and that the G100A mutation helps maintain or improve the activity of humanized antibodies.

[0165] As shown in Figures 6D and 6F, the humanized antibodies based on the human VH2VL4 framework regions had very low binding activity to VLP-hCLDN9, indicating that these antibodies maintained or improved their selectivity for CLND6.

[0166] Examples 64-91 Antibodies containing heavy chain CDR region mutations and introduced back mutations hAb-50-84

[0167] Mutations were introduced into S31 and G100 of the heavy chain, and a humanized VH2VL4 framework containing back mutations was implanted. On this basis, new mutations were introduced to obtain different humanized antibodies containing single mutations, double mutations or multiple mutations, as shown in the table below.

[0168] Examples 92-163: Generation of ADC-1-72 using antibody interchain disulfide bonds and / or cysteine ​​mutation sites

[0169] The following ADCs 1 to 15 were prepared by coupling 1 to 20 anti-tumor toxin molecules through the interchain disulfide bond sites of the antibody:

[0170] FIG7 (A) shows the overall method for preparing antibody-drug conjugates using maleimide linkers. The specific coupling process is as follows: Take a 0.5 ml centrifuge tube, add 2 mg of antibody, add 30 mM His-HAC (pH 5.5) buffer to dilute the antibody to 5 mg / ml, add 100 mM EDTA aqueous solution to a final concentration of 5 mM, and shake to mix. Add a certain volume of TCEP aqueous solution according to 2 to 15 times the molar number of the antibody, shake to mix, and incubate at 20°C in a constant temperature mixer for 1.5 hours. Add a certain volume of toxin-linker solution according to 4 to 24 times the molar number of the antibody, and supplement DMSO to 10% of the final reaction volume. Shake to mix, and incubate at 20°C in a constant temperature mixer for 0.5-24 hours. After the coupling is completed, use 300 mg / mL dextran-coated activated carbon (manufacturer: Sigma) to remove free small molecules. Then, the residual activated carbon particles in the supernatant were filtered out using a hydrophilic filter membrane, and finally the ADC storage buffer was replaced with 50 mM PBS (pH 6.0) using an ultrafiltration tube and stored at -80°C for future use.

[0171] Specifically, when synthesizing ADC-1-15, taking ADC-1-5 as an example, the TCEP dosage was 4, 4, 2, 3, and 1.5 times the molar amount of the antibody, respectively, and the toxin-linker dosage was 4, 4, 4, 5, and 4 times the molar amount of the antibody. When synthesizing IMAB027-vcMMAE, the TCEP dosage was 3.4 times the molar amount of the antibody, and the vcMMAE dosage was 10 times the molar amount of the antibody.

[0172] Figure 7(B) illustrates the overall method for preparing antibody-drug conjugates using ene linkers. 1-20 anti-tumor toxin molecules are conjugated to prepare the antibody-drug conjugate. To a 0.5ml centrifuge tube, add 2mg of antibody. Dilute the antibody to 10mg / ml with 25mM Na2B4O7, 25mM NaCl, and 1mM DTPA (pH 7.4) buffer. Add a volume of TCEP aqueous solution 2-15 times the molarity of the antibody. Vortex to mix thoroughly, and incubate in a thermomixer at 20°C for 2-24 hours. Add a volume of toxin-linker solution 4-24 times the molarity of the antibody. Vortex to mix thoroughly, and incubate in a thermomixer at 20°C for 0.5-24 hours. After conjugation, stop the reaction and purify the ADC. For ADC15, the TCEP dosage is 15 times the molarity of the antibody, and the toxin-linker dosage is 24 times the molarity of the antibody.

[0173] 7(A) and 7(C) illustrate the overall preparation method of antibody-drug conjugates, wherein 1-20 anti-tumor toxin molecules are conjugated via antibody interchain disulfide bonds and / or cysteine ​​mutation sites to prepare the following ADC-16-75:

[0174] To a 0.5ml centrifuge tube, add 2mg of antibody. Dilute the antibody to 5mg / ml with 30mM His-HAC buffer (pH 5.5). Add 100mM EDTA to a final concentration of 5mM and vortex to mix. Add 2mg / ml TCEP in water at a 3:1 TCEP to antibody molar ratio, vortex to mix, and incubate at 20°C in a thermomixer for 1.5 hours. Alternatively, use a desalting column to exchange the antibody buffer with 2mM EDTA and 100mM Tris-HCl (pH 8.0). To a 1.5ml centrifuge tube, add 1mg of antibody. Add 100mM DTT in water at a DTT to antibody molar ratio of 20:1 to 100:1. Mix by pipetting, and incubate at 22°C in a thermomixer overnight. Add 10mg / ml linker-toxin in DMSO at a 12:1 compound to antibody molar ratio, and make up DMSO to 20% of the final reaction volume. After vortexing and mixing, incubate in a thermomixer at 20°C for 1.5 hours. After coupling, remove free small molecules using 300 mg / mL dextran-coated activated carbon (Sigma). Residual activated carbon particles in the supernatant are then filtered through a hydrophilic filter membrane. Finally, replace the ADC storage buffer with 50 mM PBS (pH 7.2) using an ultrafiltration tube and freeze at -80°C until ready for storage.

[0175] Alternatively, use a desalting column to exchange the antibody buffer with 2mM EDTA, 100mM Tris-HCl (pH 8.0). In a 1.5ml centrifuge tube, add 1mg of antibody and add 100mM DTT at a molar ratio of 100:1. Mix by pipetting and incubate overnight at 22°C in a thermomixer. Use a desalting column to exchange the antibody buffer with 2mM EDTA, 150mM NaCl, 50mM Tris-HCl (pH 7.5). Add 100mM DHAADMA at a molar ratio of 20:1 DHAA to antibody. Mix by pipetting and incubate at 22°C in a thermomixer for 2 hours. Remove the DHAA using a desalting column and exchange the antibody buffer with 150mM NaCl, 50mM Tris-HCl (pH 7.5). Add a 10 mg / ml linker-toxin DMA solution at a 6:1 molar ratio of compound to antibody, and add DMA to 10% of the final reaction volume. Mix thoroughly by vortexing and incubate at 20°C in a thermomixer for 4 hours. After coupling, remove free small molecules using a desalting column.

[0176] The ADC storage buffer was replaced with 50 mM His-HAC (pH 5.5) and stored at -80°C until use.

[0177] Example 164 In vitro cytotoxicity of anti-CLDN6 antibodies and preferred linker-loaded antibody-drug conjugates on CLDN6-positive human ovarian cancer OV90 cells

[0178] 1.5×10 3 CLDN6-positive human ovarian cancer OV90 cells were seeded in a 96-well plate (excluding the edge wells filled with PBS). The cells were cultured in 80 μL of DMEM supplemented with 10% FBS (v / v), 100 U / mL penicillin, and 100 mg / mL streptomycin and cultured overnight in a humidified incubator at 37°C and 7.5% CO2.

[0179] After one day of incubation in a constant temperature incubator, the selected ADC was added to each well (20 μL) to give a final ADC concentration range of 0.01 nM to 1000 nM. After an additional 5 days of incubation, the 96-well plate was removed from the incubator and equilibrated to room temperature. Approximately 30 minutes later, 40 μL of (Promega, G7572). The plate was shaken at 450 rpm for 5 minutes and allowed to equilibrate for 10 minutes without shaking. Luminescence was then measured on a SpectraMax i3x microplate reader. Luminescence versus ADC concentration (nM) was fitted using Graphpad Prism software. IC50 values ​​were determined using the built-in "log (Inhibitor) vs. Response - Variable Slope (4 Parameters)" function in Prism software.

[0180] Figure 8A and the table below show the in vitro cytotoxicity of the antibody-drug conjugates

[0181] Note: DAR is the drug to antibody ratio

[0182] Figures 8B and 8C show the in vitro cytotoxicity of selected antibody-drug conjugates on OV90 cells.

[0183] Example 165 In vitro cytotoxicity of anti-CLDN6 antibodies and preferred linker-loaded antibody-drug conjugates on CLDN6-positive human choriocarcinoma JEG-3 cells

[0184] 1.5×10 3 CLDN6-positive human ovarian cancer JEG-3 cells were seeded in 96-well plates (excluding the PBS-filled edge wells). Cells were cultured in 80 μL of MEM supplemented with 10% FBS (v / v), 100 U / mL penicillin, 100 mg / mL streptomycin, 1% non-essential amino acids, and 1 mM sodium pyruvate and incubated overnight at 37°C in a humidified incubator with 7.5% CO2.

[0185] After one day of incubation in a constant temperature incubator, each ADC was added to each well (20 μL), with the final ADC concentration ranging from 0.01 nM to 1000 nM. After an additional 5 days of incubation, the 96-well plate was removed from the incubator and equilibrated to room temperature. Approximately 30 minutes later, 40 μL of CellTiter (Promega, G7572). The plate was shaken at 450 rpm for 5 minutes and allowed to equilibrate for 10 minutes without shaking. Luminescence was then measured on a SpectraMax i3x microplate reader. Luminescence versus ADC concentration (nM) was fitted using Graphpad Prism software. IC50 values ​​were determined using the built-in "log (Inhibitor) vs. Response - Variable Slope (4 Parameters)" function in Prism software.

[0186] FIG9 shows the in vitro cytotoxicity of the tested antibody-drug conjugates on JEG3 cells.

[0187] Example 166 Evaluation of the in vivo efficacy of anti-hCLDN6 ADC in an ovarian cancer model established using CLDN6-positive human ovarian cancer OV90 cells

[0188] On day 0 of the study, 8-10 week-old mice weighing at least 20 g each were inoculated with OV90 tumor cells (5 × 10 6 cells / animal in 200 μL PBS). On days 17 and 24 after transfer, the ADC formulated in PBS was administered (IV) at 10 mg / kg to groups of 5 mice. PBS served as a negative vehicle control. All treatment groups were injected into the tail vein once a week for two or three weeks. Tumor volume was measured to determine whether tumor growth was inhibited, delayed, or cured. Tumor size was measured twice a week with a caliper, and the average tumor volume of mice in each group was plotted over time. Volume units are mm 3 The formula is as follows: V = 0.5axb 2 , where a and b are the long and short diameters of the tumor, respectively. The use and welfare of experimental animals will comply with the rules of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.

[0189] Figure 10 shows the efficacy of ADC-2, DAR2, administered for two weeks (A) and ADC-46, DAR4, administered for three weeks (B) in a xenograft model in female BALB / C nude mice injected with hCLDN6-positive OV90 cells.

[0190] The embodiments of the present invention are only used to illustrate the present invention and do not limit the scope of the claims. Other substantially equivalent alternatives that can be thought of by those skilled in the art are all within the scope of protection of the present invention.

Claims

1. [Corrected 26.07.2024 in accordance with Rule 91] An isolated antigen binding protein capable of binding to hCLDN6, characterized in that comprising a heavy chain variable region, wherein the heavy chain variable region has: HCDR1 with the amino acid sequence X1YTMS, HCDR2 of the amino acid sequence TISSGGGX2TYYPDSVKG, X2=R, N, Q, D or E, HCDR3 with the amino acid sequence of GDX4RYDX3FAY, X4=Y, N or Q, in, (1) X1=Y, M, Q, D or E, X3=G, or (2) X1=S or D, X3=A.

2. The antigen-binding protein according to claim 1, characterized in that The heavy chain variable region has a human heavy chain framework region, and the human heavy chain framework region has a back mutation at one or more sites relative to the mouse heavy chain framework region, and the sites of the back mutation in the heavy chain variable region include R44, and the position numbering is determined by the Kabat numbering scheme.

3. The antigen-binding protein according to claim 2, characterized in that The heavy chain variable region is: HFR1 to HCDR3 have 92.59% or more identity, have the back mutation site, and have the sequence of the X1, X2, X3 and / or X4 sites.

4. The antigen-binding protein according to claim 3, characterized in that The heavy chain variable region has a sequence shown in any one of SEQ ID No. 22, 23, 24, 59, 60, 61, 62, 63, 66, 67, 133 to 153.

5. The antigen-binding protein according to claim 2, characterized in that The heavy chain framework region is a human IgG heavy chain framework region.

6. The antigen-binding protein according to claim 1, characterized in that Also included is a light chain variable region, the light chain variable region having: LCDR1 of amino acid sequence RASENI DSX5LA, X5 = Y, Q or R, LCDR2 with the amino acid sequence of ASTLLVD, LCDR3 with amino acid sequence QHYYSX6PYT, X6=I or E.

7. The antigen-binding protein according to claim 6, characterized in that The light chain variable region has a human light chain framework region, which has one or more back mutations relative to the mouse light chain framework region, and the back mutation sites of the light chain framework region include one or more of R66, Q70, and R85, and the position numbering is determined by the Kabat numbering scheme.

8. The antigen-binding protein according to claim 7, characterized in that A sequence having 95.88% or more identity with HFR1 to HCDR3 of the light chain variable region, having the back mutation site, and having the X5 and / or X6 site.

9. The antigen-binding protein according to claim 8, characterized in that The light chain variable region has a sequence shown in any one of SEQ ID No. 25, 28, 154, 155, and 156.

10. The antigen binding protein according to claim 7, characterized in that The light chain framework region is a human IgG light chain framework region.

11. The antigen-binding protein according to claim 6, characterized in that Includes human heavy chain constant region and light chain constant region.

12. The antigen binding protein according to claim 1, characterized in that Includes a heavy chain constant region of human origin.

13. The antigen binding protein according to claim 11 or 12, characterized in that The heavy chain constant region is a human IgG1 heavy chain constant region and contains one or more mutations of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.

14. The antigen binding protein according to claim 11 or 12, characterized in that The amino acid sequence of the heavy chain constant region is shown in SEQ ID No.

9.

15. The antigen binding protein according to claim 11, characterized in that The amino acid sequence of the light chain constant region is shown in SEQ ID No.

10.

16. The antigen binding protein of claim 1, characterized in that The antigen binding protein includes an antibody or an antigen binding fragment thereof.

17. The antigen binding protein according to claim 2, characterized in that The heavy chain has a sequence as shown in any one of SEQ ID Nos. 157, 159 to 167.

18. The antigen binding protein according to claim 2, characterized in that The heavy chain has a sequence as shown in any one of SEQ ID No. 158 and 171.

19. The antigen binding protein according to claim 7, characterized in that The light chain has a sequence as shown in any one of SEQ ID No. 169 and 170.

20. The antigen binding protein of claim 7, characterized in that The light chain has a sequence as shown in SEQ ID No.

168.

21. The antigen binding protein of claim 1, characterized in that The antigen binding protein is antibody hAb-13: comprising a heavy chain variable region sequence as shown in SEQ ID No.18, and a light chain variable region sequence as shown in SEQ ID No.

28.

22. The antigen binding protein of claim 1, characterized in that Selected from: Antibody hAb-26: comprising a heavy chain variable region having a sequence of SEQ ID No.59 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-27: comprising a heavy chain variable region having a sequence of SEQ ID No.60 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-28: comprising a heavy chain variable region having a sequence of SEQ ID No.61 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-29: comprising a heavy chain variable region having a sequence of SEQ ID No.62 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-30: comprising a heavy chain variable region having a sequence of SEQ ID No.63 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-33: comprising a heavy chain variable region having a sequence of SEQ ID No.66 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-34: comprising a heavy chain variable region having a sequence of SEQ ID No.67 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-44: comprising a heavy chain variable region having a sequence of SEQ ID No.22 and a light chain variable region having a sequence of SEQ ID No.25; or Antibody hAb-45: comprising a heavy chain variable region having a sequence of SEQ ID No.23 and a light chain variable region having a sequence of SEQ ID No.25; or Antibody hAb-46: comprising a heavy chain variable region having a sequence of SEQ ID No.24 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-47: comprising a heavy chain variable region having a sequence of SEQ ID No.22 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-48: comprising a heavy chain variable region having a sequence of SEQ ID No.23 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-49: comprising a heavy chain variable region having a sequence of SEQ ID No.24 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-50: comprising a heavy chain variable region having a sequence of SEQ ID No.133 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-51: comprising a heavy chain variable region having a sequence of SEQ ID No.134 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-52: comprising a heavy chain variable region of SEQ ID No.135 and a light chain variable region of SEQ ID No.28; or Antibody hAb-53: comprising a heavy chain variable region having a sequence of SEQ ID No.136 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-54: comprising a heavy chain variable region having a sequence of SEQ ID No.137 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-55: comprising a heavy chain variable region having a sequence of SEQ ID No.138 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-56: comprising a heavy chain variable region of SEQ ID No.139 and a light chain variable region of SEQ ID No.28; or Antibody hAb-57: comprising a heavy chain variable region having a sequence of SEQ ID No.140 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-58: comprising a heavy chain variable region having a sequence of SEQ ID No.141 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-59: comprising a heavy chain variable region having a sequence of SEQ ID No.142 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-60: comprising a heavy chain variable region having a sequence of SEQ ID No.143 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-61: comprising a heavy chain variable region having a sequence of SEQ ID No.144 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-62: comprising a heavy chain variable region of SEQ ID No.145 and a light chain variable region of SEQ ID No.28; or Antibody hAb-63: comprising a heavy chain variable region of SEQ ID No.146 and a light chain variable region of SEQ ID No.28; or Antibody hAb-64: comprising a heavy chain variable region of SEQ ID No.147 and a light chain variable region of SEQ ID No.28; or Antibody hAb-65: comprising a heavy chain variable region having a sequence of SEQ ID No.148 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-66: comprising a heavy chain variable region of SEQ ID No.149 and a light chain variable region of SEQ ID No.28; or Antibody hAb-67: comprising a heavy chain variable region having a sequence of SEQ ID No.150 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-68: comprising a heavy chain variable region having a sequence of SEQ ID No.151 and a light chain variable region having a sequence of SEQ ID No.28; or Antibody hAb-69: comprising a heavy chain variable region of SEQ ID No.152 and a light chain variable region of SEQ ID No.28; or Antibody hAb-70: comprising a heavy chain variable region of SEQ ID No.153 and a light chain variable region of SEQ ID No.28; or Antibody hAb-71: comprising a heavy chain variable region having a sequence of SEQ ID No.24 and a light chain variable region having a sequence of SEQ ID No.154; or Antibody hAb-72: comprising a heavy chain variable region having a sequence of SEQ ID No.24 and a light chain variable region having a sequence of SEQ ID No.155; or Antibody hAb-73: comprising a heavy chain variable region having a sequence of SEQ ID No.24 and a light chain variable region having a sequence of SEQ ID No.156; or Antibody hAb-74: comprising a heavy chain having a sequence of SEQ ID No.157 and a light chain having a sequence of SEQ ID No.168; or Antibody hAb-75: comprising a heavy chain having a sequence of SEQ ID No.158 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-76: comprising a heavy chain having a sequence of SEQ ID No.159 and a light chain having a sequence of SEQ ID No.170; or Antibody hAb-77: comprising a heavy chain having a sequence of SEQ ID No.160 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-78: comprising a heavy chain having a sequence of SEQ ID No.161 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-79: comprising a heavy chain having a sequence of SEQ ID No.162 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-80: comprising a heavy chain having a sequence of SEQ ID No.163 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-81: comprising a heavy chain having a sequence of SEQ ID No.164 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-82: comprising a heavy chain having a sequence of SEQ ID No.165 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-83: comprising a heavy chain having a sequence of SEQ ID No.166 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-84: comprising a heavy chain having a sequence of SEQ ID No.167 and a light chain having a sequence of SEQ ID No.169; or Antibody hAb-85: comprises a heavy chain with a sequence of SEQ ID No.171 and a light chain with a sequence of SEQ ID No.

169.

23. A nucleic acid molecule characterized in that Used to encode the antigen-binding protein according to any one of claims 1 to 22.

24. A carrier, characterized in that Comprising the nucleic acid molecule of claim 23.

25. A cell comprising the nucleic acid molecule of claim 23, and / or the vector of claim 24.

26. A drug molecule characterized by Comprising the antigen binding protein according to any one of claims 1 to 22.

27. The drug molecule according to claim 26, characterized in that The structure is shown in Formula I: Ab is the antigen binding protein, L is a linker containing one or more linkers, D is the therapeutic agent or detectable marker, and n is an integer selected from 1-20.

28. The drug molecule according to any one of claims 27, characterized in that n is selected from an integer of 2-8.

29. The drug molecule according to claim 27, characterized in that The therapeutic agent is selected from immunomodulators, cytotoxic agents, cytostatic agents, radioisotopes, anti-angiogenic agents or liposomes.

30. The drug molecule according to claim 29, characterized in that The cytotoxic agent is selected from one or more of maytansine alkaloids, auristatins, eribulin, taxanes, calicheamicin, cemadotine, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, α-amanitin and its derivatives, trabectedin and its derivatives, rubectedin and its derivatives.

31. The drug molecule according to claim 27, characterized in that The linker is selected from one or more of an oligopeptide linker, a hydrazine linker, a thiourea linker, a triggered self-degradation linker, a succinimidyl trans-4-(maleimidomethyl)cyclohexane-1-carboxylate linker, a maleimide linker, a disulfide linker, a thioether linker, and an olefin linker.

32. The drug molecule according to claim 27, characterized in that Any one selected from ADC1 to 72:

33. Use of the antigen binding protein of any one of claims 1 to 22, the nucleic acid molecule of claim 23, the vector of claim 24, the cell of claim 25, or the drug molecule of any one of claims 26 to 32 in the preparation of a drug, wherein the drug is used for diagnosing, preventing and / or treating hCLDN6-related diseases and / or conditions.

34. The use according to claim 33, characterized in that The diseases and / or conditions include tumors, and the tumors are ovarian cancer, testicular cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, choriocarcinoma or endometrial cancer.

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