Antigen-binding protein for HCLDN6 and its use

Abstract

An antigen-binding protein against hCLDN6 and its use are provided. The antigen-binding protein includes a heavy chain variable region. The heavy chain variable region has HCDR1 having the amino acid sequence X1YTMS, HCDR2 having the amino acid sequence TISSGGGX2TYYPDSVKG (X2=R, N, Q, D, or E), and HCDR3 having the amino acid sequence GDX4RYDX3FAY (X4=Y, N, or Q), provided that (1) X1=Y, M, Q, D, or E and X3=G, or (2) X1=S or D and X3=A. Novel humanized antibodies targeting CLDN6 and various compositions based on these antibodies are provided. These have superior cell proliferation inhibitory activity and tumor inhibitory activity, thereby providing novel means for the diagnosis and treatment of cancer.

Description

Technical Field

[0001] The present invention relates to an antigen-binding protein against hCLDN6, a nucleic acid molecule encoding the antigen-binding protein, a vector, a cell, a pharmaceutical molecule, and their uses.

Background Art

[0002] The claudin (CLDN) family includes 27 members, all of which are multi-pass transmembrane proteins. Some of these members are involved in the formation of tight junctions. Tight junctions are one of the important connection forms of cell adhesion structures. CLDN proteins, together with other extracellular and surface membrane proteins, form a barrier between adjacent polarized epithelial or endothelial cells and prevent the transport of large molecules. CLDN proteins also bind to signaling proteins and cytoskeletal proteins and are involved in cell signaling. Among the members involved in tight junctions, CLDN6 is unique in that it is expressed only in embryos and hardly expressed in normal adult tissues. On the other hand, CLDN6 is a tumor antigen specifically expressed in various cancers such as ovarian cancer, testicular cancer, non-small cell lung cancer, endometrial cancer, liver cancer, pancreatic cancer, or choriocarcinoma. CLDN6 may also be involved in the differentiation of stem cells and is associated with cancer cell proliferation, apoptosis, migration and metastasis, and drug resistance. Inhibition of CLDN6 delays 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 the antibody and its composition. CLDN9 is the most similar to CLDN6, differing in sequence only by three amino acid residues in the extracellular region (the extracellular loop regions of human CLDN6 and human CLDN9 are underlined in Figure 1). Two extracellular loop regions, EL1 and EL2, exist. EL1 spans 29-81 amino acids (AA) in human CLDN6 and CLDN9, respectively, while EL2 spans 138-160 amino acids (AA). CLDN9 is expressed in tissues such as the pituitary gland, nasal mucosa, and inner ear, and mutations in CLDN9 are associated with hereditary hearing loss. Therefore, antibodies with high selectivity for CLDN6, which recognize CLDN6 but do not bind to or only weakly bind to CLDN9 or other CLDN family members, can improve the safety of the antibody and its composition.

[0004] Furthermore, mouse antibodies are highly immunogenic, and humanization can reduce their immunogenicity, making them more suitable for clinical application. However, humanization is complex, and the resulting humanized antibodies often exhibit reduced affinity, expression, and / or stability. Currently, there is no method to guarantee that high-affinity humanized antibodies can be obtained after humanizing mouse antibodies using a specific method. [Overview of the project]

[0005] The main objective of this application is to provide an antigen-binding protein that can specifically bind to CLDN6 and has good antigen-binding activity.

[0006] A first aspect of the present invention is an isolated antigen-binding protein capable of binding to hCLDN6, comprising a heavy chain variable region, the heavy chain variable region being HCDR1 having the amino acid sequence X1YTMS, HCDR2 having the amino acid sequence TISSGGGX2TYYPDSVKG (wherein X2 = R, N, Q, D, or E), and HCDR3 has the amino acid sequence GDX4RYDX3FAY (wherein X4 = Y, N, or Q), (1) relating to antigen-binding proteins where X1=Y, M, Q, D, or E and X3=G, or (2) X1=S or D and X3=A.

[0007] In some embodiments of the first aspect of the present invention, the heavy chain variable region has a human heavy chain framework region, the human heavy chain framework region has one or more back mutations compared to the mouse heavy chain framework region. The back mutation site in the human heavy chain variable region includes R44, the position number of which is determined by the Kabat numbering scheme.

[0008] In some embodiments of the first aspect of the present invention, the heavy chain variable region has 92.59% or more identity with HFR1 to HCDR3, has a reverse mutation site, and has a sequence including X1, X2, X3, and / or X4 sites.

[0009] In some embodiments of the first aspect of the present invention, the heavy chain variable region has a sequence represented by any one of sequence numbers 22, 23, 24, 59, 60, 61, 62, 63, 66, 67, and 133-153, or a sequence having 92.59% or more identity with HFR1-HCDR3 of the heavy chain variable region.

[0010] In some embodiments of the first aspect of the present invention, the heavy chain framework region is a human IgG heavy chain framework region.

[0011] In some embodiments of the first aspect of the present invention, the heavy chain variable region has a mouse heavy chain framework region.

[0012] In some embodiments of the first aspect of the present invention, the heavy chain variable region has the sequence shown in any one of sequence numbers 29, 30, or 45.

[0013] In some embodiments of the first aspect of the present invention, the antigen-binding protein further comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 5.

[0014] In some embodiments of the first aspect of the present invention, the antigen-binding protein further comprises a light chain variable region, the light chain variable region is LCDR1 having the amino acid sequence RASENIDSX5LA (wherein X5 = Y, Q, or R), LCDR2 having the amino acid sequence of ASTLLVD, and It has LCDR3 with the amino acid sequence QHYYSX6PYT (where X6 = I or E).

[0015] 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 reverse mutations compared to the mouse light chain framework region, the reverse mutation sites of the light chain framework region include one or more of R66, Q70, and R85, and the positional numbering is determined by a Kabat numbering scheme.

[0016] In some embodiments of the first aspect of the present invention, a sequence having 95.88% or more identity with HFR1 to HCDR3 of the light chain variable region has a reverse mutation site and also has an X5 site and / or an X6 site.

[0017] In some embodiments of the first aspect of the present invention, the light chain variable region has a sequence represented by any one of sequence numbers 25, 28, 154, 155, and 156, or a sequence having 95.88% or more identity with HFR1 to HCDR3 of the light chain variable region.

[0018] In some embodiments of the first aspect of the present invention, the light chain framework region is a human IgG light chain framework region.

[0019] In some embodiments of the first aspect of the present invention, the antigen-binding protein comprises a human heavy chain constant region and a human light chain constant region.

[0020] In some embodiments of the first aspect of the present invention, the antigen-binding protein comprises a human heavy chain constant region.

[0021] In some embodiments of the first aspect of the present invention, the constant region comprises mutations at one or more sites.

[0022] In some embodiments of the first aspect of the present invention, the heavy chain constant region comprises the LALA mutation.

[0023] 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 comprises one or more mutations selected from the group consisting of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.

[0024] In some embodiments of the first aspect of the present invention, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9.

[0025] In some embodiments of the first aspect of the present invention, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 10.

[0026] In some embodiments of the first aspect of the present invention, the CDR sequences are determined according to the Kabat definition scheme.

[0027] In some embodiments of the first aspect of the present invention, the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0028] <00001m02>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.

[0029] 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.

[0030] In some embodiments of the first aspect of the present invention, the antigen-binding protein has a heavy chain having the sequence shown in any one of SEQ ID NOs: 157 and 159-167, or has a sequence having 92.59% or more identity with HFR1-HCDR3 of the heavy chain.

[0031] In some embodiments of the first aspect of the present invention, the antigen-binding protein has a heavy chain having the sequence shown in either SEQ ID NOs. 158 or 171, or a sequence having 92.59% or more identity with HFR1 to HCDR3 of the heavy chain.

[0032] In some embodiments of the first aspect of the present invention, the antigen-binding protein has a light chain having the sequence shown in either SEQ ID NOs: 169 or 170, or a sequence having 95.88% or more identity with HFR1 to HCDR3 of the light chain.

[0033] In some embodiments of the first aspect of the present invention, the antigen-binding protein has a light chain having the sequence shown in SEQ ID NO: 168, or a sequence having 95.88% or more identity with HFR1 to HCDR3 of the light chain.

[0034] In some embodiments of the first aspect of the present invention, the antigen-binding protein is an antibody hAb-13 having a heavy chain variable region sequence shown in SEQ ID NO: 18 and a light chain variable region shown in SEQ ID NO: 28.

[0035] A second aspect of the present invention relates to a nucleic acid molecule used to encode any of the antigen-binding proteins.

[0036] A third aspect of the present invention relates to a vector characterized by containing a nucleic acid molecule.

[0037] A fourth aspect of the present invention relates to a cell comprising nucleic acid molecules and / or vectors.

[0038] A fifth aspect of the present invention relates to a pharmaceutical molecule characterized by containing any one of the above-mentioned antigen-binding proteins.

[0039] In some embodiments of a fifth aspect of the present invention, the pharmaceutical molecule further comprises a therapeutic agent or a detectable marker conjugated to an antigen-binding protein.

[0040] In some embodiments of a fifth aspect of the present invention, the therapeutic agent is selected from cytotoxic agents, cell proliferation inhibitors, radioisotopes, anti-angiogenic agents, or liposomes.

[0041] In some embodiments of a fifth aspect of the present invention, the therapeutic agent is an immunomodulator.

[0042] In some embodiments of the fifth aspect of the present invention, the cytotoxic agent is a molecule derived from a plant, fungus, or bacterium, or a derivative thereof.

[0043] In some embodiments of a fifth aspect of the present invention, the cytotoxic agent is selected from peptide toxins, protein toxins, or alkylated toxins.

[0044] In some embodiments of a fifth aspect of the present invention, the cytotoxic agent is selected from one or more of the following: meitansin alkaloids, auristatin, eribulin, taxanes, calicheamycin, semadin, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, α-amanitin and its derivatives, trabectedin and its derivatives, and lurbinectidin and its derivatives.

[0045] In some embodiments of a fifth aspect of the present invention, a therapeutic agent or detectable marker is bound to an antigen-binding protein via a cleavable or incleavable linker.

[0046] In some embodiments of a fifth aspect of the present invention, the linker has one or more connectors.

[0047] In some embodiments of a fifth aspect of the present invention, the connector is selected from one or more of the following: oligopeptide connectors, hydrazine connectors, thiourea connectors, triggered self-immolative connectors, succinimidyltrans-4-(maleimidomethyl)cyclohexane-1-carboxylate connectors, maleimide connectors, disulfide connectors, thioether connectors, and olefin connectors.

[0048] In some embodiments of a fifth aspect of the present invention, the structure is as shown in formula I:

number

[0049] In the formula, Ab is an antigen-binding protein, L is a linker containing one or more connectors, D is a therapeutic agent or a detectable marker, and n is an integer selected from 1 to 20.

[0050] In some embodiments of a fifth aspect of the present invention, n is an integer selected from 2 to 8.

[0051] A sixth aspect of the present invention relates to a pharmaceutical composition characterized by comprising any of an antigen-binding protein, a nucleic acid molecule, a vector, a cell, and / or a pharmaceutical molecule, as well as a pharmaceutically acceptable carrier and / or additive.

[0052] A seventh aspect of the present invention relates to the use of any of antigen-binding proteins, nucleic acid molecules, vectors, cells, and pharmaceutical molecules in the manufacture of pharmaceuticals for diagnosing, preventing, and / or treating hCLDN6-related diseases and / or disorders.

[0053] In some embodiments of the seventh aspect of the present invention, the disease and / or disorder includes a tumor.

[0054] In some embodiments of the seventh aspect of the present invention, the disease and / or disorder includes a tumor, where the tumor is ovarian cancer, testicular cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, choriocarcinoma, or endometrial cancer.

[0055] This invention provides highly selective humanized anti-CLDN6 antibodies having affinity equivalent to or greater than that of mouse parental antibodies. Compositions such as ADC conjugates based on these antibodies have also been prepared by combining them with various linkers and toxins. These antibodies and compositions offer novel approaches to the diagnosis and treatment of tumors. In particular, anti-CLDN6 ADCs exhibit excellent antitumor activity and have potential for tumor therapy. This invention provides novel humanized antibodies targeting CLDN6 and various compositions based on these antibodies. These possess superior cell proliferation inhibitory activity and tumor inhibitory activity, thereby providing novel means for the diagnosis and treatment of cancer. [Brief explanation of the drawing]

[0056] [Figure 1] Figure 1 shows the sequence alignment of human CLDN6 (hCLDN6) and human CLDN9 (hCLDN9). Extracellular EL1 and EL2 sequences are underlined. [Figure 2] Figure 2 shows the binding activity of anti-CLDN6 mouse / human chimeric monoclonal antibody and CDR-transplanted humanized antibody to human ovarian cancer cells OV90. [Figure 3] Figure 3 shows the sequence alignment of the heavy chain variable region (A) and light chain variable region (B) of a representative humanized antibody with a chimeric antibody, a CDR-transplanted humanized antibody, and a reverse mutation introduced after CDR transplantation. [Figure 4] Figure 4 shows the endocytosis activity of selected humanized antibodies with reverse mutations introduced after CDR transplantation to JEG-3 cells. [Figure 5]Figure 5 shows the binding activity of cAb-2 to cAb-31 to VLP-hCLDN6 (A-B) and their endocytosis activity against JEG-3 cells (C). [Figure 6] Figure 6 shows the detection of binding activity between hAb-26 to hAb-49 and VLP-hCLDN6 (A to C, E) or VLP-hCLDN9 (D, F). [Figure 7] Figure 7 is a schematic diagram illustrating the process for producing antibody-drug conjugates (ADCs) using inter-antibody disulfide bonds and / or cysteine ​​mutation sites. [Figure 8] Figure 8 shows the in vitro cytotoxicity of selected ADCs against human ovarian cancer cells OV90. [Figure 9] Figure 9 shows the in vitro cytotoxicity of selected ADCs against human choriocarcinoma cells JEG3. [Figure 10] Figure 10 shows the efficacy of ADC-2, DAR2 (A) and ADC-46, DAR4 (B) in a xenograft model of female BALB / C nude mice established using hCLDN6-positive OV90 cells. [Modes for carrying out the invention]

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In the event of any conflict, the explanations and definitions herein shall prevail.

[0058] In view of the problems in the prior art, the present invention aims to obtain an antigen-binding protein with higher hCLDN6 binding activity by modifying and humanizing a mouse antibody. The heavy chain variable region of the antigen-binding protein involved in the present invention is HCDR1 having the amino acid sequence X1YTMS, HCDR2 having the amino acid sequence TISSGGGX2TYYPDSVKG (wherein X2 = R, N, Q, D, or E), and HCDR3 has the amino acid sequence GDX4RYDX3FAY (wherein X4 = Y, N, or Q), (1) X1 = Y, M, Q, D, or E and X3 = G, or (2) X1 = S or D and X3 = A.

[0059] The heavy chain variable region of the present invention relates to specific mutations at one or more sites, such as S31 and G100, in the HCDR region shown in SEQ ID NOs: 2-4. The antigen-binding protein obtained by the mutation showed improved binding activity to hCLDN6.

[0060] The aforementioned types of antigen-binding proteins include, but are not limited to, full-length antibodies, antibody fragments, immune complexes, 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 their equivalents.

[0061] A chimeric antibody generally refers to an antibody that possesses the V region (variable region) of a mouse monoclonal antibody and the C region (constant region) of a human immunoglobulin.

[0062] Humanized antibodies generally refer to antibodies formed by introducing the CDR sequence of a mouse antibody into the framework sequences of the human VH domain and VL domain, respectively.

[0063] Chimeric antibodies and humanized antibodies can be produced using known methods. For example, a V-region gene consisting of three CDRs linked to four FRs can be fully amplified by annealing at the 5' and 3' ends, to which appropriate restriction enzyme recognition sequence primers can be attached. The DNA obtained in this manner is assembled into an expression vector for in-frame fusion together with DNA encoding the human antibody C-region. This recombinant vector is introduced into a host to establish recombinant cells, which are then cultured to express the antibody-encoding DNA and generate antibodies. See Example 2 for details.

[0064] Antibody forms include, but are not limited to, IgG, IgM, IgA, IgE, and IgD, with IgG being preferred. Furthermore, examples of IgG include IgG1, IgG2, IgG3, and IgG4. For chimeric and humanized antibodies, the constant region can be Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε, and similar entities in the heavy chain, and Cκ, Cλ, and similar entities in the light chain. Introducing LALA mutations into the Fc region of an antibody may reduce the antibody's binding to the Fcγ receptor and complement. Examples of mutations in the heavy chain constant region include, but are not limited to, one or more of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.

[0065] In this invention, CDRs are used to represent the complementarity-determining regions (or hypervariable loops) of an antibody. The amino acid sequence within the CDR determines the chemical structure and characteristics of the antigen-binding site of the antibody. The CDRs of the heavy chain variable region (VH) are referred to as HCDRs, with the three heavy chain CDR regions designated as HCDR1, HCDR2, and HCDR3, respectively. The CDRs of the light chain variable region (VL) are referred to as LCDRs, with the three light chain CDR regions designated as LCDR1, LCDR2, and LCDR3, respectively. It should be noted that the CDR regions of an antibody are generally predicted and identified experimentally. Unless otherwise specified, CDR sequences in this invention are defined according to the Kabat definition scheme, and the relevant amino acid numbers are determined by the Kabat numbering scheme. It should be understood that when different CDR definition schemes are used, the predicted CDR region sequences may only partially overlap, or may be shortened or lengthened. During antibody production, CDR and FR gene sequences, defined according to different definition schemes, can be ligated, amplified to full length, and further used to produce antibodies or antibody fragments using known methods.

[0066] An antibody fragment Fab refers to a fragment consisting of the VH region of the heavy chain's variable region, the CH1 functional region of the heavy chain's first constant region, and the entire light chain connected by disulfide bonds. It primarily performs the antigen-binding function of the antibody.

[0067] scFv is a single-chain antibody. Specifically, it involves ligating the light chain variable region genes and heavy chain variable region genes of an antibody to a suitable oligonucleotide chain (linker) at the DNA level, thereby allowing it to be 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 variable region and the light chain variable region. The linker is not particularly limited. For example, any single-chain peptide containing approximately 3 to 25 residues can be used as a linker.

[0068] dsFv are disulfide-stabilized antibodies. Specifically, they include antibodies formed by mutating amino acid residues to cysteine ​​in the heavy chain variable region (VH) and light chain variable region (VL) of an antibody, and then linking VH and VL with interchain disulfide bonds.

[0069] VHH refers to heavy chain single-domain antibodies, specifically antibodies that contain only the VH functional domain and not the VL functional domain.

[0070] A bispecific antibody is an antibody that possesses binding properties for two different antigens.

[0071] Fusion proteins are products created by fusing fragments of antibody molecules with other proteins (such as antibodies, enzymes, immunotoxins, immune cytokines, and immune adhesion molecules) using genetic engineering techniques.

[0072] Methods for preparing the various antibody fragments described above are well known. One method involves enzymatically treating an antibody to produce an antibody fragment. 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 this gene into an expression vector, and expressing it in a suitable host cell to produce the antibody fragment.

[0073] In this invention, CH is used to represent the heavy chain constant region of the antibody, and CL is used to represent the light chain constant region of the antibody. The heavy chain constant region typically further includes CH1, CH2, and CH3 domains. FR is used to represent the framework of the 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.

[0074] In the present invention, an antibody derivative refers to a product obtained by substituting, mutating, modifying, exchanging, deleting, and / or adding one or more amino acid residues in an antibody while retaining the endogenous function of the antibody.

[0075] In the present invention, the binding activity of the antigen-binding protein can be detected by existing methods such as ELISA and flow cytometry, and can be detected by referring to the methods of Example 23 and Comparative Example 7.

[0076] The present invention employs existing methods for detecting the endocytosis activity of prepared antigen-binding proteins. For example, a test antigen-binding protein containing a detectable marker is co-incubated with cells expressing CLDN6, and the fluorescence intensity excited by the protein after it enters the cells is detected by flow cytometry to analyze the endocytosis activity. See Example 24 for details.

[0077] The framework region of the heavy chain variable region can be derived from a mouse or human framework region. The human heavy chain framework region has one or more reverse mutations compared to the mouse heavy chain framework region. A preferred reverse mutation site is R44, and the position numbering is determined by the Kabat numbering scheme.

[0078] In antigen-binding proteins, other heavy chain variable region sequences can also be selected that have 92.59% or more identity with the above heavy chain variable regions HFR1 to HCDR3, have a reverse mutation site, and have X1, X2, X3, and / or X4 sites.

[0079] Preferably, the antigen-binding protein further includes a light chain variable region, the light chain variable region is LCDR1 having the amino acid sequence RASENIDSX5LA (wherein X5 = Y, Q, or R), LCDR2 having the amino acid sequence of ASTLLVD, and It has LCDR3 with the amino acid sequence QHYYSX6PYT (where X6 = I or E).

[0080] The framework region of the light chain variable region can be derived from a mouse or human framework region and, in combination with the corresponding heavy chain variable region described above, can form various types of antigen-binding proteins. The human light chain framework region has one or more reverse mutations compared to the mouse light chain framework region. Preferred reverse mutation sites include one or more of R66, Q70, and R85, and the position numbering is determined by the Kabat numbering scheme.

[0081] In antigen-binding proteins, other light chain variable region sequences having 95.88% or more identity with the above-mentioned light chain variable region HFR1-HCDR3 can also be selected.

[0082] The vectors of the present invention are used for DNA or RNA replication, transcription, and / or translation. Desired expression products can be produced by culturing cells containing the vectors. The selected vectors are known, such as plasmids.

[0083] Furthermore, the present invention also relates to pharmaceutical molecules containing the aforementioned antigen-binding proteins. Pharmaceutical molecules may include, but are not limited to, proteins, peptides, small molecule drugs, antibody-drug conjugates, or any known pharmaceutical form, including various combinations of pharmaceutical forms.

[0084] An antibody-drug conjugate refers to an antigen-binding protein (usually an antibody or antibody fragment) conjugated to a therapeutic agent via a linker. Its structure is shown in formula I:

number

[0085] In the formula, Ab is an antigen-binding protein, L is a linker containing one or more connectors, D is a therapeutic agent or a detectable marker, and n is an integer selected from 1 to 20, and may further be an integer selected from 2 to 8.

[0086] The linker-drug conjugate is typically generated by known chemical synthesis methods, forming a linker-therapeutic structure that can bind to the antibody. During the preparation of the antibody-drug conjugate, the antibody is conjugated to one or more linker-therapeutic structures. The bond between the antibody and the linker-therapeutic structure is typically established at the side chain of a lysine or cysteine ​​residue of the antibody. In the case of lysine conjugation, the linker typically contains an N-hydroxysuccinimide group that reacts with the ε-amino group of lysine to form a stable amide bond, thereby forming the antibody-therapeutic conjugate. In the case of cysteine ​​conjugation, the linker typically contains a maleimide group or a functional group that can mediate the conjugation reaction, such as arenamide. The cysteine ​​is subjected to disulfide reduction using known methods to produce a reactive thiol, which is then conjugated to the maleimide group to form the antibody-therapeutic conjugate. For example, the methods illustrated in Examples 92-163 of the present invention can be referenced.

[0087] Applicable linkers include non-cleavable and cleavable types. The linker may have one or more connectors, including, but not limited to, oligopeptide connectors (including cleavable and / or non-cleavable oligopeptide connectors), hydrazine connectors, thiourea connectors, trigger-activated self-deactivating connectors, succinimidyltrans-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) connectors, maleimide connectors, disulfide connectors, thioether connectors, and / or olefin connectors.

[0088] Applicable therapeutic agents may include cytotoxic agents and cell proliferation inhibitors. Cytotoxic agents may be molecules derived from plants, fungi, or bacteria, or derivatives thereof, and include, but are not limited to, peptide toxins, protein toxins, alkylated toxins, or other types of toxins. Toxic pharmaceuticals can be selected from one or more of the following: meitansin alkaloids (e.g., meitansinol or DM1 meitansin), auristatins (e.g., monomethyl auristatin E or monomethyl auristatin F), eribulin, taxanes, calicheamycin, semadin, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, α-amanitin and its derivatives, trabectedin and its derivatives, and lurubinectedin and its derivatives. The cytotoxic substances used in the present invention may be used individually or in combination of two or more cytotoxic substances.

[0089] Applicable therapeutic agents include metabolites (e.g., folate antagonists such as methotrexate, fluoropyrimidines such as 5-fluorouracil, cytarabine, or purines or adenosine analogs); insertors (e.g., anthracyclines such as doxorubicin, nemorubicin, or preferably derivatives of PNU-159682), other insertors such as daunorubicin, epirubicin, idarubicin, mitomycin C, actinomycin D, or mithramycin, or pyrrolobenzodiazepines; DNA reagents such as calicheamicin, tiancimycin, and other enediynes; platinum derivatives (e.g., cisplatin or carboplatin); alkylating agents (e.g., chlormethine, melphalan, chlormethine, chloro These may include: rambucil, busulfan, cyclophosphamide, ifosfamide, nitrosourea, or thiotepa; RNA polymerase inhibitors such as α-amanitin; antimitotic agents (e.g., vinca alkaloids such as vincristine, or taxanes such as paclitaxel or docetaxel); topoisomerase inhibitors (e.g., etoposide, teniposide, amsacrine, topotecan, exitecan); cell cycle inhibitors (e.g., flavopyridol); or antimicrotubule agents (e.g., epothyron, tubulicin, pretubulicin, discordermolide analogs, or eleucerobin analogs). Therapeutic agents may include proteasome inhibitors or topoisomerase inhibitors 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.

[0090] Applicable therapeutic agents include immunomodulatory agents such as STING agonists, TLR7, TLR8, and / or TLR9 agonists, and PD-1 inhibitors.

[0091] Pharmaceutical molecules containing detectable markers can be used for diagnostic purposes. Examples include contrast agents. Contrast agents may be radioisotopes such as iodine (131I or 125I), indium (111In), technetium (99Tc), phosphorus (32P), carbon (14C), tritium (3H), other radioisotopes (such as radioactive ions), or any of the therapeutic radioisotopes listed above. Furthermore, contrast agents may include radiopaque materials, magnetic resonance imaging (MRI) agents, ultrasound imaging agents, and other contrast agents suitable for detection by imaging devices for animal bodies. Detectable markers may be fluorescent markers, bioactive enzyme markers, luminescent markers, or chromophore markers.

[0092] The present invention also relates to pharmaceutical compositions prepared from the above-mentioned antigen-binding proteins, nucleic acid molecules, vectors, cells, and pharmaceutically acceptable carriers and / or additives.

[0093] The aforementioned pharmaceutical compositions may be formulated according to conventional methods and may also contain pharmaceutically acceptable carriers and / or additives. Examples of additives include, but are not limited to, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow improvers, and flavorings. Examples of carriers for the pharmaceutical compositions include, but are not limited to, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like.

[0094] Multiple antibodies may be combined as needed and incorporated into the aforementioned pharmaceutical composition. For example, creating a cocktail of multiple anti-CLDN6 antibodies can enhance cytotoxicity against CLDN6-expressing cells. Alternatively, the therapeutic effect can be enhanced by incorporating antibodies that recognize other tumor-associated antigens in addition to anti-CLDN6 antibodies.

[0095] The route of administration of the pharmaceutical composition may be oral or parenteral. Examples of parenteral administration routes include injection, nasal administration, pulmonary administration, transdermal administration, and similar methods. Injections may further include intravenous, intramuscular, intraperitoneal, and subcutaneous injections. Furthermore, the dosage can be specifically adjusted according to the patient's age and symptoms. For example, the dosage may be 0.0001 mg to 1000 mg per kg of body weight per dose. As another example, the dosage may be 0.001 mg to 100,000 mg per dose for each patient. It should be noted that the above dosages are for illustrative purposes only and do not limit the range of dosages.

[0096] The types of cancer treated and diagnosed by the present invention are not particularly limited, but are typically cancers that express the CLDN6 protein, and are preferably testicular cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, choriocarcinoma, or non-small cell lung cancer. [Examples]

[0097] The present invention will be further described below with reference to examples.

[0098] Example 1. Cell line 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.

[0099] The OV90 cell line was cultured in a 1:1 MCDB105 medium:medium 199 (Thermo Scientific, Logan, Utah) supplemented with 15% (v / v) heat-inactivated FBS (Thermo Scientific, Logan, Utah).

[0100] PA-1, JEG-3, and MCF-7 cell lines were cultured in MEM (Thermo Scientific, Logan, Utah) supplemented with 10% (v / v) heat-inactivated FBS (Thermo Scientific, Logan, Utah), 1% (v / v) non-essential amino acids, and 1 mM sodium pyruvate to support adherent culture.

[0101] Example 2. Antibody expression and purification The antibody's variable region coding region was prepared by whole-gene synthesis using MHSSALLCCLVLLTGVRA as the leading signal peptide. This region, along with the human IgH-γ1 coding region and IgL-κ coding region, was incorporated into an expression vector pcDNA3.4 containing an ampicillin resistance gene as a selection marker. The CMV promoter has a NotI / XbaI restriction enzyme site that can promote high-level expression of multiple genes in mammalian cells.

[0102] CHO-K1 cells were selected as the host for antibody expression. The cells were passaged three times under appropriate conditions (120 rpm, 8% CO2, 37°C). The cells were harvested and thoroughly mixed with electroporation buffer. Plasmid was then added and thoroughly mixed, and transferred to an electroporation tube. The pcDNA3.4 expression vector was transfected into CHO-K1 cells using an electroporator. After culturing at 37°C, 270 rpm, and 8% CO2 for 24 hours, nutrients / sodium butyrate / penicillin-streptomycin were added, and the cells were cultured for 3-7 days. The collected supernatant was filtered (0.22 μm) to remove cells and used for subsequent purification.

[0103] The treated supernatant was purified using a protein A affinity chromatography column and eluted with citrate buffer (pH 3.4). The eluate was placed in a dialysis bag, buffer was changed, and finally, purified AT03-2A antibody was obtained. The concentration and amount of this antibody were determined by measuring the absorbance at 280 nM using a NanoDrop instrument. Purity was evaluated by SDS-PAGE and SEC-HPLC, and it was found to be >95%.

[0104] Each culture medium was supplemented with 100 U / mL penicillin and 100 mg / mL streptomycin (Sigma Aldrich).

[0105] Comparative Example 1. Mouse / Human Chimeric Antibody cAb-1 Mouse / human chimeric antibody cAb-1 was prepared according to the method of Example 2.

[0106] [Table 1]

[0107] The sequences of the complementarity-determining regions (CDRs) in the heavy chain variable region and light chain variable region of the above antibody are as follows.

[0108] [Table 2]

[0109] Under different definition schemes, the arrangement of the CDR region is as follows:

[0110] [Table 3]

[0111] The antibody DTDH4 (INFECTION AND IMMUNITY, June 2006, pp. 3682-3683) was used as an isotype control (isotype). This antibody is an anti-human DT antibody.

[0112] Comparative Examples 2-6. Humanized Antibodies hAb-1-hAb-5 Following the method of Example 2, the CDR of Comparative Example 1 was transplanted into a humanized framework by CDR transplantation to obtain humanized antibodies hAb-1 to hAb-5. The variable region sequences of hAb-1 to hAb-5 are shown in the table below.

[0113] [Table 4]

[0114] Comparative Example 7. Detection of binding activity of hAb-1 to hAb-5 and cAb1 to the hCLDN6-positive human ovarian cancer cell line OV90. Flow cytometry: Cells were stained using standard flow cytometry. Briefly, for the antibodies hAb-1 to hAb-5 and cAb1 to be tested, 4 × 10⁻¹⁰ hCLDN6-positive human ovarian cancer cell line OV90 was used. 5 Cells were stained on ice for 1 hour with serial dilutions of the test antibody (4-fold dilutions from 100 nM to 0.006 nM). After washing twice with ice-cold PBS containing 1% (v / v) FBS, the cells were incubated on ice for 1 hour with FITC (Abcam)-conjugated goat anti-human IgG diluted in 100 μL of PBS (1:200) containing 1% (v / v) FBS. Staining after cell conjugation was analyzed using a flow cytometer (Agilent).

[0115] As shown in Figure 2, the CLDN6 mouse / human chimeric monoclonal antibody cAb-1 showed strong binding activity to OV90 cells, while the humanized antibodies hAb-1 to hAb-5, which were transplanted with CDR, almost completely lost their binding activity to OV90 cells.

[0116] Examples 3-22. Humanized antibodies with reverse mutations introduced after CDR transplantation: hAb-6 to hAb-25 Humanization was performed by selecting appropriate human germline sequences and introducing reverse mutations. The heavy chain variable region (VH) and light chain variable region (VL) sequences into which reverse mutations were introduced include the following:

[0117] [Table 5]

[0118] Figure 3A shows the sequence alignment between the heavy chain variable regions VH, and Figure 3B shows the sequence alignment between the light chain variable regions VL.

[0119] The method for small-scale expression of humanized antibodies with reverse mutations is as follows: The LVTransm transfection reagent and antibody expression vector, removed from the refrigerator, were thawed at room temperature and thoroughly mixed by pipetting. PBS was warmed to room temperature. 2 mL of PBS was added to one well of a 6-well plate, and 2 μg of pcDNA3.4-IgG1+2 and 2 μg of pcDNA3.4-IgKc were added separately and thoroughly mixed by pipetting. Next, 12 μL of LVTransm was added and immediately mixed by pipetting. The mixture was left at room temperature for 10 minutes. The above DNA / LVTransm complex was added to 3 mL of 293F cells and thoroughly mixed by gently shaking. The cells were further cultured at 37°C in a 5% CO2 incubator. After 48 hours of continuous culture, the supernatant was collected by centrifugation and antigen binding was detected by ELISA. The antibodies are listed in the table below.

[0120] [Table 6]

[0121] Example 23. Detection of the binding activity of cAb-1 and hAb-6 to hAb-25 to VLP-hCLDN6 AT-003-ag1 and the control protein were diluted to 4 μg / mL with sterile CBS (0.035 mol / L sodium bicarbonate (NaHCO3), 0.015 mol / L sodium carbonate (Na2CO3)). 100 μL of the dilution was added to each well of a new 96-well plate and coated overnight at 4°C. The plate was left at room temperature for 10 minutes to remove the antigen coating solution, and the plate was washed once with PBS (pH 7.4). 300 μL / well of blocking buffer (PBS containing 4% skim milk powder) was added, and the plate was blocked at 37°C for 2 hours. After removing the blocking buffer, the plate was washed once with PBS (pH 7.4). The aforementioned cAb-1 and hAb-6~hAb-25 were added in various amounts (total 100 μL) and incubated at 37°C for 1 hour. The control wells were filled with PBS. The wells were drained and washed three times with PBST (PBST containing 0.1% Tween®-20, pH 7.4), followed by three washes with PBS. 100 μL of HRP-protein A (1:10,000 dilution) was added and incubated at 37°C for 1 hour. The wells were drained and washed three times with PBST (PBST containing 0.1% Tween®-20, pH 7.4), followed by three washes with PBS. 100 μL / well of TMB developing solution was added and incubated in the dark at room temperature for 10 minutes. 50 μL / well of stop solution (2M hydrochloric acid (HCl)) was added and the OD450 values ​​of the wells were read using a microplate reader. The results are shown in the table below.

[0122] [Table 7]

[0123] As shown in the table above, all of these humanized antibodies maintained good VLP-hCLDN6 activity. Compared to hAb-1 to hAb-5 (Comparative Examples 2 to 6), these humanized antibodies retained the R44 site of the mouse heavy chain framework region VH0 and the R66 site of the light chain framework region VL0 (Kabat numbering system), suggesting that reverse mutations at these two sites may be very important.

[0124] Example 24. Detection of endocytosis activity of a humanized antibody with a reverse mutation introduced into human choriocarcinoma cells (JEG3 cells). Humanized antibodies hAb-6 to hAb-25, which were introduced with reverse mutations, were expressed and purified according to the method of Example 2. Humanized antibodies with a culture volume of more than 2 mL were selected. The selection results are shown in the table below.

[0125] [Table 8]

[0126] Endocytosis activity in the human choriocarcinoma cell line JEG3 was detected using selected humanized antibodies. Human choriocarcinoma cells JEG3 were cultured overnight in a constant temperature incubator (37°C, 5% CO2). The selected antibodies were each labeled with pHrodo iFL Green human IgG reagent, and the antibody-pHrodo iFL Green human IgG conjugates were incubated with human ovarian cancer cells PA-1 and human choriocarcinoma cells JEG3, respectively, for 24 hours. The maximum antibody concentration was set to 10 μg / mL, and 4-fold serial dilutions were performed. After incubation, the cells were washed with PBS and resuspended in culture medium. The fluorescence intensity excited after pHrodo iFL Green entered the cells was measured using a NovoCyte flow cytometer to analyze the antibody's endocytosis activity.

[0127] As shown in Figure 4, all of these humanized antibodies retained good endocytotic activity. Of these, hAb-13, with the variable region combination VH2VL4, showed higher endocytotic activity than the mouse / human chimeric antibody cAb-1.

[0128] Examples 25-54. Mouse / human chimeric antibodies cAb-2 to cAb-31 containing heavy chain CDR region mutations. Based on cAb-1, different sites within 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.

[0129] [Table 9-1] [Table 9-2]

[0130] Example 55. Detection of the binding activity of cAb-2 to cAb-31 to VLP-hCLDN6 ELISA: Each well of a 96-well 3590 plate (Corning, New York) was coated overnight at 4°C with 100 μL of coating buffer (0.015 M sodium carbonate (Na2CO3), 0.035 M sodium bicarbonate (NaHCO3), pH 9.5) containing 400 ng of VLP-CLDN6. The wells were blocked at 37°C for 2 hours with 150 μL of PBS buffer containing 4% (w / v) skim milk powder per well. The supernatant was discarded, and the wells were gently patted dry with a clean paper towel. Next, 100 μL of the 4-fold diluted antibody to be tested (cAb-1, cAb-2, cAb-3, cAB-18) was added to each well as the primary antibody at 37°C. After incubation at 37°C for 1 hour, the plate was washed three times with 1% PBST, and the supernatant was discarded. After draining the water on a clean paper towel, 100 μL of PBS buffer containing 1.25 ng / mL horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc (Invitrogen, California) and 4% (w / v) skim milk powder was added to each well and incubated at 37°C for 45 minutes. After washing three times with 1% PBST and draining the water on a clean paper towel, the color reaction was performed using TMB color development solution (Beyotime, Shanghai, China) according to the manufacturer's instructions. The plates were incubated in the dark at room temperature for 15 minutes until the expected color intensity was reached, and then TMB color stop solution was added. Absorbance was measured at 450 nm using a SpectraMax i3X microplate reader (Molecular Devices, Sunnyvale, California). The results are shown in Figure 5. Some specific EC50 values ​​are shown in the table below.

[0131] [Table 10]

[0132] As shown in Figure 5 and the table above, cAb2, cAb3, and cAb18 all maintained good binding activity to VLP-hCLDN6, and among them, cAb-3, which has the S31D mutation site, showed significantly improved binding activity to VLP-hCLDN6.

[0133] Examples 56-62. Antibodies containing heavy chain CDR region mutations and introduced reverse mutations: hAb-26-hAb-30, hAb-33, hAb-34, hAb-44-hAb-49 Mutations were introduced into the heavy chains of S31 and G100 and transplanted into various humanization frameworks, including reverse mutations, to obtain a variety of humanized antibodies containing single, double, or multiple mutations, as shown in the table below.

[0134] [Table 11]

[0135] Comparative Examples 8-18. Antibodies containing other heavy chain CDR region mutations and introduced reverse mutations. Different mutations from those in Examples 56-62 were introduced into the CDR region of the heavy chain and transplanted into a humanized VH2VL4 framework including reverse mutations. Alternatively, the S31D / G100A mutation was introduced into the CDR region of the heavy chain, and additional humanization mutations were introduced into the VH2VL4 framework region to obtain various humanized antibodies containing single, double, or multiple mutations, as shown in the table below.

[0136] [Table 12]

[0137] Example 63. Detection of the binding activity of hAb-26~hAb-39 and hAb-44~hAb-49 to VLP-hCLDN6 or VLP-hCLDN9. Using the detection method from Example 28, the binding activity of cAb-1, hAb-26 to hAb-39, and hAb-44 to hAb-49 to VLP-hCLDN6 or VLP-hCLDN9 was detected and compared. The results are shown in Figure 6.

[0138] [Table 13]

[0139] As shown in Figures 6A-6C, Figure 6E, and the table above, when CDRs containing single mutations of S31Y, S31M, S31Q, S31D, S31E, or G100A were combined with the human VH2VL4 framework region, the binding activity of the antibody against VLP-hCLDN6 was improved to varying degrees compared to mouse / human chimeric antibodies. The affinity of humanized antibodies containing the S31D / G100A double mutation was also comparable to that of mouse / human chimeric antibodies. On the other hand, while CDRs containing the G100L and G100I mutations could produce antibodies with equivalent or better affinity or endocytosis when combined with the original mouse framework region, CDRs containing these mutations lost almost all binding activity against VLP-hCLDN6 when combined with the human VH2VL4 framework region. This indicates that the G100 site is very important in humanized antibodies based on the human VH2VL4 framework region, and that the G100A mutation helps maintain or improve the activity of humanized antibodies.

[0140] As shown in Figures 6D and 6F, humanized antibodies based on the human VH2VL4 framework region showed very low binding activity to VLP-hCLDN9, indicating that these antibodies maintained or improved selectivity for CLDN6.

[0141] Examples 64-91. Antibodies hAb-50-hAb-84 containing mutations in the heavy chain CDR region and introduced reverse mutations. The heavy chain was modified by introducing mutations at S31 and G100 and transplanted into a humanized VH2VL4 framework that included reverse mutations. Based on this, new mutations were introduced to obtain various humanized antibodies, including single, double, or multiple mutations, as shown in the table below.

[0142] [Table 14-1] [Table 14-2]

[0143] Examples 92-163. Generation of ADC-1 to ADC-72 using inter-antibody chain disulfide bonds and / or cysteine ​​mutation sites. The following ADC-1 to ADC-15 were prepared by attaching 1 to 20 antitumor toxin molecules via interchain disulfide bonds in the antibody. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0144] Figure 7A shows the overall method for preparing antibody-drug conjugates using maleimide connectors. The specific application process is as follows: Add 2 mg of antibody to a 0.5 mL centrifuge tube; dilute the antibody to 5 mg / mL using 30 mM His-HAC buffer (pH 5.5); add 100 mM EDTA aqueous solution to a final concentration of 5 mM and mix well by vortexing; add 2 to 15 times the molar mass of antibody in TCEP aqueous solution and mix well by vortexing; incubate in a thermostat mixer at 20°C for 1.5 hours; add 4 to 24 times the molar mass of antibody in toxin-linker solution and replenish with DMSO until the final reaction volume reaches 10%; mix well by vortexing; incubate in a thermostat mixer at 20°C for 0.5 to 24 hours; and after conjugation, remove free small molecules using 300 mg / mL dextran-coated activated carbon (Sigma). Next, residual activated carbon particles in the supernatant were filtered through a hydrophilic filter membrane, and the ADC storage buffer was replaced with 50 mM PBS (pH 6.0) using an ultrafiltration tube. The solution was then stored at -80°C for future use.

[0145] Specifically, in the synthesis of ADC-1 to ADC-15, the amounts of TCEP used were 4, 4, 2, 3, and 1.5 times the molar mass of the antibody, respectively, and the amounts of toxin-linker used were 4, 4, 4, 5, and 4 times the molar mass of the antibody, respectively. In the synthesis of IMAB027-vcMMAE, the amount of TCEP used was 3.4 times the molar mass of the antibody, and the amount of vcMMAE used was 10 times the molar mass of the antibody.

[0146] Figure 7B shows the overall method for preparing antibody-drug conjugates using olefin connectors. Antibody-drug conjugates are prepared by conjugating 1 to 20 antitumor toxin molecules. 2 mg of antibody was added to a 0.5 mL centrifuge tube. The antibody was diluted to 10 mg / mL by adding 25 mM sodium tetraborate (Na2B4O7), 25 mM sodium chloride (NaCl), and 1 mM DTPA (pH 7.4) buffer. An amount of TCEP aqueous solution equivalent to 2 to 15 times the molar mass of the antibody was added, mixed well by vortexing, and incubated in a thermostat mixer at 20°C for 2 to 24 hours. An amount of toxin-linker solution equivalent to 4 to 24 times the molar mass of the antibody was added, mixed well by vortexing, and incubated in a thermostat mixer at 20°C for 0.5 to 24 hours. After coupling was complete, the reaction was stopped and the ADC was purified. Taking ADC15 as an example, the amount of TCEP used was 15 times the molar mass of the antibody, and the amount of toxin-linker used was 24 times the molar mass of the antibody.

[0147] Figures 7A and 7C show the overall preparation method for antibody-drug conjugates. 1 to 20 antitumor toxin molecules were conjugated via inter-antibody disulfide bonds and / or cysteine ​​mutation sites to prepare ADC-16 to ADC-75.

[0148] Add 2 mg of antibody to a 0.5 mL centrifuge tube, dilute the antibody to 5 mg / mL with 30 mM His-HAC buffer (pH 5.5), add 100 mM EDTA aqueous solution to a final concentration of 5 mM, mix well by vortexing, add 2 mg / mL TCEP in a molar ratio of TCEP to antibody of 3:1, mix by vortexing, and incubate at 20°C for 1.5 hours using a thermostat mixer. Alternatively, replace the antibody buffer with 2 mM EDTA and 100 mM Tris-HCl (pH 8.0) using a desalting column. Add 1 mg of antibody to a 1.5 mL microcentrifuge tube, add 100 mM DTT aqueous solution in a molar ratio of DTT to antibody of 20:1 to 100:1, mix with a pipette, and incubate overnight at 22°C using a thermostat mixer. A 10 mg / mL linker-toxin DMSO solution was added in a compound-to-antibody molar ratio of 12:1, and DMSO was replenished to 20% of the final reaction volume. The mixture was thoroughly mixed by vortexing and incubated at 20°C for 1.5 hours using a thermostat mixer. After conjugation, free small molecules were removed using 300 mg / mL dextran-coated activated carbon (Sigma). Next, residual activated carbon particles in the supernatant were filtered through a hydrophilic filter membrane, and the ADC storage buffer was replaced with 50 mM PBS (pH 7.2) using an ultrafiltration tube, and the mixture was stored at -80°C for future use.

[0149] Alternatively, using a desalting column, the antibody buffer was replaced with 2 mM EDTA and 100 mM Tris-HCl (pH 8.0). 1 mg of antibody was added to a 1.5 mL microcentrifuge tube, 100 mM DTT was added to achieve a molar ratio of 100:1 between DTT and antibody, mixed by pipetting, and incubated overnight at 22°C using a thermostat mixer. Using a desalting column, the antibody buffer was replaced with 2 mM EDTA, 150 mM sodium chloride (NaCl), and 50 mM Tris-HCl (pH 7.5). 100 mM DHAA DMA solution was added to achieve a molar ratio of 20:1 between DHAA and antibody. Mixed thoroughly by pipetting, and incubated at 22°C for 2 hours using a thermostat mixer. Next, DHAA was removed using a desalting column, and the antibody buffer was replaced with 150 mM sodium chloride (NaCl) and 50 mM Tris-HCl (pH 7.5). A 10 mg / mL linker-toxin DMA solution was added in a compound-to-antibody molar ratio of 6:1, and the DMA was replenished to 10% of the final reaction volume. The mixture was thoroughly mixed by vortexing and incubated at 20°C for 4 hours using a thermostat mixer. After binding, free small molecules were removed using a desalting column. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0150] The ADC storage buffer was replaced with 50 mM His-HAC (pH 5.5), and the solution was stored at -80°C until use.

[0151] Example 164. In vitro cytotoxicity of antibody-drug conjugates based on anti-CLDN6 antibody and preferred linker payload against CLDN6-positive human ovarian cancer OV90 cells. 1.5 × 10 3Individual CLDN6-positive human ovarian cancer OV90 cells were seeded in a 96-well plate (excluding the wells at the ends 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.

[0152] After incubating in a constant temperature incubator for one day, the selected ADC was added to each well (20 μL), and the final ADC concentration range was 0.01 nM to 1000 nM. After further culturing for 5 days, the 96-well plate was taken out of the incubator and equilibrated to room temperature. After about 30 minutes, 40 μL of CellTiter-Glo® (Promega, G7572) was added to each well. The plate was shaken at 450 rpm for 5 minutes and equilibrated for 10 minutes without shaking, and then luminescence was measured using a SpectraMax i3x microplate reader. The luminescence curve was approximated as a function of the ADC concentration (nM) using Graphpad Prism software. The IC50 value was determined using the "log(inhibitor) vs. response - variable slope (four parameters)" function incorporated in Prism.

[0153] Figure 8A and the following table show the in vitro cytotoxicity of the antibody-drug conjugate.

[0154]

Table 15

[0155] Figure 8B and Figure 8C show the in vitro cytotoxicity of the selected antibody-drug conjugate against OV90 cells.

[0156] Example 165. In Vitro Cytotoxicity of an Antibody-Drug Conjugate Loaded with an Anti-CLDN6 Antibody and a Preferred Linker against CLDN6-Positive Human Choriocarcinoma JEG-3 Cells 1.5×10 3Individual CLDN6-positive human ovarian cancer JEG-3 cells were seeded into a 96-well plate (excluding the wells at the ends filled with PBS). The 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 in a humidified incubator at 37 °C and 7.5% CO2.

[0157] After incubation in a constant temperature incubator for 1 day, each ADC was added to each well (20 μL), and the final ADC concentration range was 0.01 nM to 1000 nM. After further culturing for 5 days, the 96-well plate was taken out of the incubator and equilibrated at room temperature. After about 30 minutes, 40 μL of CellTiter-Glo® (Promega, G7572) was added to each well. The plate was shaken at 450 rpm for 5 minutes and then equilibrated for 10 minutes without shaking, and the luminescence was measured using a SpectraMax i3x microplate reader. The luminescence curve was approximated as a function of the ADC concentration (nM) using Graphpad Prism software. The IC50 value was determined using the "log(inhibitor) vs. response - variable slope (four parameters)" function incorporated in Prism.

[0158] Figure 9 shows the in vitro cytotoxicity of the tested antibody-drug conjugates against JEG3 cells.

[0159] 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 On day 0 of the study, OV90 tumor cells (5×10 6The mice were inoculated with 200 μL of PBS containing cells / animals. ADCs compounded with PBS were intravenously administered at 10 mg / kg to groups of five mice on days 17 and 24 post-transplant. PBS served as a negative control. All treatment groups received weekly tail vein injections for 2–3 weeks. Tumor volume was measured to determine whether tumor growth was suppressed, delayed, or cured. Tumor size was measured twice weekly with calipers, and the mean tumor volume for each group was plotted over time. Volume was expressed in mm using the following formula. 3 It is expressed as: V = 0.5a × b 2 (In the formula, a and b are the longest and shortest diameters of the tumor, respectively). The use and welfare of the laboratory animals were carried out in accordance with the guidelines of the Association for Evaluation and Accreditation of Laboratory Animal Care (AAALAC).

[0160] Figure 10 shows the efficacy of administering ADC-2 and DAR2 for 2 weeks (A) and ADC-46 and DAR4 for 3 weeks (B) in a xenograft model of female BALB / C nude mice injected with hCLDN6-positive OV90 cells.

[0161] The embodiments of this invention are used solely to illustrate the invention and do not limit the scope of the claims. Any other substantially equivalent alternatives conceivable by those skilled in the art are also protected within the scope of this invention.

Claims

1. An isolated antigen-binding protein capable of binding to hCLDN6, characterized by comprising a heavy chain variable region, wherein the heavy chain variable region is X １ HCDR1 having the amino acid sequence of YTMS, TISSGGGX ２ TYYPDSVKG (in the formula, X ２ HCDR2 having an amino acid sequence of =R, N, Q, D, or E, and GDX ４ RYDX ３ FAY (in the formula, X ４ Having HCDR3 with the amino acid sequence (=Y, N, or Q), (1) X １ = Y, M, Q, D, or E, and X ３ = G, or (2) X １ = S or D, and X ３ = A, an antigen-binding protein.

2. The antigen-binding protein according to claim 1, characterized in that the heavy chain variable region has a human heavy chain framework region, the human heavy chain framework region has one or more reverse mutations compared to the mouse heavy chain framework region, the reverse mutation site of the heavy chain variable region includes R44, and the positional numbering is determined by the Kabat numbering scheme.

3. The heavy chain variable region has 92.59% or more identity with HFR1 to HCDR3, has the reverse mutation site, and X １ Part, X ２ Part, X ３ Body part, and / or X ４ The antigen-binding protein according to claim 2, characterized in that it is a sequence having a site.

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 NOs: 22, 23, 24, 59, 60, 61, 62, 63, 66, 67, and 133-153.

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. Furthermore, it includes a light chain variable region, and the light chain variable region is RASENIDSX ５ LA (wherein, X ５ =LCDR1 having an amino acid sequence of Y, Q, or R, LCDR2 having the amino acid sequence of ASTLLVD, and QHYYSX ６ PYT (in the formula ６ The antigen-binding protein according to claim 1, comprising an LCDR3 having the amino acid sequence of (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, and the light chain framework region has one or more reverse mutations compared to the mouse light chain framework region, the reverse mutation sites of the light chain framework region include one or more of R66, Q70, and R85, and the positional numbering is determined by a Kabat numbering scheme.

8. The light chain variable region has 95.88% or more identity with HFR1 to HCDR3, has the reverse mutation site, and X ５ Body part and / or X ６ The antigen-binding protein according to claim 7, characterized in that it is a sequence having a 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 sequence numbers 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 by comprising a human heavy chain constant region and a human light chain constant region.

12. The antigen-binding protein according to claim 1, characterized in that it contains a human heavy chain constant region.

13. The antigen-binding protein according to claim 11 or 12, characterized in that the heavy chain constant region is the human IgG1 heavy chain constant region and contains one or more mutations selected from the group consisting 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 according to claim 1, characterized in that the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

17. The antigen-binding protein according to claim 2, characterized by having a heavy chain having the sequence shown in any one of sequence numbers 157 and 159-167.

18. The antigen-binding protein according to claim 2, characterized by having a heavy chain having the sequence shown in either one of sequence numbers 158 and 171.

19. The antigen-binding protein according to claim 7, characterized by having a light chain having the sequence shown in either one of sequence numbers 169 and 170.

20. The antigen-binding protein according to claim 7, characterized by having a light chain having the sequence shown in Sequence ID No.

168.

21. The antigen-binding protein according to claim 1, characterized in that the antigen-binding protein is an antibody hAb-13 comprising a heavy chain variable region having the sequence shown in SEQ ID NO: 18 and a light chain variable region shown in SEQ ID NO:

28.

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

169.

23. A nucleic acid molecule characterized by being used to encode an antigen-binding protein according to any one of claims 1 to 22.

24. A vector characterized by containing the nucleic acid molecule described in claim 23.

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

26. A pharmaceutical molecule characterized by comprising an antigen-binding protein as described in any one of claims 1 to 22.

27. Formula I: [Math 1] The pharmaceutical molecule according to claim 26, characterized by having the structure shown in the formula (wherein Ab is the antigen-binding protein, L is a linker comprising one or more connectors, D is a therapeutic agent or a detectable marker, and n is an integer selected from 1 to 20).

28. The pharmaceutical molecule according to claim 27, characterized in that n is an integer selected from 2 to 8.

29. The pharmaceutical molecule according to claim 27, characterized in that the therapeutic agent is selected from an immunomodulator, a cytotoxic agent, a cell proliferation inhibitor, a radioisotope, an anti-angiogenic agent, or a liposome.

30. The pharmacokinetic molecule according to claim 29, characterized in that the cytotoxic agent is selected from one or more of the following: meitansin alkaloids, auristatin, eribulin, taxanes, calicheamycin, semadin, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, α-amanitin and its derivatives, trabectedin and its derivatives, and lurbinectidin and its derivatives.

31. The pharmaceutical molecule according to claim 27, characterized in that the connector is selected from one or more of the following: an oligopeptide connector, a hydrazine connector, a thiourea connector, a triggered self-deleting connector, a succinimidyltrans-4-(maleimidomethyl)cyclohexane-1-carboxylate connector, a maleimide connector, a disulfide connector, a thioether connector, and an olefin connector.

32. ADC1 to ADC72: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 【Chemistry 31】 【Chemistry 32】 【Transformation 33】 【Transformation 34】 【Chemistry 35】 【Transformation 36】 【Chemistry 37】 【Transformation 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 [Transformation 50] 【Chemistry 51】 【Chemistry 52】 【Chemistry 53】 【Chemistry 54】 【Transformation 55】 【Transformation 56】 【Chemistry 57】 【Chemistry 58】 【Chemistry 59】 【Transformation 60】 【Chemistry 61】 【Transformation 62】 【Transformation 63】 【Chemistry 64】 【Transformation 65】 【Chemical Formula 66】 【Transformation 67】 【Transformation 68】 【Transformation 69】 【Transformation 70】 【Chemistry 71】 【Chemistry 72】 The pharmaceutical molecule according to claim 27, characterized in that it is selected from any one of the following.

33. Use in the manufacture of a pharmaceutical product for diagnosing, preventing and / or treating hCLDN6-related diseases and / or disorders of an antigen-binding protein according to any one of claims 1 to 22, a nucleic acid molecule according to claim 23, a vector according to claim 24, a cell according to claim 25, or a pharmaceutical molecule according to any one of claims 26 to 32.

34. The use according to claim 33, characterized in that the disease and / or disorder includes a tumor, and the tumor is ovarian cancer, testicular cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, choriocarcinoma, or endometrial cancer.

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