Conjugate of Anti-CDH3 humanized antibody and drug, and use thereof

Abstract

The present invention addresses the problem of providing a novel anti-CDH3 humanized antibody-drug conjugate. Provided according to the present invention is an antibody-drug conjugate in which an anti-CDH3 humanized antibody is linked with a chemotherapeutic agent having a linker, wherein the anti-CDH3 humanized antibody is (1) an anti-CDH3 humanized antibody having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 2, (2) an anti-CDH3 humanized antibody having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 4, etc., the chemotherapeutic having a linker is represented by formula (x): (in the formula, * indicates the connection with the antibody.), etc.

Description

Conjugates of anti-CDH3 humanized antibodies and drugs, and uses thereof

[0001] The present invention relates to a conjugate of an anti-CDH3 humanized antibody and a drug. The present invention further relates to a medicament comprising the conjugate.

[0002] Cancer is a serious disease that is one of the leading causes of death, yet there is still an unmet need for treatment. In recent years, in order to solve the problem of conventional chemotherapy that also damages normal cells, there has been active research into cancer treatment using molecular targeted drugs, which are designed to target specific molecules that are specifically expressed in cancer cells.

[0003] One of the targets was identified as a cell membrane surface antigen, CDH3 (P-cadherin). CDH3 is a membrane protein discovered as a molecule involved in calcium-dependent homophilic cell adhesion. Proteins with cadherin repeats consisting of approximately 110 amino acid residues that share high homology with each other are called the cadherin superfamily, and CDH3 is a major member of this family.

[0004] Cases of elevated CDH3 expression in certain cancer cells have been reported, and cancer therapy using antibodies against cancer cells with higher CDH3 expression in cancer tissues compared to normal tissues is being investigated.

[0005] One effective way to enhance the anti-cancer activity of antibodies is to link them to highly toxic substances (drugs). When administered alone to a patient, highly toxic substances also damage normal tissues, making them ineffective therapeutic approaches. However, by linking a drug to an antibody that binds to a cancer cell-specific antigen, it is possible to kill only cancer cells without adversely affecting normal tissues. Such drugs are called antibody-drug conjugates (ADCs). That is, drugs do not exhibit any toxicity when bound to an antibody. However, certain antibodies, once bound to cells expressing the target antigen, are internalized into those cells and degraded in the lysosomes. Therefore, after such drug-conjugated antibodies are internalized, the drug is released by intracellular degradation, exerting toxicity only within specific cells, resulting in cell death.

[0006] Patent Documents 1 and 2 describe anti-CDH3 (P-cadherin) antibodies and drug conjugates thereof. Patent Documents 3 to 6 describe antibody-drug conjugates and synthetic intermediates thereof.

[0007] International Publication No. WO2013 / 150623 International Publication No. WO2014 / 126198 International Publication No. WO2023 / 163227 International Publication No. WO2023 / 163234 International Publication No. WO2023 / 234426 International Publication No. WO2023 / 234427

[0008] An object of the present invention is to provide a novel anti-CDH3 humanized antibody-drug conjugate.

[0009] As a result of intensive research to solve the above-mentioned problems, the present inventors have succeeded in producing a less immunogenic anti-CDH3 humanized antibody by combining a complementarity-determining region (CDR) sequence defined from an antibody that specifically recognizes CDH3 with various human-derived framework region (FR) sequences and by introducing appropriate amino acid mutations to improve affinity, and further in using this to produce an anti-CDH3 humanized antibody-drug conjugate that more efficiently kills CDH3-expressing cancer cells, thereby completing the present invention. According to the present invention, the following inventions are provided.

[0010] <1> An antibody-drug conjugate in which an anti-CDH3 humanized antibody and a chemotherapeutic agent having a linker are linked, wherein the anti-CDH3 humanized antibody is: (1) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2; (2) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4; (3) a fragment of the anti-CDH3 humanized antibody set forth in (1) or (2) above, which has the ability to bind to CDH3; or (4) an antibody consisting of a partial sequence of the antibody set forth in (1) or (2) above, which has the ability to bind to CDH3; and the chemotherapeutic agent having a linker is represented by formula (x): (wherein * indicates the connecting portion to the antibody) or formula (y): (wherein * represents the connecting portion to the antibody), a pharmacologically acceptable salt thereof, or a hydrate thereof. <2> The antibody-drug conjugate according to <1>, a pharmacologically acceptable salt thereof, or a hydrate thereof, wherein the anti-CDH3 humanized antibody is any of the following antibodies: (1) An anti-CDH3 humanized antibody having a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 6. (2) An anti-CDH3 humanized antibody having a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 8. <3> The antibody-drug conjugate according to <1>, a pharmacologically acceptable salt thereof, or a hydrate thereof, wherein an average of 1 to 10 molecules of a chemotherapeutic agent having a linker are bound to one molecule of the anti-CDH3 humanized antibody. <4> A pharmaceutical for treating a disease characterized by overexpression of CDH3, comprising the antibody-drug conjugate according to any one of <1> to <3>, a pharmacologically acceptable salt thereof, or a hydrate thereof. <5> The pharmaceutical according to <4>, wherein the disease characterized by overexpression of CDH3 is a tumor or cancer.

[0011] The present invention further provides a method for treating a disease characterized by overexpression of CDH3, comprising administering the above-described antibody-drug conjugate to a patient.The present invention further provides use of the above-described antibody-drug conjugate for the manufacture of a medicament for treating a disease characterized by overexpression of CDH3.The present invention further provides the above-described antibody-drug conjugate for use in treating a disease characterized by overexpression of CDH3.

[0012] Anti-CDH3 humanized antibodies are expected to have reduced immunogenicity compared to their parent antibodies. Humanized antibodies are constructed by appropriately combining the CDR sequences of the parent antibody with human-derived FR sequences. If these antibodies lack affinity for the antigen, attempts can be made to restore affinity by introducing amino acid mutations into the antibody variable region. By combining CDR sequences with appropriate FR sequences and optionally introducing amino acid mutations, anti-CDH3 humanized antibodies that specifically bind to CDH3 can be obtained. Immunoconjugates formed by linking the anti-CDH3 humanized antibody of the present invention to a chemotherapeutic agent thus obtained exhibit potent cytotoxic activity against cancer cells expressing CDH3 compared to antibodies not bound to the chemotherapeutic agent. By administering the immunoconjugate of the present invention to patients with cancer cells expressing CDH3, a strong anticancer effect can be achieved while also reducing the immunogenicity of the immunoconjugate itself. The immunoconjugates of the present invention are useful as anticancer agents.

[0013] Figure 1 shows the percentage of viable cells in a cell viability assay for ADC1 and ADC3. Figure 2 shows the percentage of viable cells in a cell viability assay for ADC1 and ADC2. Figure 3 shows the percentage of viable cells in a cell viability assay for ADC3 and ADC4. Figure 4 shows the results of an in vivo mouse efficacy study for ADC3. Figure 5 shows the weight results during an in vivo mouse efficacy study for ADC3. Figure 6 shows the results of an in vivo mouse efficacy study for ADC4. Figure 7 shows the weight results during an in vivo mouse efficacy study for ADC4. Figure 8 shows the results of an in vivo mouse efficacy study for ADC3, ADC5, and ADC7. Figure 9 shows the weight results during an in vivo mouse efficacy study for ADC3, ADC5, and ADC7. Figure 10 shows the results of an in vivo mouse pharmacokinetic study.

[0014] The present invention is described in further detail below. The present invention relates to an antibody-drug conjugate comprising an anti-CDH3 humanized antibody and a chemotherapeutic agent having a linker linked thereto, wherein the anti-CDH3 humanized antibody is: (1) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2; (2) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4; (3) a fragment of the anti-CDH3 humanized antibody set forth in (1) or (2) above, which has the ability to bind to CDH3; or (4) an antibody consisting of a partial sequence of the antibody set forth in (1) or (2) above, which has the ability to bind to CDH3; and the chemotherapeutic agent having a linker is represented by formula (x): (wherein * indicates the connecting portion to the antibody) or Formula (y): (wherein * represents the connecting portion to the antibody), or a pharmacologically acceptable salt thereof, or a hydrate thereof. In the present invention, a chemotherapeutic agent having a linker represented by the above formula (x) or (y) is used; however, it has been found that when a chemotherapeutic agent having a linker other than that represented by formula (x) or (y) (for example, a chemotherapeutic agent in which the chemical structure of a portion of the linker may be different and monomethyl auristatin E (MMAE) or eribulin, etc. is used as the chemotherapeutic agent) is used, the medicinal effect is weaker than that of the antibody-drug conjugate of the present invention.

[0015] <Anti-CDH3 humanized antibody> The antibody of the present invention is any of the following antibodies: (1) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2; (2) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4;

[0016] The antibody of the present invention is preferably any one of the following antibodies: (1) an anti-CDH3 humanized antibody having a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 6; (2) an anti-CDH3 humanized antibody having a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0017] The anti-CDH3 humanized antibodies of the present invention are provided by combining CDR sequences defined from an antibody that specifically recognizes CDH3 (preferably human CDH3) with various appropriate human-derived FR sequences, and further by introducing appropriate amino acid mutations to improve affinity.

[0018] In one embodiment, the antibody of the present invention binds to CDH3 expressed on a cell surface. In one embodiment, the antibody of the present invention binds to an epitope within a CDH3 region. Preferably, the antibody of the present invention binds to CDH3 expressed on the surface of human cells, and particularly preferably binds to CDH3 expressed on the surface of cancer cells.

[0019] As an antigen for producing the antibody of the present invention, CDH3 (preferably human CDH3) or a partial peptide thereof can be used. One example is a soluble CDH3 protein corresponding to the extracellular domain of CDH3 (corresponding to amino acids 108 to 654 of SEQ ID NO: 2 in International Publication WO 2014 / 126198), but is not limited thereto.

[0020] The antibody of the present invention is a humanized monoclonal antibody. In the present invention, hybridomas are obtained as a material for obtaining the humanized monoclonal antibody by immunizing mice. Such materials can be obtained by various methods well known in the art. For example, they can be obtained by the method described below, but are not limited to these.

[0021] To establish hybridomas that produce antibodies that specifically bind to CDH3, CDH3 or a partial peptide thereof is first administered to mice as an antigen. The dose per mouse is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is typically carried out by intravenous, subcutaneous, or intraperitoneal injection. The interval between immunizations is not particularly limited; immunizations are performed 1 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 2 to 5 weeks. Antibody-producing cells are then collected 1 to 60 days, preferably 1 to 14 days, after the final immunization. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells or local lymph node cells being preferred.

[0022] To obtain hybridomas, antibody-producing cells are fused with myeloma cells. Commonly available mouse-derived cell lines that are drug-selective for HAT medium or similar can be used as myeloma cells. Examples include P3X63-Ag. 8. U1 (P3U1) and NS-1.

[0023] Cell fusion was carried out in an animal cell culture medium such as serum-free DMEM or RPMI-1640 medium, with 1×10 ６ ~1 x 10 ７ / mL antibody-producing cells and 2 × 10 ５ ~2 x 10 ６ The antibody-producing cells can be mixed with myeloma cells at a concentration of 1000 / mL and the fusion reaction can be carried out in the presence of a cell fusion promoter. Examples of cell fusion promoters that can be used include polyethylene glycol with an average molecular weight of 1,000 to 6,000 daltons. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation.

[0024] Hybridomas can be obtained by culturing in a selective medium. The cell suspension is appropriately diluted with, for example, RPMI-1640 medium containing fetal bovine serum, and then plated at 3 x 10 cells on a microtiter plate. ５The cells are seeded at approximately 100 cells / well, and selective medium is added to each well. After that, the selective medium is replaced as needed and the cells are cultured. As a result, hybridomas can be obtained from the cells that grow from about 14 days after the start of culture in the selective medium.

[0025] Next, the culture supernatant of the grown hybridomas is screened for the presence or absence of the target antibody. Screening of hybridomas can be performed according to conventional methods and is not particularly limited. For example, a portion of the culture supernatant contained in the well in which the hybridomas have grown can be collected, and hybridomas that produce antibodies that bind to CDH3 can be screened using enzyme immunoassay, radioimmunoassay, or the like. Cloning of the fused cells can be performed using limiting dilution or the like, and ultimately hybridomas that produce monoclonal antibody can be established.

[0026] Using established hybridomas as a starting material, antibodies derived therefrom can be humanized using known methods. Specifically, a DNA sequence designed to link the CDR sequences of a mouse antibody with the FR sequences of a human antibody is synthesized by PCR from several oligonucleotides engineered to have overlapping ends. The resulting DNA is ligated to DNA encoding the constant region of a human antibody, then inserted into an expression vector, which is then introduced into a host for production (see, for example, EP 239400 and WO 96 / 02576).

[0027] CDR sequences are particularly divergent in the variable regions of antibodies and represent sequence regions that play an extremely important role in determining the specificity of the antibody. The amino acid residues in these regions are thought to contain many residues directly involved in antigen binding and specificity, and three CDR regions exist in each of the light and heavy chain variable regions. CDR sequences were defined by sequence comparison by Kabat et al. (Sequences of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and also by three-dimensional structure by Chothia et al. (J. Mol. Biol.; 196, p. 901 (1987)).

[0028] The CDRs defined by Kabat are generally considered to be located around residues 24-34, around residues 50-56, and around residues 89-97 in the light chain variable region, and around residues 31-35, around residues 50-65, and around residues 95-102 in the heavy chain variable region; however, not all residues in these regions are necessarily directly involved in antigen binding, and they do not necessarily completely match the CDR sequences defined from the three-dimensional structure.

[0029] The human FR sequences to be selected are appropriately selected at any time. In the present specification, FR sequences derived from human germline sequences selected under optimal alignment were used.

[0030] Specifically, the variable region sequences of a mouse antibody are screened against a publicly available library of human variable region sequences, and the most similar human variable region sequence can be used as the human FR sequence derived from the germline of the humanized antibody (Sims et al., J. Immunol.; 151, p. 2296 (1993); Chothia et al., J. Mol. Biol.; 196, p. 901 (1987); Tempest et al., Biotechnology; 9, p. 266 (1991)).

[0031] The germline sequence that is most similar in sequence can be confirmed by performing alignment (e.g., IMGT / V-QUEST (https: / / www.imgt.org / IMGT_vquest / vquest?livret=0&Option=humanIg)) against the sequence of the original antibody in a database in which a large number of such libraries are registered.

[0032] Whether a selected FR sequence is suitable for humanization can be determined by whether it is appropriately combined with each antibody clone CDR sequence and whether it can maintain an appropriate three-dimensional structure for the antigen to which it is intended to bind.

[0033] If a humanized antibody is expressed by flanking a CDR sequence and a human-derived FR sequence and the sequence is not selected appropriately, the affinity may often decrease. This means that some residues present in the FR sequence also play an important role in maintaining the structure. Amino acid residue substitutions can be made to address this issue. For example, as a result of alignment, the amino acid residues present in the human-derived sequence can be substituted (reshaped) with the same residue as the mouse antibody amino acid present in a position homologous to the human sequence. This may improve the affinity loss caused by humanization.

[0034] The positions of the amino acid residues to be substituted vary depending on the antibody of interest, and in many cases are not identified until the antibody is actually expressed. In the following examples, the amino acid residues at one or more positions selected from position 55 in the light chain variable region and positions 49, 71, and 78 in the heavy chain variable region, which are positions where substitutions are performed in a relatively large number of publications (e.g., Proc. Natl. Acad. Sci. USA; 89, p. 4285 (1992)), were substituted to improve affinity reduction, but the substitution positions, combinations, and types of amino acid residues after substitution are arbitrary and are not limited thereto.

[0035] While many hosts for antibody production are mammalian in origin, those skilled in the art can select a specific host cell line that best suits the gene product desired to be expressed. Common host cell lines include, but are not limited to, CHO-derived cell lines (Chinese hamster ovary cell lines), CV1 (monkey kidney cell lines), COS (a CV1 derivative expressing the SV40 T antigen), SP2 / 0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), 293 (human kidney), and 293T (a 293 derivative expressing the SV40 T antigen). Host cell lines can be obtained from various manufacturers, the American Tissue Culture Collection (ATCC), or from sources where they have been published.

[0036] Preferably, a CHO-derived cell line or SP2 / 0 deficient in the expression of the dgfr gene can be used as the host cell line (Urland, G. et al., Somat. Cell. Mol. Genet.; 12, p. 5555 (1986) (Non-Patent Document 4), and Schulman, M. et al., Nature; 276, p. 269 (1978) (Non-Patent Document 5)). Most preferably, the host cell line is a DHFR-deficient CHO.

[0037] Transfection of a plasmid into a host cell can be carried out using any technique. Specific methods include, but are not limited to, transfection (including calcium phosphate, DEAE, lipofection, and electroporation), methods of introducing DNA using an envelope such as Sendai virus, microinjection, and infection using a viral vector such as retrovirus or adenovirus (Current Protocols in Molecular Biology, Chapter 9 Introduction of DNA into Mammalian Cells, John Wiley and Sons, Inc.). The most preferred method is introduction of a plasmid into a host cell by electroporation.

[0038] These antibodies may be monovalent, bivalent, or multivalent antibodies, as long as they have the ability to bind to CDH3. Furthermore, these antibodies may be minibodies such as antibody fragments, modified antibodies, or partial antibody sequences, as long as they have the ability to bind to CDH3. Examples of minibodies such as antibody fragments include Fab, Fab', and F(ab'). ２ , Fv, ScFv (single chain Fv), diabodies, etc. Furthermore, antibody fragments and minibodies, for example, Fab, Fab', F(ab') ２ The antibody may be one in which the Fc portion is fused to Fv, ScFv (single chain Fv), diabody, etc. Such antibodies can be obtained by constructing genes encoding these antibodies, introducing them into an expression vector, and then expressing them in appropriate host cells.

[0039] <Chemotherapeutic agent having a linker> As the chemotherapeutic agent having a linker, those represented by the above formula (x) or formula (y) are used. In the chemotherapeutic agent having a linker represented by formula (x), the compound before being linked to the antibody can be synthesized using a method similar to Reference Example 3 described in WO2023163227, Example 3 described in WO2023163234, Reference Example A3 described in WO2023234426, or Example A3 described in WO2023234427. In the chemotherapeutic agent having a linker represented by formula (y), the compound before being linked to the antibody can be synthesized using a method similar to Reference Example 2 described in WO2023234426 or Example 2 described in WO2023234427.

[0040] <Method of linking an antibody to a chemotherapeutic agent having a linker> Linking an antibody to a chemotherapeutic agent having a linker can be carried out by a method similar to that described in Patent Documents 3 to 6 (WO2023 / 163227, WO2023 / 163234, WO2023 / 234426, WO2023 / 234427).

[0041] The number of chemotherapeutic agent molecules bound to one molecule of an anti-CDH3 humanized antibody, a fragment thereof, or a partial sequence thereof is not particularly limited, but preferably 1 to 16 molecules, and more preferably 6 to 16 molecules, of the chemotherapeutic agent are bound. For example, one molecule of an anti-CDH3 humanized antibody, a fragment thereof, or a partial sequence thereof may be bound to one molecule of auristatin E. Furthermore, a conjugate of an anti-CDH3 humanized antibody and a drug of the present invention can be represented by the general formula (I): A-(B-C)a (wherein A is the moiety obtained by removing sulfhydryl groups from a reduced antibody, B is an -S- group, C is a chemotherapeutic agent having a linker, and a is the average number of linker-drug units (B-C) bound per antibody molecule, which is a number from 1 to 10 that may include a decimal point. Hereinafter, with regard to ADC, the average number of drugs bound per antibody, or the drug / antibody ratio, is referred to as "DAR," which is a number from 1 to 20 that may include a decimal point, and is expressed to one or two decimal points in the present specification.) The DAR is preferably a number from 2 to 18, and more preferably a number from 3 to 15.

[0042] <Forms of Antibody-Drug Conjugates> Examples of the "pharmacologically acceptable salts" used herein include alkali metal salts such as lithium, sodium, and potassium; Group 2 metal salts such as magnesium and calcium; salts with aluminum or zinc; salts with amines such as ammonia, choline, diethanolamine, lysine, ethylenediamine, tert-butylamine, tert-octylamine, tris(hydroxymethyl)aminomethane, N-methyl-glucosamine, triethanolamine, and dehydroabietylamine; salts with inorganic acids such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, and benzenesulfonic acid; and salts with acidic amino acids such as aspartic acid and glutamic acid. Furthermore, the term "pharmacologically acceptable salts" also encompasses internal salts.

[0043] The antibody-drug conjugate of the present invention and its pharmacologically acceptable salt may be obtained as a hydrate or solvate, and both are encompassed by the present invention. The antibody-drug conjugate of the present invention or a salt thereof may absorb moisture and become adsorbed water or hydrate upon exposure to air or recrystallization, and such water-containing antibody-drug conjugates or salts thereof are also encompassed by the present invention.

[0044] When the chemotherapeutic agent moiety having a linker in the antibody-drug conjugate of the present invention contains a partial structure having an asymmetric center and optical isomerism can occur, all optical isomers are included in the present invention.

[0045] The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugate of the present invention may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium ( ２ H), tritium ( ３ H), iodine-125 ( １２５ I) or carbon-14 ( １４ Furthermore, the antibody-drug conjugate of the present invention can be used in combination with, for example, tritium ( ３ H), iodine-125 ( １２５ I) or carbon-14 ( １４ The antibody-drug conjugates of the present invention may be radiolabeled with a radioisotope such as 1,4-dichloro-2,4-dichloro- ...

[0046] <Medicine and Method of Use> The present invention provides a pharmaceutical for treating a disease characterized by overexpression of CDH3, comprising the antibody-drug conjugate of the present invention. Diseases characterized by overexpression of CDH3 are not particularly limited as long as CDH3 is expressed in the cells, and examples include tumors and cancers. Examples include hematopoietic tumors or cancers such as lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma) and leukemia, as well as solid tumors or solid cancers. Examples of hematopoietic tumors or cancers include follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myeloblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma. Examples of solid tumors or solid cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, Examples of tumor types that can be treated include sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, biliary tract cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, thyroid cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma. However, the tumor cells to be treated may be tumor cells that express CDH3.

[0047] The antibody-drug conjugate of the present invention can be administered to mammals, preferably humans. Substances used in pharmaceutical compositions containing the antibody-drug conjugate of the present invention can be arbitrarily selected from formulation additives and other substances commonly used in this field.

[0048] The antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically acceptable ingredients. For example, the pharmaceutical compositions typically contain one or more pharmaceutically acceptable carriers. Examples of such carriers include sterile liquids such as water, mineral oil, animal oil, and vegetable oil (e.g., peanut oil, soybean oil, and sesame oil). Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solution, aqueous dextrose, and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Various pharmacologically acceptable substances known in the art can be used as excipients. The pharmaceutical compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of pharmaceutically acceptable carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0049] The antibody-drug conjugate of the present invention can be administered by various routes. A preferred administration route is parenteral administration, including subcutaneous injection, intravenous injection, intramuscular injection, or intrasternal injection. Intravenous administration is more preferred, and can be administered by drip infusion or bolus injection.

[0050] Pharmaceutical compositions adapted for intravenous administration to humans are formulated according to conventional methods. A typical pharmaceutical composition for intravenous administration is a solution in sterile isotonic aqueous buffer. The pharmaceutical composition may optionally contain a local anesthetic (e.g., lignocaine) to ease pain at the injection site.

[0051] The pharmaceutical composition may be a pharmaceutical composition containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition containing the antibody-drug conjugate and one or more other antitumor drugs.

[0052] The antibody-drug conjugate of the present invention can be administered together with other antitumor drugs, thereby enhancing the antitumor effect. The other antitumor drugs used for this purpose may be administered simultaneously with the antibody-drug conjugate, separately, or sequentially, and may be administered at different administration intervals. Examples of such antitumor drugs include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, or drugs described in International Publication No. WO 2003 / 038043, as well as LH-RH analogs (e.g., leuprorelin, goserelin), estramustine phosphate, estrogen antagonists (e.g., tamoxifen, raloxifene), and aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), but are not limited thereto as long as they have antitumor activity. Such pharmaceutical compositions can be formulated as lyophilized or liquid formulations. When formulated as lyophilized formulations, appropriate formulation additives used in this field may be added. This also applies to liquid formulations.

[0053] The composition and concentration of the pharmaceutical composition will vary depending on the administration method, but may also vary depending on the affinity of the antibody-drug conjugate contained in the pharmaceutical composition for CDH3 and the antitumor activity of the antitumor drug bound to it. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0054] The present invention will be described in more detail by the following examples, which are merely illustrative and are not intended to limit the scope of the present invention. All prior art documents cited in this specification are incorporated herein by reference.

[0055] Reference Example 1: Method for producing humanized antibody (1) Production of anti-CDH3 mouse antibody Anti-CDH3 mouse antibody was produced as described in Examples 1 to 9 of Japanese Patent No. 6377601, and the sequences of the variable regions of the anti-CDH3 mouse antibody were obtained by sequence analysis.

[0056] ApE and GENETYX (Genetyx Corporation) were used for sequence analysis. CLUSTALW was used for gene alignment. HPRD, NCBI BLAST, UCSF (University of California, Santa Cruz)-Blat, and UniProt were used to obtain and confirm gene and protein information.

[0057] Of the variable regions of the anti-CDH3 mouse antibodies derived from the mouse hybridomas sequenced in this manner, the amino acid sequences corresponding to the CDR sequences of T004 are shown below. SLTSYGVH: SEQ ID NO: 9 (CDR-H1) GVIWSGGSTD: SEQ ID NO: 10 (CDR-H2) ARNSNNGFAY: SEQ ID NO: 11 (CDR-H3) NIYSNLA: SEQ ID NO: 12 (CDR-L1) LLVYAAKN: SEQ ID NO: 13 (CDR-L2) QHFYDTPWT: SEQ ID NO: 14 (CDR-L3) In addition, the amino acid sequences corresponding to the CDR sequences of T004b are shown below. GYSFTAYN: SEQ ID NO: 15 (CDR-H1) IDPYSGII: SEQ ID NO: 16 (CDR-H2) ARRGYYDGGFDY: SEQ ID NO: 17 (CDR-H3) QDITNY: SEQ ID NO: 18 (CDR-L1) YTS: SEQ ID NO: 19 (CDR-L2) QQDSKHPRT: SEQ ID NO: 20 (CDR-L3) CDR-H1, H2, and H3 represent the CDR sequences constituting the heavy chain, and CDR-L1, L2, and L3 represent the CDR sequences constituting the light chain. The amino acid sequences of the CDRs shown were obtained by searching the nucleotide sequences of the variable regions of the anti-CDH3 mouse antibody using IMGT / V-QUEST (https: / / www.imgt.org / IMGT_vquest / vquest?livret=0&Option=humanIg).

[0058] (2) Preparation of transient expression vector for anti-CDH3 antibody For chimerization, a gene for the light chain variable region of the CDH3 mouse antibody was designed to be linked to a chimeric light chain expression vector encoding human Ck. For the heavy chain, a gene for the heavy chain variable region of the CDH3 mouse antibody was designed to be linked to a vector encoding the human Cg1 region.

[0059] For humanization, mouse-derived CDR sequences and human-derived FR sequences were combined, and a human Fc region was further linked to construct a stable-producing strain using a humanized antibody expression vector. More specifically, the procedure is as follows.

[0060] The region corresponding to the mouse FR sequence was replaced with a human-derived FR sequence, and the full length was artificially synthesized by GenScript. The amino acid sequence of the region corresponding to the FR sequence was a human germline FR sequence. The germline FR sequence was designed by inputting the base sequence of the cloned anti-CDH3 mouse antibody into IMGT / V-QUEST (https: / / www.imgt.org / IMGT_vquest / vquest?livret=0&Option=humanIg) and selecting the sequence with the highest similarity. In addition, amino acid sequence replacement (reshaping) corresponding to reduced affinity was also performed.

[0061] The amino acid sequences of the heavy chain or light chain variable regions of the anti-CDH3 humanized antibodies used in this study are shown below. Note that T004 and T004b use human germline FR sequences that are most similar to the FR sequences of their respective parent mouse antibodies. Furthermore, T004 and T004b have introduced the amino acid sequence substitutions noted below.

[0062] Antibody number: T004-VH QVQLVESGGGVVQPGRSLRLSCAASGFFSLTSYGVHWVRQAPGKGLEWVGVIWSGGSTDYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARNSNNGFAYWGQGTLVTVSS (SEQ ID NO: 1) (R71K, L78V substitutions compared to the selected germline FR sequence)

[0063] Antibody number: T004-VL DIQLTQSPSSLSASVGDRVTITCRASQNIYSNLAWYQQKPGKAPKLLVYAAKNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFYDTPWTFGQGTKVEIK (SEQ ID NO: 2) (E55A substituted relative to the selected germline FR sequence)

[0064] Antibody number: T004b-VH EVQLVQSGAEVKKPGATVKISCKVSGYSFTAYNMHWVQQAPGKGLEWMGFIDPYSGIITYAEKFQGRVTITVDKSTDTAYMELSSLRSEDTAVYYCARRGYYDGGFDYWGQGTLVTVSS (SEQ ID NO: 3) (A72V and T74K have been substituted relative to the selected germline FR sequence)

[0065] Antibody number: T004b-VL DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYYTSRLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSKHPRTFGQGTKVEIK (SEQ ID NO: 4) (E55H substitution relative to the selected germline FR sequence)

[0066] The artificial synthetic sequence genes designed to have these sequences were incorporated into a pCXN vector containing a human IgG1-derived constant region gene for the heavy chain variable region, and into a pCXN vector containing a human κ chain-derived constant region gene for the light chain variable region.

[0067] The full-length amino acid sequences of the anti-CDH3 humanized antibodies obtained by expressing these antibodies are shown below. Here, the anti-CDH3 humanized antibody T004 consists of an H chain T004-H and an L chain T004-L, and the anti-CDH3 humanized antibody T004b consists of an H chain T004b-H and an L chain T004b-L. Antibody number: T004-H QVQLVESGGGVVQPGRSLRLSCAASGFSLTSYGVHWVRQAPGKGLEWVGVIWSGGSTDYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARNSNNGFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0068] Antibody number: T004-L DIQLTQSPSSLSASVGDRVTITCRASQNIYSNLAWYQQKPGKAPKLLVYAAKNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFYDTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6)

[0069] Antibody number: T004b-H EVQLVQSGAEVKKPGATVKISCKVSGYSFTAYNMHWVQQAPGKGLEWMGFIDPYSGIITYAEKFQGRVTITVDKSTDTAYMELSSLRSEDTAVYYCARRGYYDGGFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

[0070] Antibody number: T004b-L DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYYTSRLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSKHPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8)

[0071] <Synthesis Example 1> PGAP-1 ((6S,9S,12S,31S)-1-amino-6-((4-(((((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-1-phenylpropoxy)(hydroxy)phos (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-9-isopropyl-1,8,11,14,30,34-hexaoxo-12-(2-oxo-2-(((S)-2-oxotetrahydrofuran-3-yl)amino)ethyl)-17,20,23,26-tetraoxa-2,7,10,13,29,33-hexaazahexatriacontan-36-yl)phosphonic acid

[0072] The compound of Synthesis Example 1 can be synthesized using a method similar to Reference Example 3 described in WO2023163227, Example 3 described in WO2023163234, Reference Example A3 described in WO2023234426, or Example A3 described in WO2023234427. The MS of Synthesis Example 1 is shown below. MS (DUIS) m / z 1921 (M+H) ＋ MS (DUIS) m / z 1919 (MH) － (Analysis conditions) Apparatus: LCMS-2050 System, Shimadzu Corporation Column: ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 50 mm, Waters Eluent: Solution A: 0.1% formic acid aqueous solution, Solution B: 0.1% formic acid acetonitrile solution Gradient (B%): 25 (0 min) → 95 (0.75 min) → 95 (1.5 min) Temperature: 40°C Flow rate: 0.8 mL / min Injection amount: 5 μL

[0073] <Synthesis Example 2> PGAP-2

[0074] ((6S,9S,12S,41S)-1-amino-23-((6S,9S,12S)-1-amino-6-((4-(((((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy -2-methylpropanamido)-1-phenylpropoxy)(hydroxy)phosphoryl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,14-tetraoxo-12-(2-oxo-2-(((S)-2-oxotetrahydrofuran-3-yl)amino)ethyl)-17,20-dioxa-2,7,10,13-tetraazadocosan-22-yl)-6-((4-((((1S,2R )-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-1-phenylpropoxy)(hydroxy)phosphoryl)oxy)methyl)phenyl)carbamoyl)- 41-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-9-isopropyl-1,8,11,14,24,40,44-heptaoxo-12-(2-oxo-2-(((S)-2-oxotetrahydrofuran-3-yl)amino)ethyl)-17,20,27,30,33,36-hexaoxa-2,7,10,13,23,39,43-heptaazahexatetracontan-46-yl)phosphonic acid

[0075] The compound of Synthesis Example 2 can be synthesized using a method similar to that of Reference Example 2 described in WO2023234426 or Example 2 described in WO2023234427. The MS of Synthesis Example 2 is shown below. MS (DUIS) m / z 1797 (M+2H) ２＋ MS (DUIS) m / z 1795 (M-2H) ２－(Analysis conditions) Apparatus: LCMS-2050 system, Shimadzu Corporation Column: ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 50 mm, Waters Eluent: Solution A: 0.1% formic acid aqueous solution, Solution B: 0.1% formic acid acetonitrile solution Gradient (B%): 25 (0 min) → 95 (0.75 min) → 95 (1.5 min) Temperature: 40°C Flow rate: 0.8 mL / min Injection amount: 5 μL

[0076] <Synthesis of Antibody-Drug Conjugates> The antibody-drug conjugates of the present invention were synthesized by the following method. The antibody-drug conjugates obtained or obtained in each of the following examples are represented by general formula (I). General formula (I): A-(B-C)a (In the formula, A is the moiety obtained by removing sulfhydryl groups from a reduced antibody, B is an -S- group, C is a chemotherapeutic agent having a linker, and a is the average number of linker-drug units (B-C) bound per antibody molecule, which is a number from 1 to 10 that may include a decimal point. Hereinafter, with regard to ADC, the average number of drugs bound per antibody, or the drug / antibody ratio, is referred to as "DAR," which is a number from 1 to 20 that may include a decimal point and is expressed to one or two decimal points in the present specification.)

[0077] Example 1: T004-PGAP-1 (ADC1) Synthesis of an antibody-drug conjugate in which, in general formula (I), A is a moiety obtained by removing a sulfhydryl group from reduced antibody T004, B is -S-, and C is represented by the following formula (x): (In the formula, * indicates the connecting portion with B.)

[0078] <1-1> Buffer exchange of T004: EDTA (Thermo Fisher Scientific) was added to phosphate-buffered saline (hereinafter referred to as PBS) (Thermo Fisher Scientific) to a concentration of 10 mM (hereinafter referred to as PBS / EDTA). 1 mL of the original T004 solution obtained in Reference Example 1 was added to one NAP-10 column (Cytiva) previously equilibrated with PBS / EDTA, followed by elution with 1.5 mL of PBS / EDTA. The fraction containing T004 (1.5 mL) was collected to obtain a T004 solution. The absorbance at 280 nm of the T004 solution was measured using a microplate reader (FlexStation 3, Molecular Devices) to determine the antibody concentration. (Antibody concentration: 3.4mg / mL)

[0079] <1-2> Reduction of T004: The T004 solution obtained in <1-1> above was diluted with PBS / EDTA to a concentration of approximately 2.5 mg / mL. 0.99 mL of this solution was placed in a 1.5 mL microtube, and 10 μL of a PBS / EDTA solution of TCEP hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added (so that 6.9 equivalents of TCEP were used per antibody molecule). The mixture was incubated at 37°C for 2 hours to carry out a reduction reaction targeting the disulfide bond between the peptide chains constituting antibody T004, yielding a reduced T004 solution.

[0080] <1-3> Formation of a conjugate between reduced T004 and PGAP-1 (Synthesis Example 1): To 900 μL of the reduced T004 solution obtained in <1-2> above, 100 μL of a dimethyl sulfoxide solution containing PGAP-1 obtained in Synthesis Example 1 (so that PGAP-1 was 15 equivalents per antibody molecule) was added, and the mixture was allowed to react under ice-cooling for 1 hour to obtain a solution of antibody-drug conjugate (T004-PGAP-1).

[0081] <1-4> Preparation of Reduced T004 for Reference: 100 μL of dimethyl sulfoxide was added to 900 μL of the reduced T004 solution, and the mixture was allowed to stand on ice for 1 hour to obtain a reduced T004 solution for reference.

[0082] <1-5> Purification of antibody-drug conjugate: 200 μL of the antibody-drug conjugate solution obtained in 1-3 above was added to one pre-conditioned Protein A column (GL Sciences, MonoSpin 96WP ProA) and allowed to adsorb onto the column. After washing the column with the wash buffer provided with the column, the antibody-drug conjugate was eluted with the elution buffer provided with the column. The antibody-drug conjugate solution (1 mL) eluted from the Protein A column was added to a NAP-10 column previously equilibrated with PBS / EDTA and eluted with 1.5 mL of PBS / EDTA. The fraction containing the antibody-drug conjugate (1.5 mL) was collected to obtain a purified antibody-drug conjugate. The absorbance at 280 nm of the purified antibody-drug conjugate was measured using a microplate reader. (Absorbance: 1.19)

[0083] <1-5a> Purification of antibody-drug conjugate for test examples The same procedure as in <1-5> was carried out using PBS without EDTA (absorbance: 1.22). The purified antibody-drug conjugate obtained by this procedure was used in the test examples.

[0084] <1-6> Purification of Reference Reduced T004: 200 μL of the reference reduced T004 solution obtained in <1-4> above was added to one pre-conditioned Protein A column (GL Sciences, MonoSpin 96WP ProA) and allowed to adsorb onto the column. After washing the column with the wash buffer provided with the column, reduced T004 was eluted with the elution buffer provided with the column. The column was then loaded onto a NAP-10 column previously equilibrated with PBS / EDTA and eluted with 1.5 mL of PBS / EDTA. The fraction containing reduced T004 (1.5 mL) was collected to obtain a reference purified reduced T004 solution. The absorbance at 280 nm of the purified reduced T004 solution was measured using a microplate reader to determine the antibody concentration. (Antibody concentration: 0.85 mg / mL)

[0085] <1-7> DAR Calculation by Ellman's Assay 480 μL of the purified antibody-drug conjugate solution eluted with PBS / EDTA and the reference purified reduced T004 solution were placed in separate microtubes, and 20 μL of a 10 mM ethanol solution of 5,5'-dithiobis(2-nitrobenzoic acid) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and mixed, allowing the reaction to proceed at room temperature for 15 minutes. The absorbance at 412 nm of the resulting reaction solution was measured using a microplate reader. Using a standard curve obtained from a solution of L-cysteine ​​(Fujifilm Wako Pure Chemical Industries, Ltd.) with a known concentration, the free thiol group concentrations in the purified antibody-drug conjugate solution and the purified reduced T004 solution were determined. (Free thiol group concentration in the purified antibody-drug conjugate solution: 1.8 μM, free thiol group concentration in the purified reduced T004 solution: 52 μM)

[0086] (Calculation of DAR: When there is one chemotherapeutic agent present in C of general formula (I) (a=DAR)) Number of thiol groups per molecule of reduced T004, N ＳＨ、Ａｂ and the number of thiol groups per molecule of the antibody-drug conjugate, N ＳＨ、ＡＤＣ can be calculated using the following formula: ＳＨ、Ａｂ =C ＳＨ、Ａｂ / C Ａｂ Formula (1) N ＳＨ、ＡＤＣ =C ＳＨ、ＡＤＣ / C ＡＤＣ Equation (2) (where C ＳＨ、Ａｂ indicates the thiol group concentration in the reduced T004 solution, and C Ａｂ indicates the antibody concentration of the reduced T004 solution, and C ＳＨ、ＡＤＣ indicates the thiol group concentration in the antibody-drug conjugate solution, and C ＡＤＣ indicates the antibody concentration in the antibody-drug conjugate solution. ＳＨ、Ａｂ Since it is not possible for N to exceed 8, the calculation of equation (1) ＳＨ、Ａｂ If it exceeds 8, it will be considered as 8.) N ＳＨ、Ａｂ From N ＳＨ、ＡＤＣ The difference obtained by subtracting N can be considered as the DAR of the antibody-drug conjugate. That is, the DAR of the antibody-drug conjugate can be calculated by the following formula: DAR = NＳＨ、Ａｂ -N ＳＨ、ＡＤＣ Since the total absorbance at a certain wavelength is equal to the sum of all absorbing chemical species present in the system, assuming that there is no change in the molar absorption coefficients of the antibody and drug before and after conjugation of the antibody and drug, the following equation holds for the absorbance of the antibody-drug conjugate: A ＡＤＣ、２８０ =C ＡＤＣ (ε Ａｂ、２８０ + DAR × ε Ｄ、２８０ ) Equation (4) (where A ＡＤＣ、２８０ indicates the absorbance at 280 nm of the antibody-drug conjugate solution, and C ＡＤＣ denotes the antibody concentration of the antibody-drug conjugate, and ε Ａｂ、２８０ indicates the molar extinction coefficient of T004 at 280 nm, DAR indicates the DAR of the antibody-drug conjugate, and ε Ｄ、２８０ represents the molar absorption coefficient of PGAP-1 at 280 nm.) From equations (1), (2), (3), and (4), the following equation is established for DAR: DAR=(N ＳＨ、Ａｂ A ＡＤＣ、２８０ -C ＳＨ、ＡＤＣ ε Ａｂ、２８０ ) / (A ＡＤＣ、２８０ +C ＳＨ、ＡＤＣ ε Ｄ、２８０ ) Equation (5) As a result of calculation using equation (5), the calculated DAR value of the antibody-drug conjugate of Example 1 was 7.7.

[0087] Furthermore, the DAR of the antibody-drug conjugate eluted with PBS obtained in 1-5a can be considered to be equal to the DAR of the antibody-drug conjugate eluted with PBS / EDTA, and the antibody concentration of the antibody-drug conjugate solution eluted with PBS can be calculated using equation (4). (Antibody-drug conjugate concentration: 0.83 mg / mL. This concentration is shown in Table 1.)

[0088] Example 2: T004-PGAP-2 (ADC2) Synthesis of an antibody-drug conjugate in which, in general formula (I), A is a moiety obtained by removing a sulfhydryl group from reduced antibody T004, B is -S-, and C is represented by the following formula (y): (In the formula, * indicates the connecting portion with B.)

[0089] Using T004 and PGAP-2 (Synthesis Example 2) instead of PGAP-1, the same reaction and treatment procedures as in Example 1 were carried out to obtain the antibody-drug conjugate of Example 2. In Example 2, two chemotherapeutic agents are present at C in general formula (I), in which case DAR = 2a, and the DAR is calculated by doubling the value obtained by formula (5).

[0090] Example 3: T004b-PGAP-1 (ADC3), and Example 4: T004b-PGAP-2 (ADC4) Using T004b instead of T004, and PGAP-1 (Synthesis Example 1) or PGAP-2 (Synthesis Example 2), the same reactions and treatments as in Example 1 or Example 2 were carried out to obtain the antibody-drug conjugates of Example 3 and Example 4, respectively. In Example 4, two chemotherapeutic agents are present at C in general formula (I), and the DAR is calculated by doubling the value obtained by formula (5).

[0091] The results of Examples 1 to 4 are summarized in Table 1. (In the table, T004' represents the moiety obtained by removing sulfhydryl groups from reduced antibody T004, and T004b' represents the moiety obtained by removing sulfhydryl groups from reduced antibody T004b. In Examples 3 and 4, synthesis was carried out twice, and the DAR was calculated for each.)

[0092] Example 5: T004b-PGAP-1-DAR4 (ADC5) Synthesis of an antibody-drug conjugate in which, in general formula (I), A is a moiety obtained by removing a sulfhydryl group from reduced antibody T004b, B is -S-, and C is (x) described in Example 1

[0093] <5-1> Buffer exchange of T004b: EDTA (Thermo Fisher Scientific) was added to phosphate-buffered saline (hereinafter referred to as PBS) (Thermo Fisher Scientific) to a concentration of 10 mM (hereinafter referred to as PBS / EDTA). 1 mL of the original T004b solution obtained in Reference Example 1 was added to one NAP-10 column (Cytiva) previously equilibrated with PBS / EDTA, followed by elution with 1.5 mL of PBS / EDTA. A fraction containing T004b (1.5 mL) was collected to obtain a T004b solution. The absorbance of the T004b solution at 280 nm was measured using a microplate reader (FlexStation 3, manufactured by Molecular Devices) to determine the antibody concentration (antibody concentration: 3.64 mg / mL).

[0094] <5-2> Reduction of T004b: The T004b solution obtained in <1-1> above was diluted with PBS / EDTA to a concentration of approximately 2.5 mg / mL. 0.99 mL of this solution was placed in a 1.5 mL microtube, and 10 μL of a PBS / EDTA solution of TCEP hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added (so that 3.45 equivalents of TCEP were used per antibody molecule). The mixture was incubated at 37°C for 2 hours to carry out a reduction reaction targeting the disulfide bond between the peptide chains constituting antibody T004b, yielding a reduced T004b solution.

[0095] <5-3> Formation of a conjugate between reduced T004b and PGAP-1 (Synthesis Example 1): To 900 μL of the reduced T004b solution obtained in <1-2> above, 100 μL of a dimethyl sulfoxide solution containing PGAP-1 obtained in Synthesis Example 1 (so that PGAP-1 was 15 equivalents per antibody molecule) was added, and the mixture was allowed to react under ice-cooling for 1 hour to obtain a solution of the antibody-drug conjugate (T004b-PGAP-1).

[0096] <5-4> Purification of antibody-drug conjugate: 200 μL of the antibody-drug conjugate solution obtained in 1-3 above was added to one pre-conditioned Protein A column (GL Sciences, MonoSpin 96WP ProA) and allowed to adsorb onto the column. After washing the column with the wash buffer provided with the column, the antibody-drug conjugate was eluted with the elution buffer provided with the column. The antibody-drug conjugate solution (1 mL) eluted from the Protein A column was added to a NAP-10 column pre-equilibrated with PBS and eluted with 1.5 mL of PBS. The fraction containing the antibody-drug conjugate (1.5 mL) was collected to obtain a purified antibody-drug conjugate. The absorbance at 280 nm of the purified antibody-drug conjugate was measured using a microplate reader. (Absorbance: 1.33)

[0097] <5-5> Drug-Antibody Ratio (DAR) Measurement by HIC Analysis: The DAR profile of the antibody-drug conjugate solution eluted with PBS was measured using a high-performance liquid chromatograph (HPLC, Shimadzu Corporation, LC-20AD) equipped with a hydrophobic chromatography column (TOSOH, TSKgel Butyl-NPR, 4.6 x 35 mm). Mobile phase A consisted of 0.03 M phosphate buffer, 1.5 M ammonium sulfate, pH 7.0, and mobile phase B consisted of 90% v / v 0.03 M phosphate buffer (pH 7.0) and 10% v / v isopropanol. The antibody-drug conjugate solution was diluted 2-fold with a mixture of 50% v / v mobile phase A and 50% v / v mobile phase B to prepare the analytical sample. When the antibody-drug conjugate solution was highly concentrated, the sample was diluted to approximately 1 mg / mL with PBS. 10 μL of the analytical sample was injected into the column at a flow rate of 0.8 mL / min, and absorbance at 280 nm was detected in gradient mode for 35 min. The gradient was set to mobile phase A: 75% (0.0 min) - 35% (30.0 min) - 5% (30.1 min) - 5% (35.0 min). The average DAR was calculated using software (LabSolutions, Shimadzu Corporation) and the product of the area % of each DAR and the DAR number was added to obtain the average DAR. (Average DAR for antibody-drug conjugates: 3.79)

[0098] <5-6> Calculation of antibody-drug conjugate solution concentration Since the total absorbance at a certain wavelength is equal to the sum of all absorbing chemical species present in the system, assuming that there is no change in the molar absorption coefficients of the antibody and drug before and after conjugation of the antibody and drug, the following equation holds for the absorbance of the antibody-drug conjugate. A ＡＤＣ、２８０ =C ＡＤＣ (ε Ａｂ、２８０ + DAR × ε Ｄ、２８０ ) Equation (1) (where A ＡＤＣ、２８０ indicates the absorbance at 280 nm of the antibody-drug conjugate solution, and C ＡＤＣ denotes the antibody concentration of the antibody-drug conjugate, and ε Ａｂ、２８０ indicates the molar extinction coefficient of T004b at 280 nm, DAR indicates the DAR of the antibody-drug conjugate, and ε Ｄ、２８０ represents the molar absorption coefficient of PGAP-1 at 280 nm.) Using equation (1), C ＡＤＣ (C of antibody-drug conjugate) ＡＤＣ :0.95mg / mL)

[0099] Example 6 A T004b solution obtained by the same method as in Example <5-1> was diluted with PBS / EDTA to adjust the concentration to approximately 2.5 mg / mL. 0.99 mL of this solution was placed in a 1.5 mL microtube, and 10 μL of a PBS / EDTA solution of TCEP hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added (so that 3.45 equivalents of TCEP per antibody molecule were used). The mixture was incubated at 4°C for 6 hours to reduce the disulfide bond in the hinge region of antibody T004b, yielding a reduced T004b solution. The same reactions and treatments as in Example 5 were then performed to obtain the antibody-drug conjugate of Example 6. (ADC6)

[0100] Example 7 A T004b solution obtained by the same method as in Example <5-1> was diluted with PBS / EDTA to adjust the concentration to approximately 2.5 mg / mL. 0.99 mL of this solution was placed in a 1.5 mL microtube, and 10 μL of a PBS / EDTA solution of TCEP hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added (so that 3.8 equivalents of TCEP per antibody molecule were used). The mixture was incubated at 4°C for 6 hours to reduce the disulfide bond in the hinge region of antibody T004b, yielding a reduced T004b solution. The same reactions and treatments as in Example 5 were then performed to obtain the antibody-drug conjugate of Example 7. (ADC7)

[0101] The results of Examples 5 to 7 are summarized in Table 2.

[0102] (In the table, T004b' represents the moiety obtained by removing sulfhydryl groups from reduced antibody T004b. In Examples 5, 6, and 7, synthesis was carried out twice, and the DAR was calculated for each.)

[0103] Example 8 Preparation of Comparative Drug-Conjugated Antibody (Control ADC) 8-1 Drug Synthesis DM1SMe was prepared as previously described in US Pat. Nos. 5,208,020 and 6,333,410 B1.

[0104] <8-2> Reduction Treatment of Bonding Drug 0.78 mg of DM1SMe dissolved in 300 μL of ethanol was mixed with 180 μL of 50 mM potassium phosphate buffer (pH 7.5) and 20 μL of TCEP Solution (Bond Breaker, Thermo Fisher Scientific), and the mixture was reacted at room temperature for 30 minutes or more under a nitrogen atmosphere to reduce the drug. The reducing drug was purified using HPLC, the solvent was evaporated, and then the solution was dissolved in dimethylacetamide to a concentration of 10 mg / mL.

[0105] <8-3> Preparation of maleimidated antibody To 1 mg / mL T004, sulfo-SMCC (PIERCE) was added in a 30-fold molar excess, and the mixture was allowed to react for 1 hour at 30° C. To remove excess crosslinker, the mixture was desalted using a desalting column (ZebaSpinColumn, Thermo Fisher Scientific) equilibrated with 50 mM potassium phosphate, 50 mM NaCl, and 2 mM EDTA (pH 6.5).

[0106] 1 mg / mL of maleimide-modified T004 was reacted with a reducing agent in an amount equivalent to 1.7 times the number of bound maleimide groups in 50 mM potassium phosphate, 50 mM NaCl, and 2 mM EDTA (pH 6.5) at room temperature overnight. Excess agent was then removed by gel filtration.

[0107] <8-5> Quantification of antibody-drug binding amount The number of drugs bound per antibody was determined by measuring absorbance at 252 nm and 280 nm. The determination method was based on the extinction coefficient εAb described in a non-patent document (Widdison, W.C., Wilhelm, S.D., Cavanagh, E.E., et al. (2006) Semisynthetic maytansine analogues for the targeted treatment of cancer. J. Med. Chem., 49, 4392-4408). ２８０ = 223,000M －１ cm －１ , εAb ２５２ =82,510M- １ cm －１ , εDM1 ２８０ = 5,180M －１ cm －１ , εDM1 ２５２ = 26,160M －１ cm －１ was used.

[0108] Example 9: Cytotoxicity Assay The efficacy of ADCs was evaluated using human CDH3-expressing cell lines, HCC1954, BxPC3, and KLM1. First, these cell lines were seeded at 1,000 cells / well in a 384-well plate (Greiner) and cultured overnight at 37°C and 5% CO2. The following day, serially diluted ADC1, ADC2, ADC3, and ADC4 were added to each well. After 4 days of culture in an incubator, cell viability was assessed using CellTiter-Glo (Promega). The luminescence value of the well containing cells cultured in medium alone was taken as 100% viability, and the viability of each well was calculated. The treatment concentration required to inhibit 50% of cell proliferation or viability (IC50) was calculated using XLfit 5.2 (IDBS), and comparisons were made using pIC50 (Log(-IC50)).

[0109] The pIC50 results for ADC1 to ADC4 in various cell lines (HCC1954, BxPC3 KLM1) are summarized in Table 3. Both ADC1 and ADC3, which use antibodies T004 and T004b, showed strong antiproliferative activity in the cell lines used (Figure 1). Furthermore, ADC1 and ADC2, which use antibody T004, and ADC3 and ADC4, which use antibody T004b, also showed strong cell growth inhibitory effects (Figures 2 and 3).

[0110]

[0111] Example 10: In vivo drug efficacy test in mice BxPC3 cells, a human pancreatic cancer cell line, were cultured and expanded until a number approximately twice the amount required for transplantation was obtained. On the day of transplantation, the cells were washed with PBS(-), detached using a TrypleExpress (Thermofisher Scientific), and centrifuged at 1,000 rpm for 5 minutes to collect a pellet. The pellet was suspended in antibiotic- and serum-free RPMI-1640 medium and Matrigel (Corning, 356237) at a concentration of 5 x 10 ６The solution was prepared at a concentration of 0.5 mg / ml and 100 μl of the solution was implanted subcutaneously into the right side of each mouse. For the in vivo test, five mice were used in each group, and each solution was administered via the tail vein. The administration solutions were 0.5, 1, and 2 mg / kg for ADC3 and ADC4, respectively, and 2 mg / kg for the control ADC. The administration was carried out in mice with an average tumor diameter of approximately 150 mm. ３ At this point, a single dose was administered.

[0112] The results are shown in Figures 4 to 7. Both ADC3 and ADC4 showed high efficacy at the minimum dose of 0.5 mg / kg. There was no effect on body weight.

[0113] Furthermore, we conducted an in vivo study to compare the efficacy of ADC3, ADC5, and ADC7, which have different drug-antibody ratios (DAR), in mice. As shown in Figure 8, mice administered ADC5 and ADC7 (0.5 mg / kg), which have an average DAR of 4, showed significant efficacy comparable to that of ADC3 (0.5 mg / kg), which has an average DAR of 8. Furthermore, no weight loss was observed in mice administered the ADCs (Figure 9).

[0114] Example 11: Pharmacokinetic study in mice ADC3, ADC5, and ADC7 were orally administered at a single dose of 3 mg / kg to ICR mice. Blood samples were collected at 5 minutes (Day 3, Day 7, and Day 14), and plasma antibody levels (TAb) were measured by ELISA. A 100 μL solution of anti-T004b antibody (2.5 μg / mL, antibody solution, prepared in-house) in DPBS (GIBCO, 14190144) was added to each well of a 96-well polystyrene plate coated with MaxiSorp™ (Thermo Scientific, 442404). The plate was incubated overnight at 4°C. After incubation, the plate was washed three times with PBS containing 0.05% Tween-20 (PBS-T). The wells were then blocked with 100% Block Ace (UKB500, KAC) solution for at least 1 hour at room temperature. After blocking, the plate was washed three times with PBS-T. To prepare a standard curve, antibody or ADC solutions were serially diluted 2-fold starting from 10 ng / mL in TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween, prepared in-house) to prepare seven standard solutions. Plasma samples were serially diluted 2-fold starting from an arbitrary dilution factor to prepare four standard solutions. Plasma samples and standard solutions (100 μL) were added to the wells of the ELISA plate and incubated at room temperature for 1 hour. After incubation, the samples were removed, and the plate was washed three times with PBS-T. A solution of HRP-F(ab'2) Fragment anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc.) was diluted 1:10,000 in TBS-T, and 100 μL was added to each well. The plate was incubated at room temperature for 1 hour. After incubation, the samples were removed, and the plate was washed three times with PBS-T. A solution of TMB Soluble Reagent (ScyTek Laboratories) was added to each well (100 μL). The plate was incubated at room temperature for 10 to 20 minutes, and the reaction was quenched with 100 μL of TMB STOP BUFFER (ScyTek Laboratories).The plate was read at 450 nm and 620 nm using a Multiskan FC absorbance microplate reader (Thermo Fisher SCIENTIFIC). The concentration at each dilution was calculated from the calibration curve, and the average value was determined. The results are shown in Figure 10. ADC3, ADC5, and ADC7 exhibited nearly identical pharmacokinetics, which was also the same as that of the unconjugated antibody, T004b.

Claims

1. An antibody-drug conjugate comprising an anti-CDH3 humanized antibody linked to a chemotherapeutic agent having a linker, wherein the anti-CDH3 humanized antibody is: (1) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2; (2) an anti-CDH3 humanized antibody having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4; (3) a fragment of the anti-CDH3 humanized antibody set forth in (1) or (2) above, which has the ability to bind to CDH3; or (4) an antibody consisting of a partial sequence of the antibody set forth in (1) or (2) above, which has the ability to bind to CDH3; and the chemotherapeutic agent having a linker is represented by formula (x): (wherein * indicates the connecting portion to the antibody) or formula (y): (wherein * indicates the connecting portion to the antibody), a pharmacologically acceptable salt thereof, or a hydrate thereof.

2. The antibody-drug conjugate according to claim 1, a pharmacologically acceptable salt thereof, or a hydrate of either, wherein the anti-CDH3 humanized antibody is any of the following antibodies: (1) an anti-CDH3 humanized antibody having a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 6; (2) an anti-CDH3 humanized antibody having a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:

8.

3. The antibody-drug conjugate, its pharmacologically acceptable salt, or hydrate of claim 1, wherein an average of 1 to 10 molecules of a chemotherapeutic agent having a linker are bound to one molecule of the anti-CDH3 humanized antibody.

4. A pharmaceutical for treating a disease characterized by overexpression of CDH3, comprising the antibody-drug conjugate according to any one of claims 1 to 3, a pharmacologically acceptable salt thereof, or a hydrate thereof.

5. The pharmaceutical composition according to claim 4, wherein the disease characterized by overexpression of CDH3 is a tumor or cancer.

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