Anti-CDH3 humanized antibody and use thereof

Abstract

The present invention addresses the problem of producing a novel anti-CDH3 humanized antibody and providing, by using the antibody, an (anti-CDH3 humanized antibody)-drug conjugate capable of more efficiently killing CDH3-expressing cancer cells. According to the present invention, either of the following antibodies is provided. (1) An anti-CDH3 humanized antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; or (2) an anti-CDH3 humanized antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4.

Description

Anti-CDH3 humanized antibodies and their uses

[0001] The present invention relates to an anti-CDH3 humanized antibody, an immunoconjugate thereof, and a pharmaceutical.

[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] P-cadherin (CDH3), a cell membrane surface antigen, was identified as one of its targets. 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 have been reported in certain cancer cells, and cancer therapy using antibodies against cancer cells with higher CDH3 expression in cancer tissues compared to normal tissues is being considered.

[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 antibodies and drug conjugates thereof.

[0007] International Publication No. WO2013 / 150623 International Publication No. WO2014 / 126198

[0008] An object of the present invention is to create a new anti-CDH3 antibody, and further to provide an anti-CDH3 humanized antibody suitable for creating an antibody-drug conjugate that more efficiently kills cancer cells that express CDH3.

[0009] As a result of intensive research to solve the above-mentioned problems, the present inventors have succeeded in creating 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 have used this to create an anti-CDH3 humanized antibody-drug conjugate, which is an immune complex 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> 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; or (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. <2> The antibody according to <1>, which 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> A fragment of the anti-CDH3 humanized antibody according to <1> or <2>, which has the ability to bind to CDH3. <4> Fab, F(ab') ２or scFv. <5> A partial sequence of the antibody according to <1> or <2>, having the ability to bind to CDH3. <6> An immune complex in which the anti-CDH3 humanized antibody according to any one of <1> to <5>, its fragment, or its partial sequence, is linked to a chemotherapeutic agent or a radioactive substance. <7> The immune complex according to <6>, in which the chemotherapeutic agent is a cytotoxic substance. <8> The immune complex according to <6> or <7>, in which the anti-CDH3 humanized antibody, its fragment, or its partial sequence, is linked to a chemotherapeutic agent via a linker. <9> A medicament for treating a disease characterized by overexpression of CDH3, comprising the immune complex according to any one of <6> to <8>. <10> The medicament according to <9>, in which the disease characterized by overexpression of CDH3 is cancer.

[0011] The present invention further provides a method for treating a disease characterized by overexpression of CDH3, comprising administering the above-described immunoconjugate to a patient.The present invention further provides use of the above-described immunoconjugate for the manufacture of a medicament for treating a disease characterized by overexpression of CDH3.The present invention further provides the above-described immunoconjugate 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 results of imaging capillary isoelectric focusing. A: ch2012, B: hu2012c, C: hu2012d. Figure 2 shows the results of measuring thermal stability by Thermal Shift Assay. A: ch2012, B: hu2012c, C: hu2012d. Figure 3 shows the results of flow cytometry in which humanized anti-CDH3 antibodies were reacted with the lung cancer-derived cell line NCI-H358. The peak on the left indicates the negative control. Figure 4 shows a graph of the results of measuring internalization ability shown in Table 1. Figure 5 shows the results of an animal study (HCC1954 breast cancer model) using CDH3 chimeric and humanized antibody-drug conjugates.

[0014] The present invention is described in further detail below. 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; or (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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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).

[0026] 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. CDRs 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)).

[0027] The CDR sequences 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.

[0028] In the present invention, the FR sequences used were derived from human germline sequences selected under optimal alignment.

[0029] 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)).

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] <Immune Conjugate> The present invention provides an immune conjugate in which the anti-CDH3 humanized antibody of the present invention, a fragment thereof, or a partial sequence thereof is linked to a chemotherapeutic agent or a radioactive substance. That is, a preferred embodiment of the antibody of the present invention is an immune conjugate in which a chemotherapeutic agent such as a cytotoxic substance or a radioactive substance is bound to the antibody, i.e., an antibody-drug conjugate (ADC). The immune conjugate of the present invention can damage cancer cells, for example, by contacting the immune conjugate with cancer cells expressing CDH3.

[0039] The chemotherapeutic agent is preferably a cytotoxic substance. Examples of the chemotherapeutic agent include duocarmycin, duocarmycin analogs and derivatives, CC-1065, CBI-based duocarmycin analogs, MCBI-based duocarmycin analogs, CCBI-based duocarmycin analogs, doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatin, dorestatin-10, combretastatin, calicheamicin, maytansinoids or derivatives thereof (e.g., maytansine, maytansine analogs, DM1, DM4, etc.), auristatin or derivatives thereof (e.g., auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), 5- Benzoylvaleric acid AE ester (AEVB), etc.), tubulysin, disorazole, epothilone, paclitaxel, docetaxel, SN-38, topotecan, exatecan, deruxtecan, rhizoxin, echinomycin, colchicine, vinblastine, vindesine, estramustine, cemadotin, amanitin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil le, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosin, leurosidyne, actinomycin, daunorubicin, daunorubicin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate, vincristine, taxol, taxotere, retinoic acid, butyric acid, N ８ Examples include, but are not limited to, acetylspermidine and camptothecin.

[0040] 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 an average of 1 to 10 chemotherapeutic agent molecules, more preferably an average of 3 to 5 chemotherapeutic agent molecules are bound. As an example, an average of 1 to 10 DM1 molecules may be bound to one molecule of an anti-CDH3 humanized antibody, a fragment thereof, or a partial sequence thereof.

[0041] The immune complex of the present invention can be prepared by conjugating the chemotherapeutic agent and the antibody by a known method. The antibody and the chemotherapeutic agent may be directly conjugated via a linking group possessed by each of them, or may be indirectly conjugated via a linker or other substance.

[0042] Examples of linking groups for direct binding of a drug include disulfide bonds using SH groups and bonds mediated by maleimide. For example, the intramolecular disulfide bond of the Fc region of an antibody and the disulfide bond of the drug are reduced to link the two via a disulfide bond. Another method involves linking via maleimide. Another method involves genetically modifying the Fc region of an antibody to introduce the drug. For example, there is a method of genetically introducing cysteine ​​into an antibody.

[0043] The antibody and chemotherapeutic agent can also be indirectly linked via another substance (linker). The linker preferably has one or more functional groups that react with the antibody, the drug, or both. Examples of functional groups include amino groups, carboxyl groups, mercapto groups, maleimide groups, and pyridinyl groups. The linker is preferably a bifunctional cross-linking reagent.

[0044] Examples of linkers include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (Sulfo-SMCC), and N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate). (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidopropionamido)hexanoate (SMPH), Examples of the linker include, but are not limited to, N-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl 4(2-pyridylthio)pentanoate (SPP), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), p-aminobenzyloxycarbonyl (PAB), and N-succinimidyl 4(2-pyridylthio)butanoate (SPDB). For example, the linker may be a combination of para-aminobenzoic acid (PABA) and a peptide linker such as valine-citrulline (Val-Cit) or alanine-phenylalanine (ala-phe), or the above-listed linkers may be used in combination as appropriate.

[0045] In one example, the linker may be cleavable by a protease. Further, the linker may include at least one of valine-citrulline (Val-Cit), alanine-phenylalanine (ala-phe), and para-aminobenzoic acid (PABA).

[0046] The method for binding the drug to the antibody can be carried out in accordance with the method described in, for example, Cancer Res.; 52, p. 127 (1992), Cancer Res.; 68 (22), p. 9280 (2008), Nature Biotechnology; 26 (8), p. 925 (2008), Bio Conjugate Chemistry; 19, p. 1673 (2008), Cancer Res.; 68 (15), p. 6300 (2008), or JP-A No. 2008-516896.

[0047] Another embodiment of the present invention is a so-called immunotoxin, which is an antibody to which a drug is chemically or genetically linked. Examples of drugs that can be used include, but are not limited to, diphtheria toxin A chain, Pseudomonas endotoxin, ricin A chain, abrin A chain, modeccin A chain, gelonin, and saporin.

[0048] In another embodiment of the present invention, a radioactive substance can be bound to the antibody. When used as a cancer therapeutic agent, the radioactive substance is preferably a cytotoxic radioactive metal, and when used as a cancer diagnostic agent, it is preferably a non-cytotoxic radioactive metal.

[0049] Examples of such cytotoxic radioactive metals include yttrium-90 (90Y), rhenium-186 (186Re), rhenium-188 (188Re), copper-67 (67Cu), iron-59 (59Fe), strontium-89 (89Sr), gold-198 (198Au), mercury-203 (203Hg), lead-212 (212Pb), dysprosium-165 (165Dy), ruthenium-103 (103Ru), bismuth-212 (212Bi), bismuth-213 (213Bi), holmium-166 (166Ho), samarium-153 (153Sm), lutetium-177 (177Lu), astatine-211 (211At), and actinium-225 (225Ac). Among these radioactive metals, 90Y, 153Sm, and 177Lu are preferred in terms of half-life, radiation energy, ease of labeling reaction, labeling rate, stability of the complex, etc., but are not limited to these.

[0050] On the other hand, examples of suitable non-cytotoxic radioactive metals used in diagnostic agents include, but are not limited to, technetium-99m (99mTc), indium-111 (111In), indium-113m (113mIn), gallium-67 (67Ga), gallium-68 (68Ga), thallium-201 (201Tl), chromium-51 (51Cr), cobalt-57 (57Co), cobalt-58 (58Co), cobalt-60 (60Co), strontium-85 (85Sr), mercury-197 (197Hg), and copper-64 (64Cu).

[0051] To bind such a radioactive metal element to an anti-CDH3 antibody, it is preferable to react the antibody with a metal chelating agent and then react the radioactive metal element with this to form a complex. The modified antibody thus obtained has the radioactive metal element bound via the metal chelating agent.

[0052] Examples of metal chelating agents used to form such complexes include (1) 8-hydroxyquinoline, 8-acetoxyquinoline, 8-hydroxyquinaldine, oxyquinoline sulfate, O-acetyloxine, O-benzoyloxine, O-p-nitrobenzoyloxine, quinolone compounds having a quinoline skeleton such as norfloxacin, ofloxacin, enoxacin, ciprofloxacin, lomefloxacin, tosufloxacin, fleroxacin, and sparfloxacin. (2) quinoline derivatives such as oxacin; (3) compounds such as chloranilic acid, aluminone, thiourea, pyrogallol, cupferron, bismuthiol (II), galloyl gallic acid, thiolide, 2-mercaptobenzothiazole, and tetraphenylarsonium chloride; (4) compounds having a skeleton similar to these, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and dihydroxyethylglycine, diaminopropanoltetraacetic acid, ethylenediaminediacetic acid, ethylenediaminedipropionic acid hydrochloride, hydroxyethylethylenediaminetriacetic acid, ethylenediaminetetrakis(methylenephosphonic acid), glycol ether diaminetetraacetic acid, hexamethylenediaminetetraacetic acid, hydroxyethyliminodiacetic acid, iminodiacetic acid, diaminopropanetetraacetic acid, nitrilotriacetic acid, nitrilotripropionic acid, nitrilotris(methylenesulfonic acid) trisodium salt, triethylenetetraminehexaacetic acid, methyl DTPA, and cyclohexyl DTPA. , aminobenzyl EDTA, isothiocyanobenzyl EDTA, isothiocyanobenzyl DTPA, methyl isothiocyanobenzyl DTPA, cyclohexyl isothiocyanobenzyl DTPA, maleimidopropylamidobenzyl EDTA, maleimidopentylamidobenzyl EDTA, maleimidodecylamidobenzyl EDTA, maleimidopentylamidobenzyl DTPA, maleimidodecylamidobenzyl EDTA, maleimidodecylamidobenzyl DTPA;(4) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane (Cyclen), 1,4,8,11-tetraazacyclotetradecane (Cyclam), isothiocyanobenzyl DOTA, isothiocyanobenzyl NOTA, and the like;

[0053] Of these metal chelating reagents, isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, and cyclohexylisothiocyanobenzyl DTPA are preferred in terms of ease of introduction of the metal chelate into the antibody, labeling rate, stability of the complex, and the like.

[0054] The binding of a radioactive metal element to an antibody can be carried out by a conventional method, for example, by reacting the antibody with a metal chelating agent to prepare a label precursor in advance, and then reacting the antibody with a radioactive metal element.

[0055] <Drugs> The present invention provides pharmaceuticals for treating diseases characterized by overexpression of CDH3, comprising the immune complex of the present invention. The immune complexes provided by the present invention may contain pharmaceutically acceptable carriers, excipients, diluents, etc., as appropriate, to maintain the stability of the drug. The immune complexes of the present invention can be formulated, for example, as injections. The dosage of the immune complexes of the present invention depends on the severity of the patient's symptoms, age, and body weight, the method of administration, etc., and is usually in the range of about 10 ng to about 100 mg / kg body weight in terms of the weight of the antibody, which is the active ingredient.

[0056] Diseases that can be treated by the immunoconjugate of the present invention are not particularly limited as long as CDH3 is expressed in the cells, and examples thereof include cancer. Examples of cancer include, but are not limited to, colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, invasive bladder cancer, pancreatic cancer, metastatic brain cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, lung squamous cell carcinoma, cutaneous squamous cell carcinoma, melanoma, breast adenocarcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, pancreatic squamous cell carcinoma, colon squamous cell carcinoma, gastric squamous cell carcinoma, prostate cancer, osteosarcoma, and soft tissue sarcoma. The medicament of the present invention can be used as an antitumor agent.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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 CDRs are shown below: GYSFTAYN: SEQ ID NO: 9 (CDR-H1) IDPYSGII: SEQ ID NO: 10 (CDR-H2) ARRGYYDGGFDY: SEQ ID NO: 11 (CDR-H3) QDITNY: SEQ ID NO: 12 (CDR-L1) YTS: SEQ ID NO: 13 (CDR-L2) QQDSKHPRT: SEQ ID NO: 14 (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).

[0061] (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.

[0062] 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.

[0063] 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 frame sequence. The germline frame 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.

[0064] 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. Antibody numbers hu2012c and hu2012d have the same CDR sequences. Furthermore, hu2012d has the amino acid sequence substitutions described below.

[0065] Antibody number: hu2012c-VH EVQLVQSGAEVKKPGATVKISCKVSGYSFTAYNMHWVQQAPGKGLEWMGFIDPYSGIITYAEKFQGRVTITADTSDTAYMELSSLRSEDTAVYYCARRGYYDGGFDYWGQGTLVTVSS (SEQ ID NO: 1)

[0066] Antibody number: hu2012c-VL DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYYTSRLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSKHPRTFGQGTKVEIK (SEQ ID NO: 2)

[0067] Antibody number: hu2012d-VH EVQLVQSGAEVKKPGATVKISCKVSGYSFTAYNMHWVQQAPGKGLEWMGFIDPYSGIITYAEKFQGRVTITVDKSTDTAYMELSSLRSEDTAVYYCARRGYYDGGFDYWGQGTLVTVSS (SEQ ID NO: 3) (A72V and T74K are substituted relative to hu2012c-VH)

[0068] Antibody number: hu2012d-VL DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYYTSRLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSKHPRTFGQGTKVEIK (SEQ ID NO: 4) (E55H is substituted relative to hu2012c-VL)

[0069] 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.

[0070] 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 hu2012c refers to an antibody consisting of a hu2012c-H heavy chain and a hu2012c-L light chain, and the anti-CDH3 humanized antibody hu2012d refers to an antibody consisting of a hu2012d-H heavy chain and a hu2012d-L light chain, but the heavy and light chains may be interchanged and expressed. Antibody number: hu2012c-H EVQLVQSGAEVKKPGATVKISCKVSGYSFTAYNMHWVQQAPGKGLEWMGFIDPYSGIITYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARRGYYDGGFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0071] Antibody number: hu2012c-L DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYYTSRLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSKHPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6)

[0072] Antibody number: hu2012d-H EVQLVQSGAEVKKPGATVKISCKVSGYSFTAYNMHWVQQAPGKGLEWMGFIDPYSGIITYAEKFQGRVTITVDKSTDTAYMELSSLRSEDTAVYYCARRGYYDGGFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

[0073] Antibody number: hu2012d-L DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYYTSRLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSKHPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8)

[0074] Example 2: icIEF (imaging capillary isoelectric focusing) Imaging capillary isoelectric focusing (icIEF) of ch2012 and hu2012 was performed using an iCE280 analyzer (Protein Simple).

[0075] (1) Preparation of loading mixture Pharmalyte 3-10, pI marker, and 0.5% methylcellulose solution were mixed to prepare a loading mixture. The preparation amount was adjusted appropriately according to the number of samples. Pharmalyte 3-10 2.0 uL / sample Acidic side pI marker (4.22) 0.25 uL / sample Alkaline side pI marker (9.46) 0.25 uL / sample 0.5% methylcellulose 17.5 uL / sample Total 20.0 uL / sample

[0076] (2) Sample Preparation If the sample contained 20 mM or more of salt, it was replaced with 20 mM phosphate buffer (pH 6.0). Urea was dissolved in 20 mM phosphate buffer (pH 6.0) to prepare 10 M urea immediately after use. Samples were prepared in the following proportions. The sample concentration was 140-200 μg / mL for antibodies and 400 μg / mL for drug-labeled antibodies.

[0077] For n=1 measurement: 10 M urea 10 uL, antibody (140-200 ug / mL) 20 uL, loading mixture 20 uL, total 50 uL

[0078] (3) Preparation of marker A marker was prepared in the following proportions: 10 M urea 20 μL 20 mM phosphate buffer (pH 6.0) 40 μL loading mixture 40 μL Total 100 μL

[0079] The prepared sample and marker were thoroughly mixed in a Vortex mixer and centrifuged for 3 to 5 minutes in a tabletop centrifuge. The sample and marker were dispensed into dedicated glass vials, which were then briefly centrifuged in a tabletop centrifuge to remove any air bubbles, and then placed in the vial holder of the iCE280 analyzer for measurement.

[0080] (4) Results Figure 1 shows the electropherograms of the imaged capillary isoelectric focusing of the chimeric antibody ch2012 and the humanized antibodies hu2012c and hu2012d. ch2012 exhibited multiple peaks with heterogeneous acidic and basic peaks. In contrast, the isoelectric points (pI) of the major peaks of the humanized antibodies hu2012c and hu2012d were 7.99 and 8.60, respectively, and almost no peaks other than the major peak were detected. Humanization of ch2012 resulted in the production of an antibody with a nearly single peak, which facilitates equivalence assessment.

[0081] Example 3: Thermal Shift Assay (TSA) (1) Sample Preparation 1 uL of the Protein Thermal Shift Dye Kit dye (1000x) was diluted with 124 uL of Milli-Q water to prepare dye (8x) (125 uL, enough for 50 samples). The amount prepared was adjusted appropriately depending on the number of samples. The sample antibody was replaced with DPBS and adjusted to a concentration of 0.25 mg / mL. The specified 8-tube PCR tube or 96-well plate was placed on ice, and the reaction solution was prepared in the following proportions for n = 1 measurement.

[0082] Dye (8x) 2.5uL 0.25 mg / mL Ab 17.5uL (antibody concentration in reaction solution: 5.0ug) Total 20.0uL

[0083] The prepared samples were measured using real-time PCR StepOnePlus (Thermofisher Scientific, StepOnePlus-01 registered trademark).

[0084] (2) Measurement conditions: Temp. range: 20-90°C, Heating rate: 0.2°C, Interval: 10 seconds, Times: 350 times. Analysis was performed using Protein Thermal Shift Software (registered trademark).

[0085] (3) Results The results of the thermal shift assay for ch2012, hu2012c, and hu2012d are shown in Figure 2. The Tm values ​​obtained by the analysis were 59.0°C for ch2012 and 68.8°C for hu2012d, indicating that the humanized hu2012d had a higher Tm value. Humanization of ch2012 improved its thermal stability.

[0086] Example 4: Antibody binding activity Antibodies having the sequences shown in Example 1 were produced, and their binding activity was evaluated by flow cytometry. Each cell line to be tested (NCI-H358 cell line, which has been confirmed to express high levels of CDH3) was detached from the culture plate by treatment with 2 mM EDTA-PBS, and then suspended in FACS solution at a concentration of 1 x 10 cells / mL. This cell suspension was seeded into a 96-well plate at 50 μL / well, and purified hu2012c and hu2012d antibodies were added at a concentration of 0.3 μg / mL. The reaction was carried out at 4°C for 60 minutes. After washing twice with FACS solution (PBS containing 1% BSA, 2 mM EDTA, and 0.1% NaN3, 150 μL / well), 4 μg / ml of AlexaFluor488-labeled anti-human IgG goat F(ab')2 (Invitrogen) was added and incubated at 4°C for 30 minutes. After washing twice with FACS solution, flow cytometry was performed. Strong reactivity with CDH3-expressing cell lines was observed (Figure 3). Humanized antibody hu2012d exhibited stronger binding activity than hu2012c.

[0087] Example 5: Measurement of internalization activity This was performed using a saporin-labeled anti-human IgG antibody (Hum ZAP, Advanced Targeting Systems). Because cell killing requires internalization by saporin, internalization activity can be evaluated by measuring the degree of killing. Activity was expressed relative to the cell viability when no saporin-labeled anti-human IgG antibody was added, which was set at 100%. More specifically, the procedure is as follows.

[0088] (1) Measurement Method Human CDH3-expressing cells were the human breast cancer-derived cell line HCC1954 and the human lung cancer-derived cell line NCIH358. Each well was adjusted to contain 5,000 cells, 100 ng (or 0 ng) of HumZAP, and 100 ng of anti-human CDH3 chimeric antibody or anti-human CDH3 humanized antibody. The cells were added to a 96-well microplate and incubated at 37°C in a 5% CO2 incubator for 3 days.

[0089] Thereafter, 10 μL of Cell Counting Kit-8 (Dojindo Laboratories) was added to each well, stirred, and then allowed to stand. After 2 hours, A450 / 620 was measured using a plate reader to count the number of viable cells.

[0090] (2) Calculation of intrinsic activity Comparison of intrinsic activity of antibodies was performed according to the following calculation formula. Sample (ZAP+): Mean signal value of sample in the presence of HumZAP Sample (ZAP-): Mean signal value of sample in the absence of HumZAP NoAb (ZAP+): Mean signal value of wells without antibody in the presence of HumZAP NoAb (ZAP-): Mean signal value of wells without antibody in the absence of HumZAP BK: Mean signal value of wells with medium only

[0091] (3) Results The results are shown in Table 1 and Figure 4. hu2012c and hu2012d showed a lower cell viability than ch2012 in both NCI-H358 and HCC1954 cells. This indicates that the saponin-labeled anti-human IgG antibody was internalized to a greater extent, demonstrating that humanization enhances internalization ability.

[0092]

[0093] Example 6: In vivo (1) Preparation of DM1-labeled antibody When DM1SMe (CAS 138148-68-2) was used as the starting material for the conjugated drug, DM1SH was obtained by reduction treatment with TCEP and HPLC purification using a C18 column. On the other hand, a maleimide group was introduced into the antibody using sulfo-SMCC, and then the antibody was reacted with DM1SH to prepare a DM1-labeled antibody. DM1SMe structural formula

[0094] (2) Preparation of Xenografts HCC1954 cells were cultured and expanded until the number of cells required for transplantation was approximately twice the required number. The day before cell transplantation, the right flank of the mouse was shaved.

[0095] (3) Transplantation After washing with PBS(-), the cells were detached using TrypleExpress (Thermofisher Scientific) and centrifuged at 1,000 rpm for 5 minutes to collect the pellet. The pellet was suspended in antibiotic- and serum-free RPMI-1640 medium and diluted to 5 × 10 ７ The mice were treated with ch2012-DM1 and hu2012(c / d)-DM1 at a dose of 5 mg / kg each in the tail vein. ３ The first dose was administered when the antibody concentration reached 1000 mg / mL, and then the same amount was administered again one week later, for a total of two doses. The results are shown in Figure 5. hu2012c and hu2012d were shown to have higher efficacy than ch2012. This suggests that humanization improves thermal stability and internalization ability, resulting in higher efficacy.

Claims

1. 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; or (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.

2. The antibody according to claim 1, which is 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.

3. A fragment of the anti-CDH3 humanized antibody according to claim 1 or 2, which has the ability to bind to CDH3.

4. Fab, F(ab') ２ 4. The fragment of the anti-CDH3 humanized antibody of claim 3, which is an scFv or scFv.

5. A partial sequence of the antibody according to claim 1 or 2, which has the ability to bind to CDH3.

6. An immunoconjugate comprising the anti-CDH3 humanized antibody, fragment thereof, or partial sequence thereof according to any one of claims 1 to 5 linked to a chemotherapeutic agent or a radioactive substance.

7. The immunoconjugate of claim 6, wherein the chemotherapeutic agent is a cytotoxic agent.

8. The immune complex according to claim 6 or 7, wherein the anti-CDH3 humanized antibody, a fragment thereof, or a partial sequence thereof is linked to the chemotherapeutic agent via a linker.

9. A medicament for treating a disease characterized by overexpression of CDH3, comprising an immunoconjugate according to any one of claims 6 to 8.

10. The pharmaceutical according to claim 9, wherein the disease characterized by overexpression of CDH3 is cancer.

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