Antibody-drug complex

Abstract

Provided is an antibody-drug complex that has cytotoxicity and / or a growth-inhibiting property specifically against cells expressing podoplanin.　This invention is an antibody-drug complex containing a drug and a polypeptide comprising an antibody that specifically binds to podoplanin or an antigen-binding fragment thereof, in a directly or indirectly bound state.

Description

antibody-drug conjugates

[0001] The present invention relates to an antibody-drug conjugate comprising an anti-podoplanin antibody and a drug.

[0002] Platelet aggregation by cancer cells has been reported to be observed during hematogenous metastasis. Most cancer cells are destroyed by attacks from the host's immune system or physical impacts once they invade blood vessels. However, it is thought that platelet aggregation protects them from these processes, enabling them to metastasize. On the other hand, platelet aggregation is thought to promote the adhesion of cancer cells to vascular endothelial cells and also induce local proliferation of cancer cells by releasing growth factors. Furthermore, platelet aggregates produced by cancer cells clogging capillaries also contribute to the promotion of hematogenous metastasis.

[0003] By repeatedly inducing experimental lung metastasis of the murine colon cancer cell line colon26, the highly metastatic NL-17 cell line and the low-metastatic NL-14 cell line were established (Non-Patent Document 1). Furthermore, a monoclonal antibody, 8F11, was produced that exhibited high reactivity with NL-17 cells but low reactivity with NL-14 cells. In in vitro experiments, NL-17 cells induced platelet aggregation in mice, but this activity was inhibited by the 8F11 antibody. Furthermore, in in vivo experiments, experimental lung metastasis of NL-17 cells was inhibited by administration of the 8F11 antibody. These findings suggest that NL-17 cells express a platelet aggregation factor recognized by the 8F11 antibody, which aggregates mouse platelets and results in lung metastasis. This platelet aggregation factor was later named podoplanin (also known as Aggrus, T1alpha, and gp36).

[0004] Subsequently, mouse podoplanin protein was purified from NL-17 cells using an 8F11 antibody column and a WGA column (Non-Patent Document 2). Purified podoplanin induced platelet aggregation in a concentration-dependent manner in mice in the absence of plasma components, and this aggregation reaction was completely inhibited by the 8F11 antibody.

[0005] Non-Patent Document 3 reports the successful cloning of the podoplanin gene. Podoplanin is a type I transmembrane protein with a transmembrane domain at the C-terminus. Epitope analysis of the mouse podoplanin neutralizing antibody 8F11 and detailed mutation experiments revealed that the threonine (Thr) in the three repeats of the sequence EDxxVTPG (PLAG domain) is the active center of podoplanin-mediated platelet aggregation and is conserved across species (Non-Patent Document 4). Approximately half of the molecular weight of podoplanin is made up of glycans, and by using mutant CHO cells (Lec1, Lec2, Lec8) deficient in glycan synthesis, it was found that the sialic acid of the O-linked glycan attached to Thr in the PLAG domain is the active center of platelet aggregation (Non-Patent Document 5).

[0006] Non-Patent Document 6 reports that a highly specific rat monoclonal antibody, NZ-1 antibody, was produced to purify human podoplanin. It was found that the NZ-1 antibody is not only useful for Western blotting, flow cytometry, and immunohistochemical staining, but also has high sensitivity and specificity for immunoprecipitation. Furthermore, it has been reported that the NZ-1 antibody also exhibits ADCC activity and CDC activity in podoplanin-positive tumor cells (Patent Document 1, Non-Patent Documents 7 to 9).

[0007] The NZ-1 antibody inhibited the binding of podoplanin to C-type lectin-like receptor-2 (CLEC-2) and inhibited podoplanin-induced platelet aggregation in a concentration-dependent manner. Furthermore, co-injection of the NZ-1 antibody with podoplanin-expressing cancer cells via the tail vein significantly suppressed lung metastasis (Non-Patent Document 10).

[0008] Podoplanin has been reported to be highly expressed in brain tumors, mesothelioma, testicular tumors, ovarian cancer, and various squamous cell carcinomas (oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer) (Non-Patent Documents 11 to 14). In particular, it is known that podoplanin expression correlates with the malignancy of astrocytic tumors, among other brain tumors. Patent Document 2 discloses multiple anti-podoplanin antibodies that recognize an epitope other than the vicinity of the PLAG domain of podoplanin and have cytotoxic activity, as well as methods for producing the same.

[0009] International Publication No. WO 2011 / 040565 International Publication No. WO 2015 / 053381

[0010] T. Tsuruo, T. Yamori et al., Cancer Res. 43, 5437–5442, 1983Toyoshima M., Nakajima M. et al., Cancer Res. 55, 767–773, 1995Y.Kato, N. Fujita et al., J. Biol. Chem. 278, 51599-51605, 2003 Gene 378C:52-57, 2003Kaneko MK, Kato Y et al., J. Biol. Chem. 279 , 38838–38843 , 2004 Kato Y. , Kaneko MK . et al., Biochem. Biophys. Res. Commun., 349: 1301–1307, 2006Abe S. et al., J Immunol. 2013 Jun 15;190(12):6239-49Kaneko MK. et al., Cancer Sci. 2012 Nov;103(11):1913-9Hatakeyama K. et al., Thromb Res. 2012 Apr;129(4):e70-6Kato Y., Kaneko MK. et al., Cancer Sci. 99 , 54-61 , 2008 Kato Y. , Sasagawa I. et al., Oncogene 23 , 8552-8556, 2004Kato Y. , Kaneko M. et al., Tumor Biol. 26,195-200, 2005Mishima K., Kato Y. et al., Acta Neuropathol.111(5):483-488, 2006aMishima K., Kato Y. et al., Acta Neuropathol.111(6):563-568, 2006b

[0011] The scent of the snowflake is a slightly smoother smoothie Eventually, it is a nice place to stay in the woods.

[0012] The present invention provides the following: [1] An antibody-drug conjugate comprising a polypeptide consisting of an antibody or antigen-binding fragment thereof that specifically binds to podoplanin and a drug, bound directly or indirectly. [2] The antibody-drug conjugate of [1], wherein the drug is a drug for use in the treatment, diagnosis, and / or prevention of a disease involving podoplanin. [3] The antibody-drug conjugate of [1], wherein the drug is an anticancer drug. [4] The antibody-drug conjugate of any of [1] to [3], wherein the polypeptide specifically binds to tumor tissue or cancer cells that express podoplanin. [5] The antibody-drug conjugate of [4], wherein the tumor tissue or cancer cells are cancer cells derived from any of the group consisting of head and neck cancer, brain tumor, mesothelioma, testicular tumor, ovarian cancer, oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer. [6] The antibody-drug conjugate according to any one of [3] to [5], wherein the anticancer drug is at least one compound selected from the group consisting of alkylating agents, platinum compounds, antimetabolites (e.g., antifolates, pyridine metabolism inhibitors, and purine metabolism inhibitors), ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, antitumor antibiotics, antihormones, and cytokine preparations. [7] The antibody-drug conjugate according to [6], wherein the anticancer drug is at least one compound selected from the group consisting of monomethylauristatin E (MMAE), exatecan, emtansine (DM1), and eribulin. [8] The antibody-drug conjugate according to any of [1] to [7], wherein the polypeptide has at least one of six CDRs: a heavy chain complementarity-determining region (CDR) 1 consisting of the amino acid sequence represented by SEQ ID NO: 1; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6.[9] The antibody-drug conjugate according to [8], wherein the polypeptide comprises: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7 or the amino acid sequence represented by SEQ ID NO:1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, and having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8 or the amino acid sequence represented by SEQ ID NO:4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:6, and having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:8.

[10] The antibody-drug conjugate according to any of [1] to [7], wherein the polypeptide has at least one of six CDRs consisting of: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 9; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 10; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 11; a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 12; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14.

[11] The antibody-drug conjugate according to

[10] , wherein the polypeptide comprises: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 15 or the amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 10, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 11, and having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence represented by SEQ ID NO: 12, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14, and having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 16.

[12] The antibody-drug conjugate according to any of [1] to [7], wherein the polypeptide has at least one of six CDRs consisting of: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 26; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 27; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 28; a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 29; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 30; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31.

[13] The antibody-drug conjugate according to

[12] , wherein the polypeptide comprises: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 32 or the amino acid sequence represented by SEQ ID NO: 26, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 27, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 28, and having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 32; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 33 or the amino acid sequence represented by SEQ ID NO: 29, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 30, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31, and having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 33.

[14] The antibody-drug conjugate according to any of [1] to

[13] , wherein the antibody is a humanized antibody or a human chimeric antibody.

[15] A pharmaceutical composition comprising the antibody-drug conjugate according to any of [1] to

[14] as an active ingredient.

[16] The pharmaceutical composition according to

[15] , for use in the treatment, diagnosis, and / or prevention of a disease involving podoplanin in a subject.

[17] The pharmaceutical composition according to

[16] , wherein the disease is cancer.

[18] The pharmaceutical composition according to

[17] , wherein the cancer comprises cells expressing podoplanin.

[19] The pharmaceutical composition according to any of

[16] to

[18] , wherein the subject is an animal, including a human.

[20] A method for treating, diagnosing, and / or preventing a disease involving podoplanin, comprising administering the pharmaceutical composition according to any of

[15] to

[19] to a subject. This specification incorporates the disclosures of Japanese Patent Application No. 2024-082379, from which the present application claims priority.

[0013] According to the present invention, it is possible to provide an antibody-drug conjugate that has cytotoxicity and / or growth inhibitory activity specifically against cells expressing podoplanin.

[0014] 3A is a schematic diagram showing the putative drug-binding site of an antibody. It is a hydrophobic liquid chromatography chart of an antibody-drug conjugate (ADC) of the antibody hLpMab-23 and monomethyl auristatin E (MMAE). It is a plot of the concentration of the ADC (hLpMab-23-MMAE) or the antibody (hLpMab-23) alone added to the culture medium, and the cell viability in the culture medium of podoplanin-expressing cancer cells (A253 cell line in which podoplanin is forcibly expressed). FIG. 3A shows the relationship between the ADC concentration and the cell viability in the culture medium of podoplanin-expressing cancer cells, and FIG. 3B shows the relationship between the concentration of the antibody alone and the cell viability in the culture medium of podoplanin-expressing cancer cells. The cell viability was expressed as the percentage of the number of cells, with the number of cells without the addition of ADC or antibody taken as 100%. This figure is a plot of the concentration of added ADC (hLpMab-23-MMAE) versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. Cell viability is expressed as the percentage of the number of cells when the number of cells in the absence of ADC is taken as 100%. This figure is a plot of the concentration of added ADC (hLpMab-23-MMAE) versus the cell viability of the LN319 cell line (podoplanin-positive) and LN229 cell line (podoplanin-negative). Cell viability is expressed as the percentage of the number of cells when the number of cells in the absence of ADC is taken as 100%. This figure is a plot of the concentration of added ADC (hLpMab-23-MMAE) versus the cell viability of the KYSE140 cell line (podoplanin-positive) and KYSE410 cell line (podoplanin-negative). Cell viability was expressed as a percentage of the number of cells when the number of cells was not added, with the number of cells in the absence of ADC being taken as 100%. Figure 1 is a plot of the concentration of added ADC (hLpMab-23-DM1) versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. Cell viability was expressed as a percentage of the number of cells when the number of cells in the absence of ADC was taken as 100%. Figure 1 is a plot of the concentration of added ADC (hLpMab-23-exatecan) versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed.Cell viability was expressed as a percentage of the number of cells obtained without the addition of ADC, with the number of cells taken as 100%. Figure 10 shows graphs showing changes in tumor size in tumor-implanted mouse model cells after the start of administration of ADC (hLpMab-23-MMAE) or antibody (hLpMab-23) alone. Figure 10A shows a plot of the MMAE concentration versus the cell viability of the A253 cell line in which podoplanin was forcibly expressed. Figure 10B shows a plot of the MMAE concentration versus the cell viability of the LN319 cell line. Figure 10C shows a plot of the MMAE concentration versus the cell viability of the KYSE140 cell line. Cell viability was expressed as a percentage of the number of cells obtained without the addition of MMAE, with the number of cells taken as 100%. Figure 10B shows a plot of the MMAE concentration versus the cell viability of the podoplanin-negative cell line. Figure 11A shows a plot of MMAE concentration versus cell viability for the HSQ-89 cell line. Figure 11B shows a plot of MMAE concentration versus cell viability for the LN229 cell line. Figure 11C shows a plot of MMAE concentration versus cell viability for the KYSE410 cell line. Cell viability is expressed as a percentage of the cell count, with the cell count without MMAE defined as 100%. Flow cytometry was performed using an anti-MMAE antibody (IgG) and a fluorescently labeled anti-IgG antibody on the KYSE140 cell line (podoplanin-positive) and KYSE410 cell lines (podoplanin-negative) incubated with the ADC (hLpMab-23-MMAE) or the antibody (hLpMab-23) alone. Figure 12A shows flow cytometry of podoplanin-positive cells. Figure 12B shows flow cytometry of podoplanin-negative cells. The figure shows a plot of the concentration of various added ADCs (hLpMab-23-MMAE, hLpMab-23-DM1 and hLpMab-23-exatecan) or antibody (hLpMab-23) alone versus the cell viability in HUVEC culture medium.Figure 13A shows the relationship between the concentration of the hLpMab-23 antibody alone and cell viability in HUVEC culture medium. Figure 13B shows the relationship between the concentration of hLpMab-23-MMAE and cell viability in HUVEC culture medium. Figure 13C shows the relationship between the concentration of hLpMab-23-DM1 and cell viability in HUVEC culture medium. Figure 13D shows the relationship between the concentration of hLpMab-23-exatecan and cell viability in HUVEC culture medium. Cell viability is expressed as the percentage of the number of cells in the absence of ADC or antibody, taken as 100%. This is a hydrophobic liquid chromatography chart of an antibody-drug conjugate (ADC) of antibody chLpMab-23 and monomethyl auristatin E (MMAE). This is a plot of the concentration of ADC (chLpMab-23-MMAE) or antibody (chLpMab-23) alone added to the culture medium, and the cell viability in the culture medium of podoplanin-expressing cancer cells (A253 cell line in which podoplanin was forcibly expressed). Figure 15A shows the relationship between ADC concentration and cell viability in the culture medium of podoplanin-expressing cancer cells, and Figure 15B shows the relationship between the concentration of antibody alone and cell viability in the culture medium of podoplanin-expressing cancer cells. Cell viability is expressed as the percentage of the number of cells, with the number of cells in the absence of ADC or antibody being taken as 100%. This is a plot of the concentration of ADC (chLpMab-23-MMAE) added, and the cell viability of A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. Cell viability was expressed as a percentage of the number of cells without ADC, with the number of cells in the absence of ADC taken as 100%. Figure 18A shows a plot of the concentration of added ADC (chLpMab-23-MMAE) versus the cell viability of the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative). Cell viability was expressed as a percentage of the number of cells in the absence of ADC, with the number of cells in the absence of ADC taken as 100%. Flow cytometry was performed using an anti-MMAE antibody (IgG) and a fluorescently labeled anti-IgG antibody on the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative) that had been reacted with ADC (chLpMab-23-MMAE) or antibody (chLpMab-23) alone. Figure 18B shows flow cytometry of podoplanin-positive cells.Figure 18B shows flow cytometry of podoplanin-negative cells. This is a chart of hydrophobic liquid chromatography of an antibody-drug conjugate (ADC) of antibody chLpMab-2 and monomethyl auristatin E (MMAE). This is a plot of the concentration of ADC (chLpMab-2-MMAE) or antibody (chLpMab-2) alone added to the culture medium versus the cell viability in the culture medium of podoplanin-expressing cancer cells (A253 cell line in which podoplanin is forcibly expressed). Figure 20A shows the relationship between ADC concentration and cell viability in the culture medium of podoplanin-expressing cancer cells, and Figure 20B shows the relationship between the concentration of antibody alone and cell viability in the culture medium of podoplanin-expressing cancer cells. Cell viability is expressed as the percentage of the number of cells, with the number of cells without the addition of ADC or antibody taken as 100%. This figure is a plot of the concentration of added ADC (chLpMab-2-MMAE) versus the cell viability of A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. Cell viability is expressed as a percentage of the number of cells, with the number of cells in the absence of ADC being taken as 100%. This figure is a chart of hydrophobic liquid chromatography of an antibody-drug conjugate (ADC) of antibody chLpMab-7 and monomethyl auristatin E (MMAE). This figure is a plot of the concentration of ADC (chLpMab-7-MMAE) or antibody (chLpMab-7) alone added to the culture medium versus the cell viability in the culture medium of podoplanin-expressing cancer cells (A253 cell line in which podoplanin was forcibly expressed). Figure 23A shows the relationship between ADC concentration and cell viability in culture medium of podoplanin-expressing cancer cells, and Figure 23B shows the relationship between antibody concentration alone and cell viability in culture medium of podoplanin-expressing cancer cells. Cell viability is expressed as the percentage of the number of cells, with the number of cells without addition of ADC or antibody taken as 100%. This figure is a plot of the concentration of added ADC (chLpMab-7-MMAE) and the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. Cell viability is expressed as the percentage of the number of cells, with the number of cells without addition of ADC taken as 100%.This figure shows a plot of the concentration of added ADC (chLpMab-7-MMAE) versus the cell viability of the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative). Cell viability is expressed as a percentage of the cell number, with the number of cells without ADC added being 100%. Flow cytometry was performed using an anti-MMAE antibody (IgG) and a fluorescently labeled anti-IgG antibody on the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative) that had been reacted with ADC (chLpMab-7-MMAE) or antibody (chLpMab-7) alone. Figure 26A shows flow cytometry of podoplanin-positive cells. Figure 26B shows flow cytometry of podoplanin-negative cells. 1 is a plot diagram of the concentration of added ADC (hLpMab-23-eribulin) versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. Cell viability is expressed as the percentage of the number of cells, with the number of cells in the absence of ADC being taken as 100%. This is a plot diagram of the concentration of ADC (hLpMab-23-MMAE) or antibody (hLpMab-23) alone added to the culture medium versus the cell viability in the culture medium of the NCI-H226 cell line. This is a plot diagram of the concentration of ADC (hLpMab-23-MMAE) or antibody (hLpMab-23) alone added to the culture medium versus the cell viability in the culture medium of the DU145 cell line. 1 is a graph showing changes in tumor size in tumor-injected model mice after the start of administration of each dose of ADC (hLpMab-23-MMAE) or antibody (hLpMab-23) alone.

[0015] 1. Configuration and Definitions In this specification, the expression "A to B" (A and B are both numerical values) means "A or more and B or less" unless otherwise specified.

[0016] As used herein, "anti-podoplanin antibody" refers to an antibody that binds to a podoplanin protein consisting of the amino acid sequence represented by SEQ ID NO: 17 (Table 1). As used herein, the term "antibody" may refer to an intact antibody having a structure in which two heavy chains (H chains) and two light chains (L chains) stabilized by disulfide bonds are associated, or a single-domain antibody (V) consisting of only heavy chains.ＨＨ , V ＮＡＲ ). In an intact antibody comprising a heavy chain and a light chain, the heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain consists of a light chain variable region VL and a light chain constant region CL. Among these, the variable region fragment (Fv) consisting of VH and VL is the region that is directly involved in antigen binding and provides diversity to the antibody. The antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region. Within the variable region, the region that directly contacts the antigen undergoes particularly large changes and is called the complementarity-determining region (CDR). Portions other than the CDR that show relatively little mutation are called the framework region (FR). The light chain and heavy chain variable regions each contain three CDRs (heavy chain CDR1 to 3, and light chain CDR1 to 3) and four framework regions (FR1 to 4). In this specification, the sequences of VH, heavy chain CDR1 to 3, and FR1 to 4 in the heavy chain, and VL, light chain CDR1 to 3, and FR1 to 4 in the light chain of an antibody refer to those determined by the numbering scheme based on the IMGT (International ImMunoGeneTics database) scheme, unless otherwise specified.

[0017]

[0018] As used herein, an anti-podoplanin antibody may be a monoclonal antibody or a polyclonal antibody. Furthermore, the anti-podoplanin antibody of the present invention may be of any isotype, including IgG, IgM, IgA, IgD, and IgE. It may be produced by immunizing a non-human animal, such as a mouse, rat, hamster, guinea pig, rabbit, or chicken, or it may be a recombinant antibody, or it may be a chimeric antibody, humanized antibody, fully humanized antibody, or the like. The term "chimeric antibody" as used herein refers to an antibody in which antibody fragments derived from different species are linked. Furthermore, the term "humanized antibody" as used herein refers to an antibody in which the corresponding positions in a human antibody are substituted with amino acid sequences characteristic of a non-human antibody. For example, an antibody may have heavy chain CDRs 1-3 and light chain CDRs 1-3 of an antibody produced by immunizing a mouse, with all other regions, including the four framework regions (FRs) of the heavy and light chains, derived from a human antibody. Such an antibody may also be referred to as a CDR-grafted antibody. As used herein, the term "humanized antibody" also encompasses human chimeric antibodies.

[0019] As used herein, the term "antigen-binding fragment" of an anti-podoplanin antibody refers to a partial fragment of the anti-podoplanin antibody that retains the ability to specifically bind to podoplanin. Specifically, this fragment includes a Fab fragment consisting of VL, VH, CL, and CH1 domains; an F(ab') fragment in which two Fabs are linked by a disulfide bond at the hinge region; and an F(ab') fragment in which two Fabs are linked by a disulfide bond at the hinge region. ２ Examples of the antibody include, but are not limited to, Fv consisting of VL and VH; scFv, which is a single-chain antibody in which VL and VH are linked by an artificial polypeptide linker; and bispecific antibodies such as diabody type, scDb type, tandem scFv type, and leucine zipper type.

[0020] As used herein, the term "amino acid" is used in its broadest sense and includes not only naturally occurring amino acids but also artificial amino acid variants and derivatives. Amino acids may be represented by conventional single-letter or three-letter symbols. As used herein, amino acids or derivatives thereof include naturally occurring proteinaceous L-amino acids; unnatural amino acids; and chemically synthesized compounds having properties known in the art that are characteristic of amino acids. Examples of unnatural amino acids include, but are not limited to, α,α-disubstituted amino acids (e.g., α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α-hydroxy acids, which have backbone structures that differ from those of natural amino acids; amino acids whose side chain structures differ from those of natural amino acids (e.g., norleucine and homohistidine); amino acids with an extra methylene in the side chain (e.g., "homo" amino acids, homophenylalanine and homohistidine); and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid).

[0021] As used herein, the "sequence identity" of an amino acid sequence refers to the proportion (percentage) of identical amino acids relative to the total overlapping amino acid sequence in an optimal alignment when two amino acid sequences are aligned with or without gaps, and refers to the value calculated by formula (1): Sequence identity (%) = number of matches (gaps ignored) / length of the shorter sequence (length excluding gaps) × 100 ... formula (1) Sequence identity can be easily determined using BLAST (Basic Local Alignment Search Tool) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), an algorithm commonly used in this field.

[0022] As used herein, the term "drug," also referred to as "medicine" or "drug," refers to a substance that, when administered to an organism, causes changes in the physiology or psychology of the organism. More specifically, the term "drug" refers to a compound used for the treatment, diagnosis, and / or prevention of disease in a subject. As used herein, the term "anticancer agent" refers to a compound that produces at least one of the following: reduction in tumor size, inhibition of tumor metastasis, inhibition (slowing or stopping) of tumor growth, and alleviation of one or more symptoms associated with cancer. More specifically, the term refers to a compound that has cytotoxicity and / or growth-suppressing properties against cancer cells.

[0023] As used herein, the term "subject" refers to a subject that is the target of treatment, diagnosis, and / or prevention. The subject is a mammal such as a human, a primate including a chimpanzee, a pet animal such as a dog or a cat, a livestock animal such as a cow, a horse, a sheep, or a goat, a rodent such as a mouse or a rat, or an animal kept in a zoo, and is preferably a human.

[0024] As used herein, "cancer cells" refer to cells, also called malignant tumor cells or malignant neoplasms, which have abnormal proliferation and metastasis and are derived mainly from mammals, particularly humans. As used herein, "tumor tissue" or "cancer cells" refers to hematopoietic cancers (e.g., hematologic malignancies such as leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL), and lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), and multiple myeloma (MM)), as well as sarcomas (e.g., soft tissue sarcoma, uterine sarcoma, etc.), skin cancer, melanoma (e.g., malignant melanoma), bladder cancer, breast cancer, uterine cancer (e.g., uterine cancer, cervical cancer), ovarian cancer, prostate cancer, It refers to tissues or cells derived from any of the following solid cancers: lung cancer, colorectal cancer (e.g., colorectal adenocarcinoma), liver cancer (so-called hepatic cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), esophageal cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), adrenal cancer, stomach cancer (e.g., gastric adenocarcinoma), testicular cancer (e.g., testicular germ cell tumor), gallbladder cancer and biliary tract cancer, thyroid cancer (e.g., primary thyroid cancer), thymus cancer, bone cancer, soft tissue cancer (e.g., desmoid tumor), brain tumor (e.g., glioblastoma, primary malignant astrocytoma), colon cancer, embryonal carcinoma, osteosarcoma, hemangioma, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), rhabdomyosarcoma, urothelial cancer, etc. Here, "solid cancer" refers to cancer of any body tissue other than blood, bone marrow, or lymphatic system, and is classified into cancers derived from epithelial cells and cancers derived from non-epithelial cells. Examples of solid epithelial cancers include cancers of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, prostate, testes, urinary tract, bladder, and skin. Examples of solid non-epithelial cancers include sarcomas, brain tumors, and bone tumors.

[0025] As used herein, the term "antibody-drug conjugate" refers to a conjugate comprising a polypeptide consisting of an antibody or an antigen-binding fragment thereof and a drug in a bound state.

[0026] 2. Antibody-drug conjugate A first embodiment of the present invention is an antibody-drug conjugate. The antibody-drug conjugate of this embodiment is characterized by comprising a drug and a polypeptide consisting of an antibody that specifically binds to podoplanin or an antigen-binding fragment thereof, bound directly or indirectly to each other. The antibody-drug conjugate of this embodiment is capable of exerting cytotoxicity and / or growth inhibitory properties specifically against cells in which podoplanin is involved, more specifically, cancer cells that express podoplanin.

[0027] The cancer cells are not particularly limited as long as they express podoplanin, but are preferably cancer cells derived from any of the group consisting of head and neck cancer, brain tumor, mesothelioma, testicular tumor, ovarian cancer, oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer.

[0028] 2-1 Anti-podoplanin Antibody or Antigen-Binding Fragment Thereof In the antibody-drug conjugate of this embodiment, the polypeptide consisting of the antibody or antigen-binding fragment thereof (hereinafter also referred to as "anti-podoplanin antibody, etc.") is preferably a polypeptide that specifically binds to tumor tissue or cancer cells that express podoplanin. The binding region on podoplanin is not particularly limited as long as it is capable of specifically binding to podoplanin, but in particular, the antibody or antigen-binding fragment can be an antibody that recognizes the extracellular region of podoplanin as an epitope.

[0029] Podoplanin is known to be expressed in some normal cells, such as lung epithelial cells. Therefore, in the antibody-drug conjugate of this embodiment, the anti-podoplanin antibody or the like is preferably an antibody that does not bind or binds very weakly to podoplanin expressed in normal cells, but binds to podoplanin expressed in cancer cells. Here, "very weak binding" refers to a binding activity of the antibody to podoplanin in cancer cells of less than 1, less than 0.1, or less than 0.01, where the binding activity is 100. The binding activity referred to here can be measured by known methods, such as ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), fluorescent antibody technique, FACS, etc.

[0030] The anti-podoplanin antibody or the like is preferably an antibody having a heavy chain and a light chain or an antigen-binding fragment thereof. The antigen-binding fragment preferably comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of the VH and VL preferably has three complementarity-determining regions (CDRs) and four framework regions (FRs).

[0031] Examples of the anti-podoplanin antibody of this embodiment include mouse monoclonal antibodies LpMab-23, LpMab-2, LpMab-3, LpMab-7, and LpMab-9 described in Patent Document 2, recombinant antibodies thereof (rLpMab-23, rLpMab-2, rLpMab-3, rLpMab-7, and rLpMab-9), chimeric antibodies, and humanized antibodies (hLpMab Any of human LpMab-23, hLpMab-2, hLpMab-3, hLpMab-7, and hLpMab-9), human chimeric antibodies (chimeric antibodies in which only the constant region is humanized: chLpMab-23, chLpMab-2, chLpMab-3, chLpMab-7, and chLpMab-9), fully humanized antibodies, CDR-grafted antibodies, and antigen-binding fragments thereof can be used.

[0032] In one aspect, an antibody having the following six CDRs (e.g., LpMab-23, chLpMab-23) can be used as an anti-podoplanin antibody or the like: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6. Alternatively, a polypeptide having at least one of the six CDRs can be used.

[0033] The polypeptide is not particularly limited as long as it has the above-mentioned CDR regions, but preferably comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7 or the amino acid sequence represented by SEQ ID NO:1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, and has a heavy chain variable region that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO:7; and The polypeptide may comprise a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6, and the light chain variable region has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 8.

[0034] In another aspect, an antibody having the following six CDRs (e.g., LpMab-2, chLpMab-2) can be used as the anti-podoplanin antibody, etc.: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:9; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:10; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:11; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:12; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:13; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:14. Alternatively, a polypeptide having at least one of the six CDRs can be used.

[0035] The polypeptide is not particularly limited as long as it has the above-mentioned CDR regions, but preferably comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 15 or the amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 10, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 11, and has a heavy chain variable region that has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15; and The polypeptide may comprise a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence represented by SEQ ID NO: 12, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14, and the light chain variable region has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 16.

[0036] In yet another aspect, an antibody having the following six CDRs (e.g., LpMab-3, chLpMab-3) can be used as the anti-podoplanin antibody, etc.: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 18; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 19; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 20; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 21; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 22; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 23. Alternatively, a polypeptide having at least one of the six CDRs can be used.

[0037] The polypeptide is not particularly limited as long as it has the CDR regions described above, but preferably comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 24 or the amino acid sequence represented by SEQ ID NO: 18, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 20, and has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24; and The polypeptide may comprise a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:25 or the amino acid sequence represented by SEQ ID NO:21, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:22, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:23, and the polypeptide may comprise a light chain variable region having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO:25.

[0038] In yet another aspect, an antibody having the following six CDRs (e.g., LpMab-7, chLpMab-7) can be used as the anti-podoplanin antibody, etc.: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 26; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 27; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 28; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 29; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 30; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31. Alternatively, a polypeptide having at least one of the six CDRs can be used.

[0039] The polypeptide is not particularly limited as long as it has the above-mentioned CDR regions, but preferably comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 32 or the amino acid sequence represented by SEQ ID NO: 26, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 27, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 28, and has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 32; and The polypeptide may comprise a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 33 or the amino acid sequence represented by SEQ ID NO: 29, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 30, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31, and the light chain variable region has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 33.

[0040] In yet another aspect, an antibody having the following six CDRs (e.g., LpMab-9, chLpMab-9) can be used as the anti-podoplanin antibody, etc.: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 34; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 35; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 36; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 37; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 38; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 39. Alternatively, a polypeptide having at least one of the six CDRs can be used.

[0041] The polypeptide is not particularly limited as long as it has the CDR regions described above, but preferably comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40 or the amino acid sequence represented by SEQ ID NO: 34, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 35, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 36, and has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 40; and The polypeptide may comprise a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:41 or the amino acid sequence represented by SEQ ID NO:37, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:38, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:39, and having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO:41.

[0042] In each of the above polypeptide aspects, as long as the effect of the antibody-drug conjugate of this embodiment is achieved, any number of the six CDRs may be contained, for example, two or more, three or more, four or more, five or more, or all six. It is particularly preferable that all six CDRs are contained.

[0043] In each of the above aspects, at least one of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 may contain addition, substitution, or deletion of one to several amino acids. When one to several amino acids are added, substituted, or deleted, the number of amino acids to be deleted, substituted, etc. is not particularly limited as long as the resulting polypeptide retains the function of a CDR, and may be, for example, one, two, or three. The substituted or added amino acids may be natural proteinogenic amino acids as well as unnatural amino acids or amino acid analogs. The position of the deletion, substitution, or addition of the amino acid may be anywhere in the original CDR sequence, as long as the function of a CDR is retained.

[0044] In each of the above aspects, at least one of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 may have an amino acid sequence that is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more identical to the amino acid sequence of the original heavy chain CDR1 to 3 and the original light chain CDR1 to 3.

[0045] CDRs consisting of amino acid sequences in which amino acids are added, substituted, or deleted from the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3, or CDRs having 80% or more sequence identity with the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3, can be prepared using known methods such as site-directed mutagenesis, random mutagenesis, chain shuffling, and CDR walking. According to these methods, antibodies or antibody fragments having various mutations in the CDRs are displayed on the surface of phages by phage display, and then screened using an antigen, thereby obtaining CDRs with more affinity maturation, as is well known to those skilled in the art (e.g., Wu et al., PNAS, 95:6037-6042(1998); Schier, R. et al., J. Mol. Bio. 263:551-567(1996); Schier, R. et al., J. Mol. Biol. 255:28-43(1996); Yang, WP et al., J. Mol. Biol., 254:392-403(1995)).

[0046] Table 2 shows the sequences of the six CDRs, VH and VL of each of the above antibodies (LpMab-23, LpMab-2, LpMab-3, LpMab-7 and LpMab-9).

[0047]

[0048] In this embodiment, the polypeptide itself may have cytotoxicity (antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity) against cancer cells that express podoplanin.

[0049] 2-2 Drug In the antibody-drug conjugate of this embodiment, the drug is preferably a drug used in the treatment, diagnosis, and / or prevention of a disease involving podoplanin. Furthermore, it is more preferably an anticancer drug. The anticancer drug referred to here is preferably an anticancer drug particularly suitable for the treatment, diagnosis, and / or prevention of any cancer in the group consisting of head and neck cancer, brain tumor, mesothelioma, testicular tumor, ovarian cancer, oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer.

[0050] The anticancer agent is not particularly limited as long as it is used in the treatment, diagnosis, and / or prevention of cancer. Examples of the anticancer agent include alkylating agents (e.g., ifosfamide, cyclophosphamide, dacarbazine, temodar, nimustine, busulfan, procarbazine, melphalan, ranimustine, nitrogen mustard, etc.), platinum agents (e.g., oxaliplatin, carboplatin, cisplatin, nedaplatin, etc.), antimetabolites (e.g., folate metabolic antagonists (e.g., pemetrexed, methotrexate, etc.), pyridine metabolism inhibitors (e.g., 5-fluorouracil, capecitabine, cytarabine, gemcitabine, etc.), ribonucleotide reductase inhibitors (e.g., hydroxyurea, etc.), nucleotide analogs, topoisomerase inhibitors (e.g., exatecan, irinotecan, nogitecan, etoposide, anthracyclines, etc.), microtubule heavy low molecular weight compounds such as microtubule depolymerization inhibitors (e.g., auristatins (monomethyl auristatin E (MMAE) and the like), maytansinoids (emtansine (DM1) and the like), eribulin, daunorubicin, doxorubicin, vincristine, vinblastine, vindesine and the like), microtubule depolymerization inhibitors (e.g., paclitaxel, docetaxel and the like), antitumor antibiotics (e.g., adriamycin, daunomycin, mitomycin, aclacinomycin, bleomycin and the like), and antihormonal drugs (e.g., tamoxifen and the like); high molecular weight compounds such as cytokine preparations (e.g., human interleukin 2, human granulocyte macrophage colony-stimulating factor, human macrophage colony-stimulating factor, human interleukin 12 and the like), toxins having anticancer activity (e.g., Pseudomonas aeruginosa exotoxin (PE) or its cytotoxic fragments (e.g., PE38), diphtheria toxin, ricin A chain and the like); ３２ P. １４ C. １２５ I, ３ H. １３１ I, ２１１ At, ９０ The compound may be at least one compound selected from the group consisting of radioisotopes having anticancer activity, such as Y. In particular, the compound may be at least one compound selected from the group consisting of MMAE, exatecan, DM1, and eribulin.

[0051] 2-3 Antibody-drug conjugate The antibody-drug conjugate of this embodiment is a conjugate in which a polypeptide described in the section "2-1 Anti-podoplanin antibody or antigen-binding fragment thereof" is directly or indirectly conjugated to a drug described in "2-2 Drug."

[0052] The bond between the polypeptide and the drug may be cleavable or non-cleavable. They may be directly or indirectly bonded. More specifically, an indirect bond refers to a bond via a linker. The linker referred to here may be a cleavable linker or a non-cleavable linker, and any known linker may be used. It is particularly preferable to use a cleavable linker.

[0053] Cleavable linkers have a structure that releases a drug from an ADC under acidic or reducing conditions, or in response to tumor-associated factors such as proteolytic enzymes (e.g., cathepsins). Examples of cleavable linkers include, but are not limited to, pH-sensitive hydrazone linkers, reducible disulfide linkers, and enzyme-cleavable linkers (e.g., peptide linkers). Examples of non-cleavable linkers that can be used include thioether linkers and maleimide-based linkers. Linkers can be selected appropriately depending on the type of cancer cells to be targeted, the site of the cancer to be targeted, and the drug to be used.

[0054] Figure 1 shows the putative drug-binding site of an antibody. In the illustrated example, the linker used is MC-VC-PAB-linker, and the drug used is monomethyl auristatin E (MMAE). There are eight putative drug-binding sites in the antibody, allowing up to eight drug molecules to be bound. After the antibody binds to a target cell, the VC portion of the linker is cleaved by an enzyme, allowing the bound drug to act on the cell in a free state.

[0055] 3. Pharmaceutical Composition A second embodiment of the present invention is a pharmaceutical composition. The pharmaceutical composition of this embodiment is characterized by comprising, as an active ingredient, an antibody-drug conjugate described in Section "2. Antibody-drug conjugate." The pharmaceutical composition of this embodiment is used for the treatment, diagnosis, and / or prevention of a disease associated with podoplanin, preferably cancer, in a subject.

[0056] The cancer is preferably a cancer containing tumor tissue or cancer cells that express podoplanin, and the tumor tissue or cancer cells may be tumor tissue or cancer cells derived from any of the group consisting of head and neck cancer, brain tumor, mesothelioma, testicular tumor, ovarian cancer, oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer.

[0057] The subject of treatment, diagnosis, and / or prevention using the pharmaceutical composition of this embodiment is not particularly limited, but is preferably an animal including a human, particularly a human, and more preferably a human suffering from cancer.

[0058] The pharmaceutical composition of this embodiment may further contain pharmaceutically acceptable carriers and additives in addition to the antigen-drug complex.

[0059] Examples of carriers and additives include, but are not limited to, water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants and the like.

[0060] The pharmaceutical composition of this embodiment can be in various forms, such as a liquid (e.g., an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, etc. A preferred embodiment is an injection, which is preferably administered parenterally (e.g., intravenously, transdermally, intraperitoneally, intramuscularly).

[0061] The pharmaceutical composition of this embodiment is effective in treating podoplanin-associated diseases, particularly cancer, including, but not limited to, head and neck cancer, brain tumor, mesothelioma, testicular tumor, ovarian cancer, oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer.

[0062] The pharmaceutical composition of this embodiment contains an antigen-drug conjugate as an active ingredient, and the content of the antibody-drug conjugate in the pharmaceutical composition can be, but is not limited to, an amount such that the amount administered to a subject is, for example, 0.025 to 50 mg / kg, preferably 0.1 to 50 mg / kg, more preferably 0.1 to 25 mg / kg, and even more preferably 0.1 to 10 mg / kg or 0.1 to 3 mg / kg.

[0063] The pharmaceutical composition of this embodiment may be administered either once or multiple times. The frequency of administration in the case of multiple administration is not particularly limited, but can be once every three days to once every two months, particularly once a week to once every three weeks. The administration period of the pharmaceutical composition of this embodiment can be appropriately adjusted depending on the condition of the subject, the presence or absence of side effects, etc., and is not particularly limited, but can be, for example, 3 days to 6 months, particularly 1 week to 3 months, or 1 week to 1 month.

[0064] 4. Method for treating, diagnosing, and / or preventing a disease associated with podoplanin A third embodiment of this embodiment is a method for treating, diagnosing, and / or preventing a disease associated with podoplanin. The disease associated with podoplanin herein is preferably cancer. The method of this embodiment is characterized by comprising administering to a subject the pharmaceutical composition described in Section "3. Pharmaceutical composition."

[0065] In this embodiment, the subject of treatment, diagnosis, and / or prevention is a mammal such as a human, a primate including a chimpanzee, a pet animal such as a dog or a cat, a livestock animal such as a cow, a horse, a sheep, or a goat, a rodent such as a mouse or a rat, or an animal kept in a zoo, preferably a human, and most preferably a human suffering from cancer or a human being under follow-up after treatment.

[0066] In the method of this embodiment, the dose of the antibody-drug conjugate, which is the active ingredient, can be, for example, but is not limited to, 0.025 to 50 mg / kg, preferably 0.1 to 50 mg / kg, more preferably 0.1 to 25 mg / kg, and even more preferably 0.1 to 10 mg / kg or 0.1 to 3 mg / kg.

[0067] In the method of this embodiment, the pharmaceutical composition may be administered once or multiple times. In the case of multiple administrations, the frequency of administration is not particularly limited, but can be once every three days to once every two months, particularly once a week to once every three weeks. The administration period of the pharmaceutical composition of this embodiment is not particularly limited, but can be, for example, three days to six months, particularly one week to three months, or one week to one month.

[0068] In the method of this embodiment, other treatments, diagnoses, and / or prophylaxis may be administered in addition to the administration of a pharmaceutical composition containing an antibody-drug conjugate. When other treatments are administered in combination, they can be appropriately selected from the following: administration of an additional anticancer drug, administration of a known single antibody drug (e.g., cetuximab, nivolumab, pembrolizumab, etc.), administration of a drug expected to have a combined effect (e.g., a hormone therapy agent for cancer of the reproductive system), immunotherapy (e.g., immune cell therapy such as CAR-T), surgical therapy, radiation therapy, etc.

[0069] The present invention will be specifically described below by showing examples, but the following description is not intended to limit the scope of the present invention to the scope of the examples.

[0070] Example 1: Humanization of antibody LpMab-23 A humanized antibody was prepared by replacing the CDRs of the murine anti-podoplanin antibody LpMab-23 described in Patent Document 2, i.e., the framework regions of the heavy chain variable region and light chain variable region, and the heavy chain constant region and light chain constant region, with amino acid sequences derived from a human antibody, according to standard methods. Hereinafter, humanized LpMab-23 will be referred to as hLpMab-23. The amino acid sequence of the heavy chain variable region of hLpMab-23 had 83% sequence identity to the amino acid sequence of SEQ ID NO:7. Furthermore, the amino acid sequence of the light chain variable region of hLpMab-23 had 83% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0071] Example 2 Preparation of Various Antibody-Drug Conjugates (ADCs) (2-1) Preparation of hLpMab-23-MMAE An ADC conjugated with the humanized anti-podoplanin antibody hLpMab-23 prepared in Example 1 and monomethyl auristatin E (MMAE) (a microtubule polymerization inhibitor) was prepared by the following procedure. 40 equivalents of a TCEP solution serving as a reducing agent were added to an aqueous hLpMab-23 solution, mixed, and stirred at room temperature for 2 hours, after which the reducing agent was removed by ultrafiltration. 20 equivalents of maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl-MMAE (MC-VC-PAB-MMAE) were then added, and the mixture was stirred at room temperature for 1 hour, followed by ultrafiltration and purification by preparative liquid chromatography (LC) (size exclusion chromatography). The purified product was dissolved in a buffer solution of pH 9.0 and then recovered again to obtain ADC (hLpMab-23-MMAE).

[0072] (2-2) Preparation of hLpMab-23-DM1 An ADC conjugated with hLpMab-23 and emtansine (DM1) (a microtubule polymerization inhibitor) was prepared by the following procedure. 20 equivalents of TCEP solution as a reducing agent were added to an hLpMab-23 solution, mixed, and stirred at room temperature for 2 hours. Then, 10 equivalents of MC-VC-PAB-DM1 was added and stirred at room temperature for 1 hour. After ultrafiltration, the mixture was purified by preparative LC (size exclusion chromatography) and the antibody peak fraction was collected. The collected solution was concentrated to obtain the ADC.

[0073] (2-3) Preparation of hLpMab-23-Exatecan An ADC in which exatecan (a DNA topoisomerase I inhibitor) was conjugated to hLpMab-23 was prepared by the following procedure. 20 equivalents of TCEP solution as a reducing agent were added to an hLpMab-23 solution, mixed, and stirred at room temperature for 2 hours, after which the reducing agent was removed by ultrafiltration. 10 equivalents of MC-VC-PAB-Exatecan was added to the solution after removal, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the product was purified by ultrafiltration and preparative LC (size exclusion chromatography), and the antibody peak fraction was collected. The collected solution was concentrated to obtain the ADC.

[0074] Example 3 Confirmation of Antibody-Drug Binding by Liquid Chromatography Hydrophobic liquid chromatography (HPLC) measurement was performed on the ADC prepared in Example 2 (2-1) under the following conditions. As a control, hLpMab-23 alone before binding was similarly measured by HPLC. - Column: TSKgel Butyl-NPR (particle size 2.5 μm, 4.6 mm × 3.5 cm) - Temperature: 25°C - Flow rate: 0.7 ml / min - Mobile phase: Solution A = 1.25 M ammonium sulfate, 20 mM phosphate buffer (pH = 7.0), Solution B = 25% IPA, 20 mM phosphate buffer (pH = 7.0) 0 to 8 minutes: Solution B 0 to 100% 8 to 13 minutes: Solution B 100% 13 to 18 minutes: Solution B 0%

[0075] An HPLC chart is shown in Figure 2. DAR (Drug Antibody Ratio) refers to the number of drug molecules bound per antibody molecule, and "DAR2," "DAR4," "DAR6," and "DAR8" refer to the peaks of complexes in which 2, 4, 6, and 8 drug molecules are bound per antibody molecule, respectively. In Example 2 (2-1), as shown in Figure 2, it was confirmed that ADCs in which 2, 4, 6, and 8 drug molecules are bound per antibody molecule were obtained.

[0076] Example 4: Evaluation of cytotoxicity of podoplanin-expressing cancer cells by hLpMab-23-MMAE Cytotoxicity was evaluated using the hLpMab-23-MMAE obtained in Example 2 (2-1) according to the following procedure. A253 cell line (human submandibular gland cancer cell line) overexpressing podoplanin was seeded onto a 96-well microplate and cultured overnight. After overnight culture, ADC (hLpMab-23-MMAE) or hLpMab-23 antibody alone was added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The cell viability of samples to which ADC or antibody alone was added at each concentration was calculated, with the control measurement value taken as 100%.

[0077] Figure 3 shows a plot of the concentration of added ADC or antibody alone versus the cell viability of podoplanin-expressing cancer cells in culture medium. Figure 3A shows the relationship between ADC concentration and cell viability of podoplanin-expressing cancer cells in culture medium, and Figure 3B shows the relationship between antibody alone concentration and cell viability of podoplanin-expressing cancer cells in culture medium. In culture medium to which antibody alone was added, there was almost no change in the viability of podoplanin-expressing cancer cells depending on the added concentration, whereas in culture medium to which ADC was added, it was confirmed that the viability of podoplanin-expressing cancer cells decreased depending on the added concentration.

[0078] Example 5 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to hLpMab-23-MMAE I Following the procedure below, the A253 cell line, in which podoplanin was forcibly expressed, and the podoplanin-negative HSQ-89 cell line (oral squamous cell carcinoma cell line) were each reacted with the ADC hLpMab-23-MMAE. The hLpMab-23-MMAE prepared in Example 2 (2-1) was used. The A253 cell line and HSQ-89 cell line, in which podoplanin was forcibly expressed, were each seeded onto a 96-well microplate and cultured overnight. After overnight culture, hLpMab-23-MMAE was added at eight different concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition, the absorbance at 450 nm was measured using a plate reader with Cell Counting Kit 8. As a control, each cell line was cultured without adding ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0079] Figure 4 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was overexpressed. The A253 cell line, in which podoplanin was overexpressed, showed a significant decrease in cell viability with 0.1 to 1 nM ADC, whereas the HSQ-89 cell line required an ADC concentration of 30 to 100 nM to reduce cell viability. This demonstrates that the ADC is specifically cytotoxic to podoplanin-positive cells.

[0080] Example 6 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to hLpMab-23-MMAE II LN319 cell line (human brain epithelial carcinoma cell line, podoplanin-positive) or LN229 cell line (human brain glioblastoma cell line, podoplanin-negative) was seeded onto a 96-well microplate and cultured overnight. Then, hLpMab-23-MMAE, the ADC obtained in Example 2 (2-1), was added at eight different concentrations by serial dilution (3-fold serial dilution), and cultured. Six days after the addition, Cell Counting Kit 8 was added, and the absorbance at 450 nm was measured using a plate reader. As a control, each cell was cultured without the addition of ADC. The measured value of the control for each cell line was set to 100%, and the cell viability of the sample to which the ADC was added at each concentration was calculated.

[0081] Figure 5 shows a plot of the concentration of added ADC versus the cell viability of the LN319 cell line (podoplanin-positive) and the LN229 cell line (podoplanin-negative). The LN319 cell line showed a decrease in cell viability with 0.3 to 3 nM of ADC, whereas the LN229 cell line required a concentration of ADC greater than 30 nM to decrease cell viability. This indicates that the ADC has specific cytotoxicity against podoplanin-positive cells.

[0082] Example 7 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to hLpMab-23-MMAE III KYSE140 cell line (human esophageal cancer cell line, podoplanin-positive) or KYSE410 cell line (human esophageal squamous cell carcinoma cell line, podoplanin-negative) was seeded onto a 96-well microplate and cultured overnight. After overnight culture, hLpMab-23-MMAE, the ADC obtained in Example 2 (2-1), was added at eight different concentrations by serial dilution (3-fold serial dilution) and cultured. Six days after the addition, Cell Counting Kit 8 was added, and the absorbance at 450 nm was measured using a plate reader. As a control, each cell line was cultured without the addition of ADC. The measured value of the control for each cell line was set to 100%, and the cell viability of the samples to which the ADC was added at each concentration was calculated.

[0083] Figure 6 shows a plot of the concentration of added ADC versus the cell viability of the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative). It was confirmed that the cell viability of the KYSE140 cell line was reduced at lower concentrations of ADC compared to the KYSE410 cell line. This indicates that the ADC has specific cytotoxicity against podoplanin-positive cells.

[0084] Example 8 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to hLpMab-23-DM1 The A253 cell line, in which podoplanin was forcibly expressed, and the podoplanin-negative HSQ-89 cell line were each reacted with hLpMab-23-DM1 according to the following procedure. hLpMab-23-DM1 prepared in Example 2 (2-2) was used. The A253 cell line and the HSQ-89 cell line, in which podoplanin was forcibly expressed, were each seeded into a 96-well microplate and cultured overnight. After overnight culture, hLpMab-23-DM1 was added at eight different concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition, the absorbance at 450 nm was measured with a plate reader using Cell Counting Kit 8. As a control, each cell line was cultured without the addition of ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0085] Figure 7 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. The A253 cell line, in which podoplanin was forcibly expressed, showed a significant decrease in cell viability with 1 to 100 nM ADC, whereas no decrease was observed in the HSQ-89 cell line. This demonstrates that the ADC has specific cytotoxicity against podoplanin-positive cells.

[0086] Example 9 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to hLpMab-23-Exatecan The A253 cell line, in which podoplanin was overexpressed, and the podoplanin-negative HSQ-89 cell line were each reacted with hLpMab-23-Exatecan according to the following procedure. The hLpMab-23-Exatecan prepared in Example 2 (2-3) was used. The A253 cell line and the HSQ-89 cell line, in which podoplanin was overexpressed, were each seeded into a 96-well microplate and cultured overnight. After overnight culture, hLpMab-23-Exatecan was added at eight different concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition, the absorbance at 450 nm was measured with a plate reader using Cell Counting Kit 8. As a control, each cell line was cultured without adding ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0087] Figure 8 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. The cell viability of the A253 cell line in which podoplanin was forcibly expressed was reduced by 1 to 10 nM of ADC, whereas a concentration of approximately 100 nM of ADC was required to reduce the cell viability of the HSQ-89 cell line. This demonstrates that the ADC is specifically cytotoxic to podoplanin-positive cells.

[0088] Example 10 Evaluation of tumor suppression effect in tumor-transplanted model mice I Podoplanin-expressing A253 cells were subcutaneously injected into the left dorsal region of BALB / cAJcl-nu / nu mice at a concentration of 4 × 10 per mouse. ６ After transplantation, the tumor volume was 100-200 mm ３ Mice were cultured until the average tumor volume reached 150-200 mm ３The animals were divided into groups of 5 animals each so that the tumor volume was 10 mg / kg. Each group was administered 10 mg / kg of ADC or hLpMab-23 prepared in Example 2 (2-1) into the tail vein once every 3 or 4 days, a total of 4 times. As a control, PBS was administered into the tail vein in place of ADC or hLpMab-23 in the same manner. The tumor volume was measured 7, 14, 21, 28, and 35 days after the start of administration.

[0089] The change in tumor size after the start of ADC or hLpMab-23 administration is shown in Figure 9. An increase in tumor size was observed in the hLpMab-23 administration group, but the increase in tumor size was shown to be suppressed in the ADC administration group.

[0090] Example 11: Evaluation of Responsiveness of Various Cell Lines to MMAE The responsiveness of podoplanin-positive and podoplanin-negative cancer cells to MMAE alone was evaluated. Podoplanin-positive cell lines (podoplanin-expressing A253, LN319, and KYSE140 cell lines) and podoplanin-negative cell lines (HSQ-89, LN229, and KYSE410 cell lines) were seeded into 96-well microplates and cultured overnight. MMAE was then added at eight different concentrations by serial dilution (3-fold serial dilution) and cultured. Six days after the addition, Cell Counting Kit 8 was added, and the absorbance at 450 nm was measured using a plate reader. As a control, each cell line was cultured without the addition of MMAE. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which MMAE was added at each concentration was calculated.

[0091] FIG. 10 shows plots of MMAE concentration versus cell viability for each podoplanin-positive cell line. FIG. 10A shows plots of MMAE concentration versus cell viability for the A253 cell line in which podoplanin was overexpressed. FIG. 10B shows plots of MMAE concentration versus cell viability for the LN319 cell line. FIG. 10C shows plots of MMAE concentration versus cell viability for the KYSE140 cell line. FIG. 11 shows plots of MMAE concentration versus cell viability for each podoplanin-negative cell line. FIG. 11A shows plots of MMAE concentration versus cell viability for the HSQ-89 cell line. FIG. 11B shows plots of MMAE concentration versus cell viability for the LN229 cell line. FIG. 11C shows plots of MMAE concentration versus cell viability for the KYSE410 cell line. Regardless of the cell type, the higher the MMAE concentration, the lower the cell viability was confirmed, indicating that MMAE alone does not have specific cytotoxicity.

[0092] [Example 12] Confirmation of binding of ADC to podoplanin-positive cells by flow cytometry 1 × 10 KYSE140 cell line (podoplanin-positive) and KYSE410 cell line (podoplanin-negative) were used. ５The cell suspension was dispensed into 1.5 mL tubes so that there were 1 cell / tube, and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of flow cytometry solution (FCM solution) was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 50 μL of 1 μg / mL hLpMab-23 antibody or hLpMab-23-MMAE prepared in Example 2 (2-1) was added, suspended, and allowed to stand on ice for 30 minutes or more. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. This procedure was repeated again. The supernatant was removed, and 50 μL of anti-MMAE antibody solution (1 μL of anti-MMAE antibody added to 1000 μL of FCM solution) was added, suspended, and the mixture was allowed to stand on ice for 30 minutes or more. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 50 μL of AF488-labeled anti-rabbit IgG antibody solution (1 μL of AF488-labeled anti-rabbit IgG antibody was added to 1000 μL of FCM solution) was added and suspended, and the mixture was left on ice for at least 30 minutes. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. This procedure was repeated again. The supernatant was removed, and the mixture was suspended in 400 μL of FCM solution and passed through a Cell Strainer. Fluorescence measurement analysis was performed using a flow cytometer (Becton Dickinson FACS Verse).

[0093] The results of flow cytometry are shown in Figure 12. Figure 12A shows flow cytometry of podoplanin-positive cells. Figure 12B shows flow cytometry of podoplanin-negative cells. In the system in which hLpMab-23-MMAE was added to podoplanin-positive cells, the number of cells with increased fluorescence intensity, indicating binding to the anti-MMAE antibody, increased. On the other hand, it was confirmed that no significant changes were observed in podoplanin-negative cells even when hLpMab-23-MMAE was added. The above results confirmed that hLpMab-23-MMAE prepared in Example 2 (2-1) is an ADC in which the podoplanin antibody hLpMab-23 is conjugated to the drug MMAE, and that the ADC specifically binds to podoplanin-positive cells.

[0094] Example 13 Cytotoxicity Test of ADCs on Human Umbilical Vein Endothelial Cells (HUVEC) Using human umbilical vein endothelial cells (HUVEC) as normal cells, the cytotoxicity of hLpMab-23-MMAE, hLpMab-23-DM1, and hLpMab-23-exatecan obtained in Examples 2(2-1) to (1-3) was evaluated.

[0095] HUVECs were seeded onto 96-well microplates and cultured overnight. Various ADCs (hLpMab-23-MMAE, hLpMab-23-DM1, and hLpMab-23-exatecan) or hLpMab-23 antibody alone were added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The cell viability of samples containing ADC or antibody alone at each concentration was calculated, with the control measurement value taken as 100%.

[0096] Figure 13 shows plots of the concentration of each ADC or antibody alone added versus cell viability in HUVEC culture medium. Figure 13A shows the relationship between the concentration of hLpMab-23 antibody alone and cell viability in HUVEC culture medium. Figure 13B shows the relationship between the concentration of hLpMab-23-MMAE and cell viability in HUVEC culture medium. Figure 13C shows the relationship between the concentration of hLpMab-23-DM1 and cell viability in HUVEC culture medium. Figure 13D shows the relationship between the concentration of hLpMab-23-exatecan and cell viability in HUVEC culture medium. It was confirmed that in both the culture medium containing the antibody alone and the various ADCs, almost no change in cell viability was observed up to an addition amount of 30 nM or more. These results confirmed that the ADC specifically damages cancer cells.

[0097] Example 14: Preparation of chLpMab-23-MMAE An ADC conjugated with chLpMab-23, a human chimeric anti-podoplanin antibody described in Patent Document 2, and monomethyl auristatin E (MMAE) (a microtubule polymerization inhibitor) was prepared by the following procedure. 14.5 equivalents of TCEP solution as a reducing agent were added to an aqueous chLpMab-23 solution, mixed, and stirred at room temperature for 2 hours, after which the reducing agent was removed by ultrafiltration. Thereafter, 20 equivalents of EDTA (final concentration 1 mM) and maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl-MMAE (MC-VC-PAB-MMAE) were added, stirred at room temperature for 30 minutes, and then ultrafiltered. Purification was carried out by preparative liquid chromatography (LC) (size exclusion chromatography).

[0098] Example 15 Confirmation of Antibody-Drug Binding by Liquid Chromatography Hydrophobic liquid chromatography (HPLC) measurement was performed on the ADC prepared in Example 14 under the following conditions: Column: BioPro HIC HT (particle size 2.3 μm, 4.6 mm × 10 cm) Temperature: 25°C Flow rate: 0.4 mL / min Mobile phase: Solution A = 1.25 M ammonium sulfate, 20 mM phosphate buffer (pH = 7.0), Solution B = 25% IPA, 20 mM phosphate buffer (pH = 7.0) 0 to 15 minutes: Solution B 0 to 100% 15 to 19 minutes: Solution B 100% 19 to 24 minutes: Solution B 0%

[0099] An HPLC chart is shown in Figure 14. DAR (Drug Antibody Ratio) refers to the number of drug molecules bound per antibody molecule. In Example 15, as shown in Figure 14, it was confirmed that ADCs in which 2, 4, 6, and 8 drug molecules were bound per antibody molecule were obtained.

[0100] Example 16: Evaluation of Cytotoxicity of Podoplanin-Expressing Cancer Cells by chLpMab-23-MMAE Using the chLpMab-23-MMAE obtained in Example 14, cytotoxicity was evaluated according to the following procedure. A253 cell lines (human submandibular gland cancer cell lines) in which podoplanin was forcibly expressed were seeded onto a 96-well microplate and cultured overnight. After overnight culture, ADC (chLpMab-23-MMAE) or chLpMab-23 antibody alone was added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The cell viability of samples to which ADC or antibody alone was added at each concentration was calculated, with the control measurement value taken as 100%.

[0101] Figure 15 shows plots of the concentration of added ADC or antibody alone versus the cell viability of podoplanin-expressing cancer cells in culture medium. Figure 15A shows the relationship between ADC concentration and cell viability of podoplanin-expressing cancer cells in culture medium, and Figure 15B shows the relationship between antibody alone concentration and cell viability of podoplanin-expressing cancer cells in culture medium. In culture medium to which antibody alone was added, there was almost no change in the viability of podoplanin-expressing cancer cells depending on the added concentration, whereas in culture medium to which ADC was added, it was confirmed that the viability of podoplanin-expressing cancer cells decreased depending on the added concentration.

[0102] Example 17 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to chLpMab-23-MMAE I In the following procedure, the A253 cell line in which podoplanin was forcibly expressed and the podoplanin-negative HSQ-89 cell line (oral squamous cell carcinoma cell line) were each reacted with the ADC chLpMab-23-MMAE. chLpMab-23-MMAE prepared in Example 14 was used. The A253 cell line and HSQ-89 cell line in which podoplanin was forcibly expressed were each seeded into a 96-well microplate and cultured overnight. Then, chLpMab-23-MMAE was added to eight concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition treatment, the absorbance at 450 nm was measured using a plate reader with Cell Counting Kit 8. As a control, each cell line was cultured without adding ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0103] Figure 16 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. The A253 cell line, in which podoplanin was forcibly expressed, showed a significant decrease in cell viability with approximately 0.1 nM ADC, whereas the HSQ-89 cell line required an ADC concentration of 5 to 50 nM to decrease cell viability. This demonstrates that the ADC is specifically cytotoxic to podoplanin-positive cells.

[0104] Example 18 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to chLpMab-23-MMAE II KYSE140 cell line (human esophageal cancer cell line, podoplanin-positive) or KYSE410 cell line (human esophageal squamous cell carcinoma cell line, podoplanin-negative) was seeded onto a 96-well microplate and cultured overnight. Then, chLpMab-23-MMAE, the ADC obtained in Example 14, was added at eight different concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition, Cell Counting Kit 8 was added, and the absorbance at 450 nm was measured using a plate reader. As a control, each cell was cultured without the addition of ADC. The measured value of the control for each cell line was set to 100%, and the cell viability of the sample to which the ADC was added at each concentration was calculated.

[0105] Figure 17 shows a plot of the concentration of added ADC versus the cell viability of the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative). The cell viability of the KYSE140 cell line was significantly reduced by approximately 1 nM of ADC, whereas the cell viability of the KYSE410 cell line required an ADC concentration of 5 to 50 nM. This indicates that the ADC has specific cytotoxicity against podoplanin-positive cells.

[0106] [Example 19] Confirmation of binding of ADC to podoplanin-positive cells by flow cytometry 1 × 10 KYSE140 cell line (podoplanin-positive) and KYSE410 cell line (podoplanin-negative) were used. ５The cell suspension was dispensed into 1.5 mL tubes so that there were 1 cell / tube, and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of flow cytometry solution (FCM solution: PBS containing 2% FBS) was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 50 μL of 1 μg / mL chLpMab-23 antibody or chLpMab-23-MMAE prepared in Example 14 was added and suspended, and the mixture was allowed to stand on ice for 30 minutes or more. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. This procedure was repeated again. The supernatant was removed, and 50 μL of anti-MMAE antibody solution (1 μL of anti-MMAE antibody added to 1000 μL of FCM solution) was added and suspended, and the mixture was allowed to stand on ice for 30 minutes or more. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 50 μL of AF488-labeled anti-rabbit IgG antibody solution (1 μL of AF488-labeled anti-rabbit IgG antibody was added to 1000 μL of FCM solution) was added and suspended, and the mixture was left on ice for at least 30 minutes. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. This procedure was repeated again. The supernatant was removed, and the mixture was suspended in 400 μL of FCM solution and passed through a Cell Strainer. Fluorescence measurement analysis was performed using a flow cytometer (Becton Dickinson FACS Verse).

[0107] The results of flow cytometry are shown in Figure 18. Figure 18A shows flow cytometry of podoplanin-positive cells. Figure 18B shows flow cytometry of podoplanin-negative cells. In the system in which chLpMab-23-MMAE was added to podoplanin-positive cells, the number of cells with increased fluorescence intensity, indicating binding with the anti-MMAE antibody, increased. On the other hand, it was confirmed that no significant changes were observed in podoplanin-negative cells even when chLpMab-23-MMAE was added. From the above results, it was confirmed that chLpMab-23-MMAE prepared in Example 14 is an ADC in which the podoplanin antibody chLpMab-23 and the drug MMAE are bound, and that the ADC specifically binds to podoplanin-positive cells.

[0108] [Example 20] Preparation of chLpMab-2-MMAE An ADC was prepared by conjugating chLpMab-2, a human chimeric anti-podoplanin antibody described in Patent Document 2, with monomethyl auristatin E (MMAE) (a microtubule polymerization inhibitor). The ADC was prepared in the same manner as in "[Example 14] Preparation of chLpMab-23-MMAE," except that chLpMab-2 was used as the anti-podoplanin antibody instead of chLpMab-23.

[0109] Example 21 Confirmation of Antibody-Drug Binding by Liquid Chromatography (2) The ADC prepared in Example 20 was subjected to hydrophobic liquid chromatography (HPLC) measurement under the same conditions as in Example 15.

[0110] An HPLC chart is shown in Figure 19. DAR (Drug Antibody Ratio) refers to the number of drug molecules bound per antibody molecule. In Example 21, as shown in Figure 19, it was confirmed that ADCs in which 2, 4, 6, and 8 drug molecules were bound per antibody molecule were obtained.

[0111] Example 22: Evaluation of Cytotoxicity of Podoplanin-Expressing Cancer Cells by chLpMab-2-MMAE Using the chLpMab-2-MMAE obtained in Example 20, cytotoxicity was evaluated according to the following procedure. A253 cell lines (human submandibular gland cancer cell lines) in which podoplanin was forcibly expressed were seeded onto a 96-well microplate and cultured overnight. After overnight culture, ADC (chLpMab-2-MMAE) or chLpMab-2 antibody alone was added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The cell viability of samples to which ADC or antibody alone was added at each concentration was calculated, with the control measurement value taken as 100%.

[0112] Figure 20 shows plots of the concentration of added ADC or antibody alone versus the cell viability of podoplanin-expressing cancer cells in culture medium. Figure 20A shows the relationship between ADC concentration and cell viability of podoplanin-expressing cancer cells in culture medium, and Figure 20B shows the relationship between antibody alone concentration and cell viability of podoplanin-expressing cancer cells in culture medium. In culture medium to which antibody alone was added, there was almost no change in the viability of podoplanin-expressing cancer cells depending on the added concentration, whereas in culture medium to which ADC was added, it was confirmed that the viability of podoplanin-expressing cancer cells decreased depending on the added concentration.

[0113] Example 23 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to chLpMab-2-MMAE In the following procedure, the A253 cell line in which podoplanin was forcibly expressed and the podoplanin-negative HSQ-89 cell line (oral squamous cell carcinoma cell line) were each reacted with the ADC chLpMab-2-MMAE. chLpMab-2-MMAE prepared in Example 20 was used. The A253 cell line and HSQ-89 cell line in which podoplanin was forcibly expressed were each seeded into a 96-well microplate and cultured overnight. Then, chLpMab-2-MMAE was added to eight concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition treatment, the absorbance at 450 nm was measured using a plate reader with Cell Counting Kit 8. As a control, each cell line was cultured without adding ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0114] Figure 21 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. The A253 cell line, in which podoplanin was forcibly expressed, showed a significant decrease in cell viability with 1 to 3 nM ADC, whereas the HSQ-89 cell line required an ADC concentration of 30 to 100 nM to decrease cell viability. This demonstrates that the ADC is specifically cytotoxic to podoplanin-positive cells.

[0115] [Example 24] Preparation of chLpMab-7-MMAE An ADC was prepared by conjugating chLpMab-7, a human chimeric anti-podoplanin antibody described in Patent Document 2, with monomethyl auristatin E (MMAE) (a microtubule polymerization inhibitor). The ADC was prepared in the same manner as in "[Example 14] Preparation of chLpMab-23-MMAE," except that chLpMab-7 was used as the anti-podoplanin antibody instead of chLpMab-23.

[0116] Example 25 Confirmation of Antibody-Drug Binding by Liquid Chromatography (3) The ADC prepared in Example 24 was subjected to hydrophobic liquid chromatography (HPLC) measurement under the same conditions as in Example 15.

[0117] An HPLC chart is shown in Figure 22. DAR (Drug Antibody Ratio) refers to the number of drug molecules bound per antibody molecule. In Example 25, as shown in Figure 22, it was confirmed that ADCs in which 2, 4, 6, and 8 drug molecules were bound per antibody molecule were obtained.

[0118] Example 26: Evaluation of Cytotoxicity of Podoplanin-Expressing Cancer Cells by chLpMab-7-MMAE Using the chLpMab-7-MMAE obtained in Example 24, cytotoxicity was evaluated according to the following procedure. A253 cell lines (human submandibular gland cancer cell lines) in which podoplanin was forcibly expressed were seeded onto a 96-well microplate and cultured overnight. After overnight culture, ADC (chLpMab-7-MMAE) or chLpMab-7 antibody alone was added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The cell viability of samples to which ADC or antibody alone was added at each concentration was calculated, with the control measurement value taken as 100%.

[0119] Figure 23 shows plots of the concentration of added ADC or antibody alone versus the cell viability of podoplanin-expressing cancer cells in culture medium. Figure 23A shows the relationship between ADC concentration and cell viability of podoplanin-expressing cancer cells in culture medium, and Figure 23B shows the relationship between antibody alone concentration and cell viability of podoplanin-expressing cancer cells in culture medium. In culture medium to which antibody alone was added, there was almost no change in the viability of podoplanin-expressing cancer cells depending on the added concentration, whereas in culture medium to which ADC was added, it was confirmed that the viability of podoplanin-expressing cancer cells decreased depending on the added concentration.

[0120] Example 27 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to chLpMab-7-MMAE I In the following procedure, the A253 cell line in which podoplanin was forcibly expressed and the podoplanin-negative HSQ-89 cell line (oral squamous cell carcinoma cell line) were each reacted with the ADC chLpMab-7-MMAE. chLpMab-7-MMAE prepared in Example 24 was used. The A253 cell line and HSQ-89 cell line in which podoplanin was forcibly expressed were each seeded into a 96-well microplate and cultured overnight. Then, chLpMab-7-MMAE was added to eight concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition treatment, the absorbance at 450 nm was measured using a plate reader with Cell Counting Kit 8. As a control, each cell line was cultured without adding ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0121] Figure 24 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. The A253 cell line, in which podoplanin was forcibly expressed, showed a significant decrease in cell viability with 0.03 to 0.1 nM ADC, whereas the HSQ-89 cell line required an ADC concentration of 10 to 30 nM to decrease cell viability. This demonstrates that the ADC is specifically cytotoxic to podoplanin-positive cells.

[0122] Example 28 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to chLpMab-7-MMAE II KYSE140 cell line (human esophageal cancer cell line, podoplanin-positive) or KYSE410 cell line (human esophageal squamous cell carcinoma cell line, podoplanin-negative) was seeded onto a 96-well microplate and cultured overnight. Then, chLpMab-7-MMAE, the ADC obtained in Example 24, was added at eight different concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition, Cell Counting Kit 8 was added, and the absorbance at 450 nm was measured using a plate reader. As a control, each cell was cultured without the addition of ADC. The measured value of the control for each cell line was set to 100%, and the cell viability of the sample to which the ADC was added at each concentration was calculated.

[0123] Figure 25 shows a plot of the concentration of added ADC versus the cell viability of the KYSE140 cell line (podoplanin-positive) and the KYSE410 cell line (podoplanin-negative). The cell viability of the KYSE140 cell line was significantly reduced by 0.1 to 0.3 nM of ADC, whereas the cell viability of the KYSE410 cell line required an ADC concentration of 10 to 30 nM. This indicates that the ADC has specific cytotoxicity against podoplanin-positive cells.

[0124] [Example 29] Confirmation of binding of ADC to podoplanin-positive cells by flow cytometry 1 × 10 KYSE140 cell line (podoplanin-positive) and KYSE410 cell line (podoplanin-negative) were used. ５The cell suspension was dispensed into 1.5 mL tubes so that there were 1 cell / tube, and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of flow cytometry solution (FCM solution) was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 50 μL of 1 μg / mL chLpMab-7 antibody or chLpMab-7-MMAE prepared in Example 24 was added, suspended, and allowed to stand on ice for 30 minutes or more. 1 mL of FCM solution was added, and the mixture was centrifuged at 1200 rpm for 5 minutes. This procedure was repeated again. The supernatant was removed, and 50 μL of anti-MMAE antibody solution (1 μL of anti-MMAE antibody (Levena Biopharma) added to 1000 μL of FCM solution) was added, suspended, and allowed to stand on ice for 30 minutes or more. 1 mL of FCM solution was added and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of FCM solution was added and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 50 μL of AF488-labeled anti-rabbit IgG antibody solution (1 μL of AF488-labeled anti-rabbit IgG antibody was added to 1000 μL of FCM solution) was added and suspended, and the mixture was left on ice for at least 30 minutes. 1 mL of FCM solution was added and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 1 mL of FCM solution was added and centrifuged at 1200 rpm for 5 minutes. This procedure was repeated three more times. The supernatant was removed, and the mixture was suspended in 400 μL of FCM solution and passed through a Cell Strainer. Fluorescence measurement analysis was performed using a flow cytometer (Becton Dickinson FACS Verse).

[0125] Figure 26 shows the results of flow cytometry. Figure 26A shows flow cytometry of podoplanin-positive cells. Figure 26B shows flow cytometry of podoplanin-negative cells. In the system in which chLpMab-7-MMAE was added to podoplanin-positive cells, the number of cells with increased fluorescence intensity, indicating binding with the anti-MMAE antibody, increased. On the other hand, it was confirmed that no significant changes were observed in podoplanin-negative cells even when chLpMab-7-MMAE was added. From the above results, it was confirmed that chLpMab-7-MMAE prepared in Example 24 is an ADC in which the podoplanin antibody chLpMab-7 and the drug MMAE are bound, and that the ADC specifically binds to podoplanin-positive cells.

[0126] Example 30 Preparation of hLpMab-23-Eribulin An ADC was prepared by conjugating eribulin (a microtubule inhibitor) to the humanized antibody hLpMab-23 prepared in Example 1 using the following procedure. 10 equivalents of a TCEP solution serving as a reducing agent were added to an hLpMab-23 solution, mixed, and stirred at room temperature for 2 hours, after which the reducing agent was removed by ultrafiltration. 20 equivalents of MC-VC-PAB-Eribulin was added to the solution after removal, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was purified by ultrafiltration and preparative LC (size exclusion chromatography), and the antibody peak fraction was collected. The collected solution was concentrated to obtain an ADC.

[0127] Example 31 Comparison of the Responsiveness of Podoplanin-Expressing and Non-Expressing Cancer Cells to hLpMab-23-Eribulin The A253 cell line, in which podoplanin was forcibly expressed, and the podoplanin-negative HSQ-89 cell line (oral squamous cell carcinoma cell line), were each reacted with the ADC hLpMab-23-Eribulin according to the following procedure. The hLpMab-23-Eribulin prepared in Example 30 was used. The A253 cell line and the HSQ-89 cell line, in which podoplanin was forcibly expressed, were each seeded into a 96-well microplate and cultured overnight. After overnight culture, hLpMab-23-Eribulin was added at 11 concentrations by serial dilution (3-fold serial dilution), and the culture was continued. Six days after the addition, the absorbance at 450 nm was measured with a plate reader using Cell Counting Kit 8. As a control, each cell line was cultured without adding ADC. The control measurement value for each cell line was set to 100%, and the cell viability of the sample to which ADC was added at each concentration was calculated.

[0128] Figure 27 shows a plot of the concentration of added ADC versus the cell viability of the A253 cell line (podoplanin-positive) and HSQ-89 cell line (podoplanin-negative) in which podoplanin was forcibly expressed. The A253 cell line, in which podoplanin was forcibly expressed, showed a significant decrease in cell viability with 0.01 to 0.1 nM ADC, whereas the HSQ-89 cell line required an ADC concentration of 3 to 10 nM to decrease cell viability. This demonstrates that the ADC is specifically cytotoxic to podoplanin-positive cells.

[0129] Example 32: Evaluation of Cytotoxicity of Podoplanin-Expressing Cancer Cells by hLpMab-23-MMAE IV Cytotoxicity was evaluated using the hLpMab-23-MMAE obtained in Example 2 (2-1) according to the following procedure. Podoplanin-positive NCI-H226 cell line (human lung squamous cell carcinoma cell line) was seeded onto a 96-well microplate and cultured overnight. After overnight culture, ADC (hLpMab-23-MMAE) or hLpMab-23 antibody alone was added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The measured value of the control was set as 100%, and the cell viability of the samples to which the ADC or antibody alone was added at each concentration was calculated.

[0130] 28 shows a plot of the cell viability of podoplanin-expressing cancer cells in culture medium against the concentrations of added ADC and antibody alone. In culture medium to which antibody alone was added, there was almost no change in the viability of podoplanin-expressing cancer cells depending on the added concentration, whereas in culture medium to which ADC was added, it was confirmed that the viability of podoplanin-expressing cancer cells decreased depending on the added concentration.

[0131] Example 33: Evaluation of Cytotoxicity of Podoplanin-Expressing Cancer Cells by hLpMab-23-MMAE V Using the hLpMab-23-MMAE obtained in Example 2 (2-1), cytotoxicity was evaluated according to the following procedure. Podoplanin-positive DU145 cell lines (human prostate cancer-derived cell lines) were seeded onto a 96-well microplate and cultured overnight. After overnight culture, ADC (hLpMab-23-MMAE) or hLpMab-23 antibody alone was added at eight different concentrations by serial dilution (3-fold serial dilution), and the cells were cultured. Six days after the addition, Cell Counting Kit 8 (Dojindo Laboratories, Inc.) was added, and the absorbance at 450 nm was measured using a plate reader. As a control, cells were cultured without the addition of ADC or antibody alone. The cell viability of samples to which ADC or antibody alone was added at each concentration was calculated, with the control measurement value taken as 100%.

[0132] A plot of the cell viability of podoplanin-expressing cancer cells in culture medium against the concentrations of added ADC and antibody alone is shown in Figure 29. In culture medium to which antibody alone was added, there was almost no change in the viability of podoplanin-expressing cancer cells depending on the added concentration, whereas in culture medium to which ADC was added, it was confirmed that the viability of podoplanin-expressing cancer cells decreased depending on the added concentration.

[0133] Example 34 Evaluation of tumor suppression effect of hLpMab-23-MMAE in tumor-transplanted model mice KYSE140 cells (human esophageal cancer cell line, podoplanin-positive) were subcutaneously injected into the left dorsal region of BALB / cAJcl-nu / nu mice at a concentration of 6 × 10 per mouse. ６ After transplantation, the tumor volume was 100-200 mm ３ Mice were cultured until the average tumor volume reached 150-200 mm ３The mice were divided into groups of 8 or 9 so that the tumor volume was 1000 mg / kg body weight. Each group was administered with the ADC or hLpMab-23 prepared in Example 2 (2-1), or PBS as a control, as follows: Group 1: PBS administered once Group 2: hLpMab-23 10 mg / kg body weight, once a week, for a total of 2 doses Group 3: ADC 10 mg / kg body weight, administered once a week Group 4: ADC 10 mg / kg body weight, once a week, for a total of 2 doses Group 5: ADC 5 mg / kg body weight, once a week, for a total of 2 doses Group 6: ADC 1 mg / kg body weight, twice a week, for a total of 4 doses The tumor volume was measured 7, 14, 21, and 28 days after the start of administration.

[0134] The changes in tumor size after the start of ADC or hLpMab-23 administration are shown in Figure 30. While a significant increase in tumor size was observed in Groups 1 and 2, the ADC-administered group demonstrated a dose-dependent suppression of tumor size increase. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An antibody-drug conjugate comprising a polypeptide consisting of an antibody that specifically binds to podoplanin or an antigen-binding fragment thereof and a drug, bound directly or indirectly.

2. The antibody-drug conjugate according to claim 1, wherein the drug is for use in the treatment, diagnosis and / or prevention of a disease in which podoplanin is involved.

3. The antibody-drug conjugate of claim 1, wherein the drug is an anticancer drug.

4. The antibody-drug conjugate of claim 1, wherein the polypeptide specifically binds to tumor tissue or cancer cells that express podoplanin.

5. The antibody-drug conjugate of claim 4, wherein the tumor tissue or cancer cells are cancer cells derived from any one of the group consisting of head and neck cancer, brain tumor, mesothelioma, testicular tumor, ovarian cancer, oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, skin cancer, osteosarcoma, hemangioma, thyroid cancer, prostate cancer, and cervical cancer.

6. The antibody-drug conjugate of claim 3, wherein the anticancer drug is at least one compound selected from the group consisting of alkylating agents, platinum compounds, antimetabolites (e.g., antifolates, pyridine metabolism inhibitors, and purine metabolism inhibitors), ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, antitumor antibiotics, antihormones, and cytokine agents.

7. The antibody-drug conjugate of claim 6, wherein the anticancer drug is at least one compound selected from the group consisting of monomethylauristatin E (MMAE), exatecan, emtansine (DM1), and eribulin.

8. The antibody-drug conjugate of claim 1, wherein the polypeptide has at least one of six CDRs: heavy chain complementarity-determining region (CDR) 1 consisting of the amino acid sequence represented by SEQ ID NO: 1; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

6.

9. The antibody-drug conjugate of claim 8, wherein the polypeptide comprises: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7 or the amino acid sequence represented by SEQ ID NO:1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, said heavy chain variable region having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8 or the amino acid sequence represented by SEQ ID NO:4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:6, said light chain variable region having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

8.

10. The antibody-drug conjugate of claim 1, wherein the polypeptide has at least one of six CDRs: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:9; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:10; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:11; light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:12; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:13; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

14.

11. The antibody-drug conjugate of claim 10, wherein the polypeptide comprises: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:15 or the amino acid sequence represented by SEQ ID NO:9, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:10, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:11, said heavy chain variable region having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:15; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:16 or the amino acid sequence represented by SEQ ID NO:12, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:13, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:14, said light chain variable region having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

16.

12. The antibody-drug conjugate of claim 1, wherein the polypeptide has at least one of six CDRs: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:26; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:27; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:28; a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:29; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:30; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

31.

13. The antibody-drug conjugate of claim 12, wherein the polypeptide comprises: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:32 or the amino acid sequence represented by SEQ ID NO:26, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:27, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:28, said heavy chain variable region having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:32; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:33 or the amino acid sequence represented by SEQ ID NO:29, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:30, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:31, said light chain variable region having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

33.

14. The antibody-drug conjugate of claim 1, wherein the antibody is a humanized antibody or a human chimeric antibody.

15. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 as an active ingredient.

16. The pharmaceutical composition according to claim 15, for use in the treatment, diagnosis and / or prevention of a disease associated with podoplanin in a subject.

17. The pharmaceutical composition of claim 16, wherein the disease is cancer.

18. The pharmaceutical composition according to claim 17, wherein the cancer is a cancer containing cells that express podoplanin.

19. The pharmaceutical composition of claim 16, wherein the subject is an animal, including a human.

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